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In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording

Nature Protocols volume 3, pages 249–255 (2008)Cite this article

Abstract

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded Vm can accurately represent population Vm changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.

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Figure 1: VSD setup, validation and calibration of whole-cell current-clamp recordings (I-clamp recording) with VSD response.
Figure 2: Analysis and display of VSD data.
Figure 3: Unlike other electrophysiological measures of neuronal population activity, VSD imaging can directly measure the spatiotemporal dynamics of inhibitory responses by reporting hyperpolarization.

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Acknowledgements

We thank Brian M. Salzberg and the reviewers for critically reading the paper and for useful comments. We also thank Chyze-Whee Ang for his part in developing these techniques. NIH NINDS Grants NS-32403 and NS38572 supported this work.

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Authors and Affiliations

  1. Translational Research Laboratory, University of Pennsylvania School of Medicine, Room 2226, 125 S 31st Street, Philadelphia, 19104-3403, Pennsylvania, USA

    Greg C Carlson

  2. Departments of Pediatrics and Neurology, University of Pennsylvania School of Medicine, Philadelphia, 19104-3403, Pennsylvania, USA

    Douglas A Coulter

  3. Abramson Pediatric Research Center, The Children's Hospital of Philadelphia, 3516 Civic Center Boulevard, Room 410D, Philadelphia, 19104-4318, Pennsylvania, USA

    Douglas A Coulter

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Correspondence to Douglas A Coulter.

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Carlson, G., Coulter, D. In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording. Nat Protoc 3, 249–255 (2008). https://doi.org/10.1038/nprot.2007.539

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