Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

High-yield in vitro recordings from neurons functionally characterized in vivo

Nature Protocols volume 13, pages 1275–1293 (2018)Cite this article

Subjects

Abstract

In vivo two-photon calcium imaging provides detailed information about the activity and response properties of individual neurons. However, in vitro methods are often required to study the underlying neuronal connectivity and physiology at the cellular and synaptic levels at high resolution. This protocol provides a fast and reliable workflow for combining the two approaches by characterizing the response properties of individual neurons in mice in vivo using genetically encoded calcium indicators (GECIs), followed by retrieval of the same neurons in brain slices for further analysis in vitro (e.g., circuit mapping). In this approach, a reference frame is provided by fluorescent-bead tracks and sparsely transduced neurons expressing a structural marker in order to re-identify the same neurons. The use of GECIs provides a substantial advancement over previous approaches by allowing for repeated in vivo imaging. This opens the possibility of directly correlating experience-dependent changes in neuronal activity and feature selectivity with changes in neuronal connectivity and physiology. This protocol requires expertise both in in vivo two-photon calcium imaging and in vitro electrophysiology. It takes 3 weeks or more to complete, depending on the time allotted for repeated in vivo imaging of neuronal activity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental paradigm and surgical procedures.
Figure 2: IOS imaging through the mouse skull and targeted virus injection into the cortical region of interest.
Figure 3: Two-photon calcium imaging and structural image stack.
Figure 4: Preparation of acute coronal brain slices containing functionally characterized cells.
Figure 5: In vivo/in vitro matching of neurons.
Figure 6: In vitro circuit analysis of functionally characterized cells.
Figure 7: Applications of the in vivo/in vitro protocol.

Similar content being viewed by others

References

  1. Keller, G.B., Bonhoeffer, T. & Hübener, M. Sensorimotor mismatch signals in primary visual cortex of the behaving mouse. Neuron 74, 809–815 (2012).

    CAS  PubMed  Google Scholar 

  2. Poort, J. et al. Learning enhances sensory and multiple non-sensory representations in primary visual cortex. Neuron 86, 1478–1490 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Li, N., Chen, T.-W., Guo, Z.V., Gerfen, C.R. & Svoboda, K. A motor cortex circuit for motor planning and movement. Nature 519, 51–56 (2015).

    CAS  PubMed  Google Scholar 

  4. Pastalkova, E., Itskov, V., Amarasingham, A. & Buzsáki, G. Internally generated cell assembly sequences in the rat hippocampus. Science 321, 1322–1327 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Lisman, J. The challenge of understanding the brain: where we stand in 2015. Neuron 86, 864–882 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Falkai, P. et al. Kraepelin revisited: schizophrenia from degeneration to failed regeneration. Mol. Psychiatry 20, 671–676 (2015).

    CAS  PubMed  Google Scholar 

  7. Sigurdsson, T. Neural circuit dysfunction in schizophrenia: insights from animal models. Neuroscience 321, 42–65 (2016).

    CAS  PubMed  Google Scholar 

  8. White, J.G., Southgate, E., Thomson, J.N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. B 314, 1 (1986).

    CAS  Google Scholar 

  9. Prevedel, R. et al. Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat. Methods 11, 727–730 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

    CAS  PubMed  Google Scholar 

  11. Denk, W., Strickler, J. & Webb, W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  PubMed  Google Scholar 

  12. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Svoboda, K. & Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).

    CAS  PubMed  Google Scholar 

  14. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).

    CAS  PubMed  Google Scholar 

  16. Rose, T., Goltstein, P.M., Portugues, R. & Griesbeck, O. Putting a finishing touch on GECIs. Front. Mol. Neurosci. 7, 88 (2014).

