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Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses

Nature Protocols volume 16, pages 2947–2967 (2021)Cite this article

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Abstract

Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre–postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system.

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Fig. 1: Experimental setup for MFB–CA3 pyramidal neuron paired recordings.
Fig. 2: Videomicroscopy-guided targeting of pre- and postsynaptic structures for MFB–CA3 pyramidal neuron paired recordings.
Fig. 3: MFB–CA3 pyramidal neuron paired recordings combined with post hoc morphological analysis.
Fig. 4: MFB–CA3 pyramidal neuron paired recordings in mice and rats.
Fig. 5: MFB–CA3 pyramidal neuron paired recordings combined with mossy fiber tract stimulation.
Fig. 6: MFB–CA3 pyramidal neuron paired recordings combined with optogenetic stimulation in Prox1-Cre mice.
Fig. 7: Miniature EPSCs evoked by depolarization of a single identified presynaptic terminal.

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Data availability

Original data are available in ref. 16 (https://doi.org/10.1016/j.neuron.2020.05.013). Further original data are stored in the scientific repository of the Institute of Science and Technology Austria and will be provided by the corresponding author upon reasonable request.

Code availability

Analysis software is available via GitHub (Stimfit version 0.15.8; https://github.com/neurodroid/stimfit). Further code for analysis of miniature EPSCs can be provided by the corresponding author upon reasonable request.

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Acknowledgements

This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 692692 to P.J.) and the Fond zur Förderung der Wissenschaftlichen Forschung (Z 312-B27, Wittgenstein award to P.J., V 739-B27 to C.B.M.). We are grateful to F. Marr and C. Altmutter for excellent technical assistance and cell reconstruction, E. Kralli-Beller for manuscript editing, and the Scientific Service Units of IST Austria, especially T. Asenov and Miba machine shop, for maximally efficient support.

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  1. These authors contributed equally: David Vandael, Yuji Okamoto.

Authors and Affiliations

  1. IST Austria (Institute of Science and Technology Austria), Klosterneuburg, Austria

    David Vandael, Yuji Okamoto, Carolina Borges-Merjane, Victor Vargas-Barroso, Benjamin A. Suter & Peter Jonas

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  1. David Vandael
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Contributions

D.V. and Y.O. performed the experiments and analyzed the data. P.J. and D.V. conceived the protocol and wrote the text. All authors analyzed data and jointly revised the protocol.

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Correspondence to Peter Jonas.

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The authors declare that they have the following competing financial interests: industrial collaboration with Leica Microsystems.

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Vandael, D. et al. Neuron 107, 509–521.e7 (2020): https://doi.org/10.1016/j.neuron.2020.05.013

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Supplementary Video 1

MFB–CA3 pyramidal neuron paired recording under experimental conditions.

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Vandael, D., Okamoto, Y., Borges-Merjane, C. et al. Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses. Nat Protoc 16, 2947–2967 (2021). https://doi.org/10.1038/s41596-021-00526-0

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