Abstract
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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References
Wojcik, S.M. & Brose, N. Regulation of membrane fusion in synaptic excitation-secretion coupling: speed and accuracy matter. Neuron 55, 11–24 (2007).
Richmond, J.E. Synaptic function. In WormBook (ed., The C. elegans Research Community) (doi/10.1895/wormbook.1.69.1; 2005).
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).
Guerrero, G. et al. Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nat. Neurosci. 8, 1188–1196 (2005).
Miller, K.G. et al. A genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proc. Natl. Acad. Sci. USA 93, 12593–12598 (1996).
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).
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).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).
Schuske, K.R. et al. Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin. Neuron 40, 749–762 (2003).
Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940–13945 (2003).
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).
Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).
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).
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).
Zhang, Y.P. & Oertner, T.G. Optical induction of synaptic plasticity using a light-sensitive channel. Nat. Methods 4, 139–141 (2007).
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).
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).
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).
McIntire, S.L., Jorgensen, E., Kaplan, J. & Horvitz, H.R. The GABAergic nervous system of Caenorhabditis elegans. Nature 364, 337–341 (1993).
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).
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).
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).
Hammarlund, M., Palfreyman, M.T., Watanabe, S., Olsen, S. & Jorgensen, E.M. Open syntaxin docks synaptic vesicles. PLoS Biol. 5, e198 (2007).
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).
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).
Jorgensen, E.M. et al. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature 378, 196–199 (1995).
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).
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).
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).
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).
Acknowledgements
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.
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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.
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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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DOI: https://doi.org/10.1038/nmeth.1252
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