    PubMed  PubMed Central  Google Scholar 

  17. Goldey, G.J. et al. Removable cranial windows for long-term imaging in awake mice. Nat. Protoc. 9, 2515–2538 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ko, H. et al. Functional specificity of local synaptic connections in neocortical networks. Nature 473, 87–91 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Ko, H. et al. The emergence of functional microcircuits in visual cortex. Nature 496, 96–100 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Cossell, L. et al. Functional organization of excitatory synaptic strength in primary visual cortex. Nature 518, 399–403 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Lien, A. & Scanziani, M. In vivo labeling of constellations of functionally identified neurons for targeted in vitro recordings. Front. Neural Circuits http://dx.doi.org/10.3389/fncir.2011.00016 (2011).

  22. Peter, M. et al. Transgenic mouse models enabling photolabeling of individual neurons in vivo. PLoS One 8, e62132 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Rose, T., Jaepel, J., Hübener, M. & Bonhoeffer, T. Cell-specific restoration of stimulus preference after monocular deprivation in the visual cortex. Science 352, 1319–1322 (2016).

    CAS  PubMed  Google Scholar 

  24. Driscoll, L.N., Pettit, N.L., Minderer, M., Chettih, S.N. & Harvey, C.D. Dynamic reorganization of neuronal activity patterns in parietal cortex. Cell 170, 986–999 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kim, T.H. et al. Long-term optical access to an estimated one million neurons in the live mouse cortex. Cell Rep. 17, 3385–3394 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Karnani, M.M. et al. Cooperative subnetworks of molecularly similar interneurons in mouse neocortex. Neuron 90, 86–100 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ringach, D.L. et al. Spatial clustering of tuning in mouse primary visual cortex. Nat. Commun. 7, 12270 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Kim, E.J. et al. Three types of cortical layer 5 neurons that differ in brain-wide connectivity and function. Neuron 88, 1253–1267 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Petreanu, L. et al. Activity in motor-sensory projections reveals distributed coding in somatosensation. Nature 489, 299–303 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kato, H.K., Gillet, S.N. & Isaacson, J.S. Flexible sensory representations in auditory cortex driven by behavioral relevance. Neuron 88, 1027–1039 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Scott, B.B., Brody, C.D. & Tank, D.W Cellular resolution functional imaging in behaving rats using voluntary head restraint. Neuron 80, 371–384 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Huber, D. et al. Multiple dynamic representations in the motor cortex during sensorimotor learning. Nature 484, 473–478 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Goncalves, J.T., Anstey, J.E., Golshani, P. & Portera-Cailliau, C. Circuit level defects in the developing neocortex of fragile X mice. Nat. Neurosci. 16, 903–909 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Liebscher, S., Keller, G.B., Goltstein, P.M., Bonhoeffer, T. & Hübener, M. Selective persistence of sensorimotor mismatch signals in visual cortex of behaving Alzheimer's disease mice. Curr. Biol. 26, 956–964 (2016).

    CAS  PubMed  Google Scholar 

  35. Hamm, J.P., Peterka, D.S., Gogos, J.A. & Yuste, R. Altered cortical ensembles in mouse models of schizophrenia. Neuron 94, 153–167 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Smith, G.B. et al. The development of cortical circuits for motion discrimination. Nat. Neurosci. 18, 252–261 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Ohki, K. et al. Highly ordered arrangement of single neurons in orientation pinwheels. Nature 442, 925–928 (2006).

    CAS  PubMed  Google Scholar 

  38. Heider, B., Nathanson, J.L., Isacoff, E.Y., Callaway, E.M. & Siegel, R.M. Two-photon imaging of calcium in virally transfected striate cortical neurons of behaving monkey. PLoS One 5, e13829 (2010).

    PubMed  PubMed Central  Google Scholar 

  39. Sadakane, O. et al. Long-term two-photon calcium imaging of neuronal populations with subcellular resolution in adult non-human primates. Cell Rep. 13, 1989–1999 (2015).

    CAS  PubMed  Google Scholar 

  40. Li, M., Liu, F., Jiang, H., Lee, T.S. & Tang, S. Long-term two-photon imaging in awake macaque monkey. Neuron 93, 1049–1057 (2017).

