- Protocol
- Published:
- Veronika Csillag1,
- J. C. Noble ORCID: orcid.org/0000-0002-4450-429X2,
- Daniela Calvigioni1,
- Björn Reinius2 &
- …
- János Fuzik ORCID: orcid.org/0000-0002-5408-48821
Nature Protocols (2024)Cite this article
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Subjects
- Electrophysiology
- Molecular neuroscience
- Neuronal physiology
- Synaptic plasticity
Abstract
Neuronal pathways recruit large postsynaptic populations and maintain connections via distinct postsynaptic response types (PRTs). Until recently, PRTs were accessible as a selection criterion for single-cell RNA sequencing only through probing by low-throughput whole-cell electrophysiology. To overcome these limitations and target neurons on the basis of specific PRTs for soma collection and subsequent single-cell RNA sequencing, we developed Voltage-Seq using the genetically encoded voltage indicator Voltron in acute brain slices from mice. We also created an onsite analysis tool, VoltView, to guide soma collection of specific PRTs using a classifier based on a previously acquired database of connectomes from multiple animals. Here we present our procedure for preparing the optical path, the imaging setup and detailing the imaging and analysis steps, as well as a complete procedure for sequencing library preparation. This enables researchers to conduct our high-throughput all-optical synaptic assay and to obtain single-cell transcriptomic data from selected postsynaptic neurons. This also allows researchers to resolve the connectivity ratio of a specific pathway and explore the diversity of PRTs within that connectome. Furthermore, combining high throughput with quick analysis gives unique access to find specific connections within a large postsynaptic connectome. Voltage-Seq also allows the investigation of correlations between connectivity and gene expression changes in a postsynaptic cell-type-specific manner for both excitatory and inhibitory connections. The Voltage-Seq workflow can be completed in ~6 weeks, including 4–5 weeks for viral expression of the Voltron sensor. The technique requires knowledge of basic laboratory techniques, micromanipulator handling skills and experience in molecular biology and bioinformatics.
Key points
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This protocol describes Voltage-Seq, a method for all-optical voltage imaging-guided postsynaptic single-cell transcriptomics. Combining the Voltron voltage indicator with the onsite analysis tool VoltView enables collection of neuronal somas for single-cell RNA sequencing on the basis of their postsynaptic response types.
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Voltage-Seq enables high-throughput analysis that was not possible with patch-clamp approaches. Moreover, the use of a genetically encoded voltage indicator increases the ability to resolve postsynaptic response types compared with GCaMP calcium indicators.
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Data availability
Voltage-Seq scRNA-seq data are available for key publication at https://www.ebi.ac.uk/biostudies/arrayexpress with accession code E-MTAB-13104. Example all-optical VMH-PAG Voltron recordings are included with the MATLAB code of VoltView v.1.0 analysis at https://zenodo.org/record/8030176. Example VoltView output.mat file can be found as supplementary data.
Code availability
VoltView v.1.0 used to analyze all-optical voltage imaging and to select neurons for Voltage-Seq neuronal soma collection is available at https://zenodo.org/record/8030176 under Creative Commons Attribution v.4.0 International Public License (with installation guide, user’s guide and example data).
References
Fuzik, J. et al. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nat. Biotechnol. 34, 175–183 (2016).
Cadwell, C. R. et al. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nat. Biotechnol. 34, 199–203 (2016).
Linders, L. E. et al. Studying synaptic connectivity and strength with optogenetics and patch-clamp electrophysiology. Int. J. Mol. Sci. 23, 11612 (2022).
Abdelfattah, A. S. et al. Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science 365, 699–704 (2019).
Bando, Y., Sakamoto, M., Kim, S., Ayzenshtat, I. & Yuste, R. Comparative evaluation of genetically encoded voltage indicators. Cell Rep. 26, 802–813.e804 (2019).
Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D. & Cohen, A. E. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat. Methods 9, 90–95 (2011).
Zhang, Y. et al. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature 615, 884–891 (2023).
Adam, Y. et al. Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics. Nature 569, 413–417 (2019).
Csillag, V., Bizzozzero, M. H., Noble, J. C., Reinius, B. & Fuzik, J. Voltage-Seq: all-optical postsynaptic connectome-guided single-cell transcriptomics. Nat. Methods 20, 1409–1416 (2023).
Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18, 100–106 (2021).
Park, P. et al. Dendritic voltage imaging reveals biophysical basis of associative plasticity rules. Preprint at bioRxiv https://doi.org/10.1101/2023.06.02.543490 (2023).
Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).
Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).
Picelli, S. et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24, 2033–2040 (2014).
Oliveira, L. S., Sumera, A. & Booker, S. A. Repeated whole-cell patch-clamp recording from CA1 pyramidal cells in rodent hippocampal slices followed by axon initial segment labeling. STAR Protoc. 2, 100336 (2021).
Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotactic Coordinates 4th edn. (Academic, 2013).
Ting, J. T. et al. Preparation of acute brain slices using an optimized N-methyl-d-glucamine protective recovery method. J. Vis. Exp. 26, 53825 (2018).
Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods 10, 1096–1098 (2013).
Bagnoli, J. W. et al. Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nat. Comm. 9, 2937 (2018).
Trombetta, J. J. et al. Preparation of single-cell RNA-seq libraries for next generation sequencing. Curr. Protoc. Mol. Biol. 107, 4.22.21–24.22.17 (2014).
Acknowledgements
We thank J. Szabadics, M. Rózsa and G. Oláh, for many helpful discussions and for their technical expertise and A. Wolthon for help with technical service in mouse work. J.F. acknowledges funding from the Swedish Research Council (Starting Grant in Medicine no. 2019-02052), StratNeuro (Start-up program 2020 and Grant for New Technologies 2024), Hjärnfonden (grant nos. FO2020-0162 and FO2022-0323), BBRF NARSAD Young Investigator Grant (no. 29079), Jeanssons Foundation (grant no. J2020-0122) and Åke Wiberg Foundation (grant nos. M21-0220 and M22-0223). B.R. acknowledges funding from the Swedish Society for Medical Research (grant no. CG-22-0260-H-02), the Swedish Research Council (grant no. 2022-01620) and the Knut and Allice Wallenberg Foundation (grant no. 2021.0142; 2022.0146).
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Authors and Affiliations
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Veronika Csillag,Daniela Calvigioni&János Fuzik
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
J. C. Noble&Björn Reinius
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- Veronika Csillag
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- J. C. Noble
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Contributions
J.F., V.C., D.C., B.R. and J.C.N. wrote the manuscript. J.F., V.C. and D.C. prepared the figures.
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Correspondence to János Fuzik.
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Nature Protocols thanks Yoav Adam, Eran Mukamel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference
Csillag et al. Nat. Methods 20, 1409–1416 (2023): https://doi.org/10.1038/s41592-023-01965-1
Supplementary information
Supplementary Data
Sample VoltView output data
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Csillag, V., Noble, J.C., Calvigioni, D. et al. All-optical voltage imaging-guided postsynaptic single-cell transcriptome profiling with Voltage-Seq. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01005-y
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DOI: https://doi.org/10.1038/s41596-024-01005-y
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Associated content
Voltage-Seq: all-optical postsynaptic connectome-guided single-cell transcriptomics
- Veronika Csillag
- Marianne Hiriart Bizzozzero
- János Fuzik
Nature Methods Article Open Access
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