Optogenetic analysis of synaptic function

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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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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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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Authors

Contributions

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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