    CAS  PubMed  Google Scholar 

  41. Kuhlman, S.J. et al. A disinhibitory microcircuit initiates critical-period plasticity in the visual cortex. Nature 501, 543–546 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Barnes, S.J. et al. Subnetwork-specific homeostatic plasticity in mouse visual cortex in vivo. Neuron 86, 1290–1303 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Keck, T. et al. Synaptic scaling and homeostatic plasticity in the mouse visual cortex in vivo. Neuron 80, 327–334 (2013).

    CAS  PubMed  Google Scholar 

  44. Clopath, C., Bonhoeffer, T., Hübener, M. & Rose, T. Variance and invariance of neuronal long-term representations. Philos. Trans. R. Soc. B 372, 20160161 (2017).

    Google Scholar 

  45. Thestrup, T. et al. Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat. Methods 11, 175 (2014).

    CAS  PubMed  Google Scholar 

  46. Mittmann, W. et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat. Neurosci. 14, 1089–1093 (2011).

    CAS  PubMed  Google Scholar 

  47. Pakan, J.M.P. et al. Behavioral-state modulation of inhibition is context-dependent and cell type specific in mouse visual cortex. eLife 5, e14985 (2016).

    PubMed  PubMed Central  Google Scholar 

  48. Resendez, S.L. et al. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat. Protoc. 11, 566 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Ziv, Y. et al. Long-term dynamics of CA1 hippocampal place codes. Nat. Neurosci. 16, 264–266 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Zong, W. et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat. Methods 14, 713–719 (2017).

    CAS  PubMed  Google Scholar 

  51. Hofer, S.B. et al. Differential connectivity and response dynamics of excitatory and inhibitory neurons in visual cortex. Nat. Neurosci. 14, 1045–1052 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Petreanu, L., Mao, T., Sternson, S.M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Yasuda, R. et al. Imaging calcium concentration dynamics in small neuronal compartments. Sci. STKE 2004, pl5 (2004).

    PubMed  Google Scholar 

  54. Kisfali, M., Lőorincz, T. & Vizi, E.S. Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non-fast-spiking GABAergic hippocampal interneurons using two-photon laser microscopy and high- and low-affinity dyes. J. Physiol. 591, 5541–5553 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Cadwell, C.R. et al. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nat. Biotechnol. 34, 199–203 (2016).

    CAS  PubMed  Google Scholar 

  56. Langer, D. & Helmchen, F. Post hoc immunostaining of GABAergic neuronal subtypes following in vivo two-photon calcium imaging in mouse neocortex. Pflugers Arch. 463, 339–354 (2012).

    CAS  PubMed  Google Scholar 

  57. Wekselblatt, J.B., Flister, E.D., Piscopo, D.M. & Niell, C.M. Large-scale imaging of cortical dynamics during sensory perception and behavior. J. Neurophysiol. 115, 2852–2866 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Itah, R., Gitelman, I., Tal, J. & Davis, C. Viral inoculation of mouse embryos in utero. J. Virol. Methods 120, 1–8 (2004).

    CAS  PubMed  Google Scholar 

  59. Potter, H. & Heller, R. Transfection by electroporation. Curr. Protoc. Mol. Biol. 62, 9.3.1–9.3.6 (2003).

    Google Scholar 

  60. Portera-Cailliau, C., Weimer, R.M., De Paola, V., Caroni, P. & Svoboda, K. Diverse modes of axon elaboration in the developing neocortex. PLoS Biol. 3, e272 (2005).

    PubMed  PubMed Central  Google Scholar 

  61. Pinto, L. & Dan, Y. Cell-type-specific activity in prefrontal cortex during goal-directed behavior. Neuron 87, 437–450 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Dana, H. et al. Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo. PLoS One 9, e108697 (2014).

    PubMed  PubMed Central  Google Scholar 

  63. Grinvald, A. et al. In-vivo optical imaging of cortical architecture and dynamics. in Modern Techniques in Neuroscience Research (eds. Windhorst, U. & Johansson, H.) 893–969 (Springer, 1999).

