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Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system

Nature Protocols volume 7, pages 1351–1365 (2012)Cite this article

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Abstract

Neurotransmitter release is triggered by membrane depolarization, Ca2+ influx and Ca2+ sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca2+ dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca2+ concentrations via UV photolysis of caged Ca2+. We developed a culture protocol for Ca2+ uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca2+-chelator nitrophenyl-EGTA and Ca2+ indicators, and a UV flash is used to trigger Ca2+-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.

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Figure 1
Figure 2: Autaptic hippocampal neuronal culture.
Figure 3: Ca2+ uncaging and kinetics of RRP depletion in wild-type (WT) and Syt-1–deficient (Syt-1 KO) neurons.
Figure 4: Experimental setup.
Figure 5: Experimental dissection of fast- and slow-release components from total UV-flash release traces.

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Acknowledgements

We thank A. Galinski, I. Beulshausen and M. Dörre (Göttingen, Germany) for excellent technical support, and M. Krohn, F. Schütte, R. Otremba and E. Özdemir (Göttingen, Germany) for fine mechanics support. We are particularly grateful to C. Rosenmund (Berlin, Germany) for advice on microisland culture, to T.M. Boeckers (Ulm, Germany) for providing the rabbit polyclonal antibody against ProSap-1 and to J. Sørensen (Copenhagen, Denmark) for help with the [Ca2+]i calibration. This work was supported by the Max Planck Society (Munich, Germany), the German Research Foundation (SFB889, B1) and the European Union (EUROSPIN, SynSys; Brussels, Belgium). K.M.M. is a recipient of a fellowship of the German Academic Exchange Service (DAAD; Bonn, Germany).

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Author notes

  1. Andrea Burgalossi & Wolf J Jockusch

    Present address: Present addresses: Bernstein Center for Computational Neuroscience, Humboldt University, Berlin, Germany (A.B.); Carl Zeiss Microscopy, Göttingen, Germany (W.J.J.).,

Authors and Affiliations

  1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany

    Andrea Burgalossi, SangYong Jung, Kwun-nok Mimi Man, Ramya Nair, Wolf J Jockusch, Sonja M Wojcik, Nils Brose & Jeong-Seop Rhee

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Contributions

J.-S.R. conceived the method and supervised the experiments. A.B. and W.J.J. performed experiments in the establishment of the protocol. R.N., S.Y.J. and K.M.M. performed additional experiments. A.B., J.-S.R., W.J.J., S.M.W. and N.B. wrote the manuscript.

Corresponding authors

Correspondence to Nils Brose or Jeong-Seop Rhee.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Technical drawing of the stamp used for generating the growth surface for microisland cultures. (a-b) Bottom (a) and lateral (b) views of the stamp. Insets show higher magnifications of the micro-dotted thermoplastic surface. (PDF 66 kb)

Supplementary Fig. 2

Linear relationship between F1 and F2. The isocoefficient α can be calculated from the linear relationship between F1 and F2. R is the correlation coefficient. (PDF 63 kb)

Supplementary Fig. 3

Mag-Fura-2/Fura-4F calibration curve from Supplementary Table 2. (PDF 55 kb)

Supplementary Table 1

Solutions for purity determination of NP-EGTA, Ca2+ calibration of Fura-4F, and photolysis efficiency (PLE) of NP-EGTA. (PDF 49 kb)

Supplementary Table 2

Ca2+ calibration solutions for Mag-Fura-2 and Fura-4F. (PDF 61 kb)

Supplementary Methods

Ca2+ calibration protocol. (PDF 109 kb)

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Burgalossi, A., Jung, S., Man, Kn. et al. Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system. Nat Protoc 7, 1351–1365 (2012). https://doi.org/10.1038/nprot.2012.074

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