Abstract
Sparse, single-cell labeling approaches enable high-resolution, high signal-to-noise recordings from subcellular compartments and intracellular organelles and allow precise manipulations of individual cells and local circuits while minimizing complex changes associated with global network manipulations. However, thus far, only a limited number of approaches have been developed to label single cells with unique combinations of genetically encoded indicators, target deep cortical structures or sustainably use the same chronic preparation for weeks. Here we developed a method to deliver plasmids selectively to single pyramidal neurons in the mouse dorsal hippocampus using two-photon visually guided in vivo single-cell electroporation to address these limitations. This method allows long-term plasmid expression in a controlled number of individual pyramidal neurons, facilitating subcellular resolution imaging, intracellular organelle tracking, monosynaptic input mapping, plasticity induction and targeted whole-cell patch-clamp recordings.
Key points
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The delivery of plasmids selectively to single pyramidal neurons in the mouse dorsal hippocampus via two-photon visually guided in vivo single-cell electroporation.
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This method allows long-term plasmid expression in a controlled number of individual pyramidal neurons, facilitating subcellular resolution imaging, intracellular organelle tracking, monosynaptic input mapping, plasticity induction and targeted whole-cell patch-clamp recordings for up to 2 months.
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Data availability
Further information and requests for resources and reagents should be directed to the lead contact Attila Losonczy (al2856@columbia.edu). All unique resources generated in this study are available from the lead contact with a completed Materials Transfer Agreement.
The authors declare that the main data discussed in this protocol are available in the supporting primary research papers (https://doi.org/10.1016/j.neuron.2021.12.003; https://doi.org/10.1126/science.abm1670; https://doi.org/10.1038/s41586-021-04169-9; https://doi.org/10.1101/2023.10.04.560848; https://doi.org/10.1038/s41467-024-50546-z; https://doi.org/10.1101/2024.02.26.582144; https://doi.org/10.1038/s41467-024-46463-w). The raw datasets are available for research purposes from the corresponding authors upon reasonable request.
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Acknowledgements
A.L. was supported by National Institute of Mental Health (NIMH) grants R01MH124047 and R01MH124867, National Institute of Neurological Disorders and Stroke (NINDS) grants R01NS121106, U01NS115530, R01NS133381 and R01NS131728, and National Institute on Aging grant RF1AG080818. F.P. was supported by NINDS grant R35NS127232 and the Nomis Foundation. K.C.G. was supported by NINDS grant 5T32NS064928-13. A. Noguchi was supported by Overseas Research Fellowships (JSPS) and an HFSP postdoctoral fellowship. J.O.H. was supported by NIMH grant F32MH118716 and NINDS grant K99NS127815.
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Conceptualization: A. Negrean, T.G. and A.L. Methodology: A. Negrean, T.G., J.O.H., K.C.G. and A. Noguchi. Investigation: A. Negrean, T.G., J.O.H. and K.C.G., A. Noguchi, M.S. and S.T. Visualization: K.C.G., A. Noguchi and G.Z. Data curation: K.C.G., A. Noguchi, G.Z. and H.C.Y. Funding acquisition: A.L. Resources: A.L. Project administration: A.L. and F.P Supervision: A.L. and F.P. Writing—original draft: K.C.G., A. Noguchi, G.Z., H.C.Y. and A.L. Writing—review and editing: all authors.
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Nature Protocols thanks Kurt Haas and Takafumi Inoue for their contribution to the peer review of this work.
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Key references
Rolotti, S. V. et al. Neuron 110, 783–794.e6 (2022): https://doi.org/10.1016/j.neuron.2021.12.003
O’Hare, J. K. et al. Science 375, eabm1670 2022: https://doi.org/10.1126/science.abm1670
Geiller, T. et al. Nature 601, 105–109 (2022): https://doi.org/10.1038/s41586-021-04169-9
Liao, Z. et al. Nat. Commun. 15, 6295 (2024): https://doi.org/10.1038/s41467-024-50546-z
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Gonzalez, K.C., Noguchi, A., Zakka, G. et al. Visually guided in vivo single-cell electroporation for monitoring and manipulating mammalian hippocampal neurons. Nat Protoc 20, 1468–1484 (2025). https://doi.org/10.1038/s41596-024-01099-4
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DOI: https://doi.org/10.1038/s41596-024-01099-4