  64. Cang, J., Kalatsky, V.A., Löwel, S. & Stryker, M.P. Optical imaging of the intrinsic signal as a measure of cortical plasticity in the mouse. Vis. Neurosci. 22, 685–691 (2005).

    PubMed  PubMed Central  Google Scholar 

  65. Wang, T. & Kass, I.S. Preparation of brain slices. in Neurotransmitter Methods (ed. Rayne, R.C.) 1–14 (Springer, 1997).

  66. Qi, G., Radnikow, G. & Feldmeyer, D. Electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patch-clamp recordings. J. Vis. Exp. (95)52358 (2015).

  67. Pologruto, T.A., Sabatini, B.L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

    PubMed  PubMed Central  Google Scholar 

  68. Pelli, D.G. The VideoToolbox software for visual psychophysics: transforming numbers into movies. Spat. Vis. 10, 437–442 (1997).

    CAS  PubMed  Google Scholar 

  69. Brainard, D.H. & Vision, S. The psychophysics toolbox. Spat. Vis. 10, 433–436 (1997).

    CAS  PubMed  Google Scholar 

  70. Suter, B. et al. Ephus: multipurpose data acquisition software for neuroscience experiments. Front. Neural Circuits 4, 100 (2010).

    PubMed  PubMed Central  Google Scholar 

  71. Niell, C.M. & Stryker, M.P. Highly selective receptive fields in mouse visual cortex. J. Neurosci. 28, 7520–7536 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Cuntz, H., Forstner, F., Borst, A. & Häusser, M. The TREES toolbox—probing the basis of axonal and dendritic branching. Neuroinformatics 9, 91–96 (2011).

    PubMed  PubMed Central  Google Scholar 

  73. Leinweber, M. et al. Two-photon calcium imaging in mice navigating a virtual reality environment. J. Vis. Exp. (84)e50885 (2014).

  74. Canfield, J.G. Dry beveling micropipettes using a computer hard drive. J. Neurosci. Methods 158, 19–21 (2006).

    PubMed  Google Scholar 

  75. Shepherd, G.M.G. Circuit mapping by ultraviolet uncaging of glutamate. Cold Spring Harb. Protoc. 2012, 998–1004 (2012).

    PubMed  PubMed Central  Google Scholar 

  76. Chen, T.-W. et al. Ultra-sensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Gökçe, O., Bonhoeffer, T. & Scheuss, V. Clusters of synaptic inputs on dendrites of layer 5 pyramidal cells in mouse visual cortex. eLife 5, e09222 (2016).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to V. Staiger for excellent technical assistance and to M. Myoga for helping to build the in vitro setup. This study was supported by the Deutsche Forschungsgemeinschaft (CRC 870; V.S., T.B., T.R., and M.H.) and the Max Planck Society.

Author information

Author notes

  1. Volker Scheuss

    Present address: Present address: Institute of Physiology, Biomedical Center, Ludwig-Maximilians-University München, München-Martinsried, Germany.,

Authors and Affiliations

  1. Max Planck Institute of Neurobiology, München-Martinsried, Germany

    Simon Weiler, Joel Bauer, Mark Hübener, Tobias Bonhoeffer, Tobias Rose & Volker Scheuss

Authors
  1. Simon Weiler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joel Bauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark Hübener
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tobias Bonhoeffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tobias Rose
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Volker Scheuss
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.W. and V.S. developed the idea. S.W., V.S., T.R. and M.H. planned the experiments. S.W. performed all experiments and the analysis, except for the in vivo long-term imaging experiments, which were performed by J.B. T.R. developed the viral construct and in vivo data analysis. S.W., V.S., T.R., M.H. and T.B. wrote the paper.

Corresponding author

Correspondence to Volker Scheuss.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Weiler, S., Bauer, J., Hübener, M. et al. High-yield in vitro recordings from neurons functionally characterized in vivo. Nat Protoc 13, 1275–1293 (2018). https://doi.org/10.1038/nprot.2018.026

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2018.026

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing