Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal ('shadowpatching'). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.
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We thank T. Branco, M. Rizzi and Y. Goda (UCL) for the pEGFP-C1 plasmid; T. Tabata and K. Powell for technical help; A. Roth for advice on image processing; T. Branco, I. Duguid, M. Rizzi, S. Smith and C. Wilms for discussions and comments on the manuscript. This work was supported by the Wellcome Trust (M.H.), Gatsby Foundation (M.H.), Japan Society for the Promotion of Science (K.K.), Uehara Foundation (K.K.), MEXT (grants-in-aid for scientific research nos. 18680034, 18650086 and 18019025 to K.K., and 17023021 and 17100004 to M.K.), and the Boehringer Ingelheim Fonds (B.J.).
Supplementary Methods, Supplementary Figures 1 and 2, and Supplementary Table 1 (PDF 1004 kb)
Movie of neocortical layer 2/3 neurons visualized using shadowimaging. (MOV 1570 kb)
Same as Supplementary Movie 1, except image has been inverted. (MOV 3407 kb)
Movie of neurons in the molecular layer of cerebellar cortex visualized using shadowimaging. Note that the dendrites of individual Purkinje cells are clearly visible, as well as the cell bodies of molecular layer interneurons. (MOV 3431 kb)
Movie showing targeted patching of a Purkinje cell using the negative image. Note the appearance of the dimple just prior to GΩ seal formation. (MOV 1926 kb)
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Kitamura, K., Judkewitz, B., Kano, M. et al. Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo. Nat Methods 5, 61–67 (2008). https://doi.org/10.1038/nmeth1150
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