Optogenetic analysis of synaptic function


We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or γ-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for γ-aminobutyric acid) or contraction (for acetylcholine), which we analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy. In dissected worms, photostimulation evoked neurotransmitter-specific postsynaptic currents that could be triggered repeatedly and at various frequencies. Light-evoked behaviors and postsynaptic currents were significantly (P ≤ 0.05) altered in mutants with pre- or postsynaptic defects, although the behavioral phenotypes did not unambiguously report on synaptic function in all cases tested. OptIoN facilitates the analysis of neurotransmission with high temporal precision, in a neurotransmitter-selective manner, possibly allowing future investigation of synaptic plasticity in C. elegans.

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Figure 1: Photoactivation of ChR2-YFP in cholinergic and GABAergic neurons causes muscle contraction and relaxation.
Figure 2: Light-induced ACh or GABA release evokes postsynaptic currents at the neuromuscular junction.
Figure 3: GABA photo-ePSCs and GABA-mediated behaviors are altered in neurotransmission mutants.
Figure 4: ACh photo-ePSCs and ACh-mediated behaviors are altered in neurotransmission mutants.
Figure 5: ACh and GABA photo-ePSCs and ACh-electro-ePSCs during repeated stimulation.
Figure 6: ACh and GABA photo-ePSCs during repeated photostimulation are altered in neurotransmission mutants implicated in synaptic-vesicle priming and recycling.

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  1. 1

    Wojcik, S.M. & Brose, N. Regulation of membrane fusion in synaptic excitation-secretion coupling: speed and accuracy matter. Neuron 55, 11–24 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Richmond, J.E. Synaptic function. In WormBook (ed., The C. elegans Research Community) (doi/10.1895/wormbook.1.69.1; 2005).

  3. 3

    Richmond, J.E. & Jorgensen, E.M. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci. 2, 791–797 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Guerrero, G. et al. Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nat. Neurosci. 8, 1188–1196 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Miller, K.G. et al. A genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proc. Natl. Acad. Sci. USA 93, 12593–12598 (1996).

    CAS  Article  Google Scholar 

  6. 6

    Richmond, J.E. Electrophysiological recordings from the neuromuscular junction of C. elegans. In WormBook (ed., The C. elegans Research Community) (doi/10.1895/wormbook.1.112.1; 2006).

    Google Scholar 

  7. 7

    Francis, M.M., Mellem, J.E. & Maricq, A.V. Bridging the gap between genes and behavior: recent advances in the electrophysiological analysis of neural function in Caenorhabditis elegans. Trends Neurosci. 26, 90–99 (2003).

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Schuske, K.R. et al. Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin. Neuron 40, 749–762 (2003).

    CAS  Article  Google Scholar 

  10. 10

    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 

  11. 11

    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 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Li, X. et al. Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc. Natl. Acad. Sci. USA 102, 17816–17821 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Zhang, Y.P. & Oertner, T.G. Optical induction of synaptic plasticity using a light-sensitive channel. Nat. Methods 4, 139–141 (2007).

    CAS  Article  Google Scholar 

  16. 16

    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 

  17. 17

    McIntire, S.L., Reimer, R.J., Schuske, K., Edwards, R.H. & Jorgensen, E.M. Identification and characterization of the vesicular GABA transporter. Nature 389, 870–876 (1997).

    CAS  Article  Google Scholar 

  18. 18

    Alfonso, A., Grundahl, K., Duerr, J.S., Han, H.P. & Rand, J.B. The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter. Science 261, 617–619 (1993).

    CAS  Article  Google Scholar 

  19. 19

    McIntire, S.L., Jorgensen, E., Kaplan, J. & Horvitz, H.R. The GABAergic nervous system of Caenorhabditis elegans. Nature 364, 337–341 (1993).

    CAS  Article  Google Scholar 

  20. 20

    Bamber, B.A., Beg, A.A., Twyman, R.E. & Jorgensen, E.M. The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor. J. Neurosci. 19, 5348–5359 (1999).

    CAS  Article  Google Scholar 

  21. 21

    Stephens, G.J., Johnson-Kerner, B., Bialek, W. & Ryu, W.S. Dimensionality and dynamics in the behavior of C. elegans. PLoS Comput. Biol. 4, e1000028 (2008).

