Optical interrogation of neural circuits in Caenorhabditis elegans

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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Figure 1: Challenges facing an all-optical interrogation of neural circuits.
Figure 2: Activation of ChR2 in ASH neurons by whole-body illumination.
Figure 3: Optical setup for ChR2 stimulation and simultaneous G-CaMP imaging.
Figure 4: Validation of specific stimulation in vivo.
Figure 5: Stimulation of ChR2 in ASH neuron activates AVA and AVD neurons.
Figure 6: Specific stimulation of ChR2 in RIM activates AVA and generates an avoidance response.

References

  1. 1

    Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    White, J.G., Southgate, E., Thomson, J.N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).

    CAS  Article  Google Scholar 

  3. 3

    Chalfie, M. et al. The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci. 5, 956–964 (1985).

    CAS  Article  Google Scholar 

  4. 4

    Bargmann, C.I. & Avery, L. Laser killing of cells in Caenorhabditis elegans. Methods Cell Biol. 48, 225–250 (1995).

    CAS  Article  Google Scholar 

  5. 5

    de Bono, M. & Maricq, A.V. Neuronal substrates of complex behaviors in C. elegans. Annu. Rev. Neurosci. 28, 451–501 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Goodman, M.B., Hall, D.H., Avery, L. & Lockery, S.R. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron 20, 763–772 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Chronis, N., Zimmer, M. & Bargmann, C.I. Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat. Methods 4, 727–731 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Kerr, R. et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940–13945 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Zhang, F. et al. Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat. Neurosci. 11, 631–633 (2008).

    Article  Google Scholar 

  13. 13

    Han, X. & Boyden, E.S. Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution. PLoS One 2, e299 (2007).

    Article  Google Scholar 

  14. 14

    Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15, 2279–2284 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Liewald, J.F. et al. Optogenetic analysis of synaptic function. Nat. Methods 5, 895–902 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Hilliard, M.A. et al. In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. EMBO J. 24, 63–72 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Lee, R.Y., Sawin, E.R., Chalfie, M., Horvitz, H.R. & Avery, L. EAT-4, a homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is necessary for glutamatergic neurotransmission in Caenorhabditis elegans. J. Neurosci. 19, 159–167 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Berger, A.J., Hart, A.C. & Kaplan, J.M. G alphas-induced neurodegeneration in Caenorhabditis elegans. J. Neurosci. 18, 2871–2880 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Gray, J.M., Hill, J.J. & Bargmann, C.I. A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 102, 3184–3191 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Alkema, M.J., Hunter-Ensor, M., Ringstad, N. & Horvitz, H.R. Tyramine Functions independently of octopamine in the Caenorhabditis elegans nervous system. Neuron 46, 247–260 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Troemel, E.R., Chou, J.H., Dwyer, N.D., Colbert, H.A. & Bargmann, C.I. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207–218 (1995).

    CAS  Article  Google Scholar 

  23. 23

    Bacaj, T., Tevlin, M., Lu, Y. & Shaham, S. Glia are essential for sensory organ function in C. elegans. Science 322, 744–747 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Tobin, D. et al. Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 35, 307–318 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Ward, A., Liu, J., Feng, Z. & Xu, X.Z. Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nat. Neurosci. 11, 916–922 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Edwards, S.L. et al. A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans. PLoS Biol. 6, e198 (2008).

    Article  Google Scholar 

  27. 27

    Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Brockie, P.J., Madsen, D.M., Zheng, Y., Mellem, J. & Maricq, A.V. Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42. J. Neurosci. 21, 1510–1522 (2001).

    CAS  Article  Google Scholar 

  29. 29

    Liu, Q., Chen, B., Gaier, E., Joshi, J. & Wang, Z.W. Low conductance gap junctions mediate specific electrical coupling in body-wall muscle cells of Caenorhabditis elegans. J. Biol. Chem. 281, 7881–7889 (2006).

    CAS  Article  Google Scholar 

  30. 30

    Kotlikoff, M.I. Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. J. Physiol. (Lond.) 578, 55–67 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    CAS  Article  Google Scholar 

  32. 32

    McIntire, S.L., Jorgensen, E. & Horvitz, H.R. Genes required for GABA function in Caenorhabditis elegans. Nature 364, 334–337 (1993).

    CAS  Article  Google Scholar 

  33. 33

    Clark, D.A., Gabel, C.V., Gabel, H. & Samuel, A.D. Temporal activity patterns in thermosensory neurons of freely moving Caenorhabditis elegans encode spatial thermal gradients. J. Neurosci. 27, 6083–6090 (2007).

    CAS  Article  Google Scholar 

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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.

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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

Supplementary Text and Figures

Supplementary Figures 1–20, Supplementary Tables 1,2 and Supplementary Note (PDF 1969 kb)

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. (MOV 730 kb)

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. (MOV 668 kb)

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'. (MOV 350 kb)

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. (MOV 939 kb)

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. (MOV 225 kb)

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. (MOV 877 kb)

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Guo, Z., Hart, A. & Ramanathan, S. Optical interrogation of neural circuits in Caenorhabditis elegans. Nat Methods 6, 891–896 (2009). https://doi.org/10.1038/nmeth.1397

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