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Silencing neurotransmission with membrane-tethered toxins

Nature Methods volume 7, pages 229–236 (2010)Cite this article

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Abstract

At synaptic terminals, high voltage–activated Cav2.1 and Cav2.2 calcium channels have an essential and joint role in coupling the presynaptic action potential to neurotransmitter release. Here we show that membrane-tethered toxins allowed cell-autonomous blockade of each channel individually or simultaneously in mouse neurons in vivo. We report optimized constitutive, inducible and Cre recombinase–dependent lentiviral vectors encoding fluorescent recombinant toxins, and we also validated the toxin-based strategy in a transgenic mouse model. Toxins delivered by lentiviral vectors selectively inhibited the dopaminergic nigrostriatal pathway, and transgenic mice with targeted expression in nociceptive peripheral neurons displayed long-lasting suppression of chronic pain. Optimized tethered toxins are tools for cell-specific and temporal manipulation of ion channel-mediated activities in vivo, including blockade of neurotransmitter release.

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Figure 1: Expression of t-toxins in neurons.
Figure 2: Electrophysiological analysis of t-toxin activity.
Figure 3: Inducible and Cre-dependent t-toxins.
Figure 4: In vivo inhibition of dopaminergic neurotransmission by t-toxins.
Figure 5: Inhibition of Cav2.2 in nociceptors of t-toxin transgenic mice.
Figure 6: Reduction of pain in transgenic mice.

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Acknowledgements

We thank P. Osten (Cold Spring Harbor Laboratory) for providing the original lentiviral plasmid; R.Y. Tsien (University of California, San Diego) for providing mCherry cDNA; P. Aebischer (École Polytechnique Fédéral de Lausanne) for providing pLVUT-tTR-KRAB plasmid; I.M. Verma (Salk Institute) for providing LV-Cre-SD plasmid; J. Duyster (Technical University Munich) for floxed DsRed cDNA; M. Missler (Westfälische Wilhelms-Universität Münster) for providing HEK293-Cav2.2 cells; J. Meyer for support with the hippocampus culture protocol, and F. Rathjen, G.R. Lewin, C. Birchmeier, T. Kuner, C. Scharff and G. Dittmar for helpful discussions. This work was supported by Helmholtz Association (31-002), Sonderforschungsbereich (SFB 665) and by Deutsche Forschungsgemeinschaft (DFG RA 424/5-1).

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Author notes

  1. Beate Liehl

    Present address: Present address: Novartis Pharma AG, Basel, Switzerland.,

  2. Sebastian Auer and Annika S Stürzebecher: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Neurobiology group, Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Sebastian Auer, Annika S Stürzebecher, Julio Santos-Torres, Christina Hanack, Silke Frahm, Beate Liehl & Inés Ibañez-Tallon

  2. Department of Neuroscience, Developmental Neurobiology group, Max Delbrück Center for Molecular Medicine, Berlin, Germany

    René Jüttner

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Contributions

S.A. and A.S.S. performed and analyzed most of the experiments. R.J. and J.S.-T. performed electrophysiology and analyzed data. C.H. and S.F. assisted with experiments. B.L. helped design tethers and established lentivirus protocol. I.I.-T. conceived and supervised the project and wrote the paper with S.A.

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Correspondence to Inés Ibañez-Tallon.

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The authors declare no competing financial interests.

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Auer, S., Stürzebecher, A., Jüttner, R. et al. Silencing neurotransmission with membrane-tethered toxins. Nat Methods 7, 229–236 (2010). https://doi.org/10.1038/nmeth.1425

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