    Article  Google Scholar 

  22. 22

    Liu, Q. et al. Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction. J. Neurosci. 25, 6745–6754 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Hammarlund, M., Palfreyman, M.T., Watanabe, S., Olsen, S. & Jorgensen, E.M. Open syntaxin docks synaptic vesicles. PLoS Biol. 5, e198 (2007).

    Article  Google Scholar 

  24. 24

    Richmond, J.E., Weimer, R.M. & Jorgensen, E.M. An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature 412, 338–341 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Nonet, M.L., Saifee, O., Zhao, H., Rand, J.B. & Wei, L. Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants. J. Neurosci. 18, 70–80 (1998).

    CAS  Article  Google Scholar 

  26. 26

    Jorgensen, E.M. et al. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature 378, 196–199 (1995).

    CAS  Article  Google Scholar 

  27. 27

    Zhang, J.Z., Davletov, B.A., Sudhof, T.C. & Anderson, R.G. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78, 751–760 (1994).

    CAS  Article  Google Scholar 

  28. 28

    Richmond, J.E., Davis, W.S. & Jorgensen, E.M. UNC-13 is required for synaptic vesicle fusion in C. elegans. Nat. Neurosci. 2, 959–964 (1999).

    CAS  Article  Google Scholar 

  29. 29

    Harris, T.W., Hartwieg, E., Horvitz, H.R. & Jorgensen, E.M. Mutations in synaptojanin disrupt synaptic vesicle recycling. J. Cell Biol. 150, 589–600 (2000).

    CAS  Article  Google Scholar 

  30. 30

    Nonet, M.L. et al. UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol. Biol. Cell 10, 2343–2360 (1999).

    CAS  Article  Google Scholar 

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We thank M. Nonet for helpful comments on the manuscript, J. Rand (Oklahoma Medical Research Foundation) for the Punc-17 plasmid, D. Miller III (Vanderbilt University) for the Punc-4 plasmid, E. Jorgensen (University of Utah) and the Caenorhabditis Genetics Center for strains, and K. Zehl for expert technical assistance. We thank the lab of Prof. R. Tampé for hospitality and ongoing support. This work was funded by the Goethe University, Frankfurt, grants from the Deutsche Forschungsgemeinschaft to A.G. (SFB628 and GO 1011/2-1), and the Cluster of Excellence Frankfurt, Macromolecular Complexes, and grants from Canadian Institute of Health Research (MOP-79404 and MOP-74530) to M.Z.; G.J.S. was supported in part by the US National Institutes of Health (R01 EY017241, P50 MH062196) and by the Swartz Foundation.

Author information




J.F.L., Mar.B., Mag.B. and A.G. designed the experiments; J.F.L., Mar.B., Mag.B. and C.S. performed the experiments; G.J.S. wrote software and performed automated analysis of worm shape; J.F.L., Mar.B., Mag.B. and A.G. performed all other data analysis; and Mar.B., J.F.L., M.Z., Mag.B. and A.G. wrote the manuscript.

Corresponding author

Correspondence to Alexander Gottschalk.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–2, Supplementary Results, Supplementary Methods (PDF 1141 kb)

Supplementary Video 1

Light-induced inhibition of swimming behaviour in transgenic animal expressing ChR2-YFP in GABAergic neurons (transgene zxIs3). (MOV 1103 kb)

Supplementary Video 2

Light-induced paralysis and elongation of transgenic animal expressing ChR2-YFP in GABAergic neurons (transgene zxIs3) in response to a 10 s stimulus. (MOV 2094 kb)

Supplementary Video 3

Light-induced GABA release evoked in transgenic unc-47(e307); zxIs3 mutant animal that lacks the vesicular GABA transporter; animal fails to respond to the 10 s light stimulus. (MOV 1439 kb)

Supplementary Video 4

Light-induced contraction and coiling of transgenic animal expressing ChR2-YFP in cholinergic neurons (transgene zxIs6) in response to a 10 s stimulus. (MOV 1852 kb)

Supplementary Video 5

Light-induced contractions of transgenic animal expressing ChR2-YFP in cholinergic neurons (transgene zxIs6) can be repeatedly stimulated. (MOV 2460 kb)

Supplementary Video 6

Enhanced light-induced contraction without coiling of transgenic unc-49(e407); zxIs6 animal expressing ChR2-YFP in cholinergic neurons. (MOV 1620 kb)

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Liewald, J., Brauner, M., Stephens, G. et al. Optogenetic analysis of synaptic function. Nat Methods 5, 895–902 (2008). https://doi.org/10.1038/nmeth.1252

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