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  • Protocol Extension
  • Published:

Opto-juxtacellular interrogation of neural circuits in freely moving mice

Nature Protocols volume 18, pages 2415–2440 (2023)Cite this article

Subjects

Abstract

Neural circuits are assembled from an enormous variety of neuronal cell types. Although significant advances have been made in classifying neurons on the basis of morphological, molecular and electrophysiological properties, understanding how this diversity contributes to brain function during behavior has remained a major experimental challenge. Here, we present an extension to our previous protocol, in which we describe the technical procedures for performing juxtacellular opto-tagging of single neurons in freely moving mice by using Channelrhodopsin-2–expressing viral vectors. This method allows one to selectively target molecularly defined cell classes for in vivo single-cell recordings. The targeted cells can be labeled via juxtacellular procedures and further characterized via post-hoc morphological and molecular analysis. In its current form, the protocol allows multiple recording and labeling attempts to be performed within individual animals, by means of a mechanical pipette micropositioning system. We provide proof-of-principle validation of this technique by recording from Calbindin-positive pyramidal neurons in the mouse hippocampus during spatial exploration; however, this approach can easily be extended to other behaviors and cortical or subcortical areas. The procedures described here, from the viral injection to the histological processing of brain sections, can be completed in ~4–5 weeks.

This protocol is an extension to: Nat. Protoc. 9, 2369–2381 (2014): https://doi.org/10.1038/nprot.2014.161

Key points

  • This Protocol Extension is for targeting, recording and labeling individual genetically defined neurons in freely moving mice. Juxtacellular labeling of in vivo opto-tagged neurons enables morphological and molecular analysis of recorded cells.

  • Opto-juxtacellular recordings are not restricted by brain area and depth, and the use of freely moving animals allows the study of natural behaviors. Moreover, pairing presynaptic (optogenetic) manipulations with postsynaptic single-cell stimulation enables the study of single-cell plasticity mechanisms.

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Fig. 1: Implant components for obtaining juxtacellular opto-tagging recordings in freely moving animals.
Fig. 2: Implant assembly steps for obtaining juxtacellular opto-tagging recordings in freely moving mice.
Fig. 3: In vivo juxtacellular opto-tagging and light-evoked responses.
Fig. 4: Morphological identification of a juxtacellularly recorded and opto-tagged Calb1-positive CA1 pyramidal cell.
Fig. 5: Morphological identification of a juxtacellularly recorded and opto-tagged Calb1-negative CA1 pyramidal cell.

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Data availability

The data associated with this protocol are available as Source Data and from the supporting primary research article37. Technical drawings for key custom equipment (see Equipment setup) are available in the Supplementary Data.

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Acknowledgements

This work was supported by the Werner Reichardt Centre for Integrative Neuroscience (EXC 307), the Eberhard Karls University of Tübingen, DFG grant BU 3126/2-1 (to A.B.) and the Athene Grant (to P.P.-F.). We thank A. Eritja and F. Monteiro for their excellent technical assistance, S. Ishiyama for illustrations and K. Vollmer and the UKT Fine-Mechanics workshop for excellent support.

Author information

Author notes

  1. Maria Diamantaki

    Present address: Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology–Hellas, Heraklion, Greece

  2. These authors contributed equally: Lingjun Ding, Giuseppe Balsamo.

Authors and Affiliations

  1. Institute of Neurobiology, Eberhard Karls University of Tübingen, Tübingen, Germany

    Lingjun Ding, Giuseppe Balsamo, Maria Diamantaki, Patricia Preston-Ferrer & Andrea Burgalossi

  2. Werner-Reichardt Centre for Integrative Neuroscience, Tübingen, Germany

    Lingjun Ding, Giuseppe Balsamo, Maria Diamantaki, Patricia Preston-Ferrer & Andrea Burgalossi

  3. Graduate Training Centre of Neuroscience—International Max-Planck Research School (IMPRS), Tübingen, Germany

    Lingjun Ding, Giuseppe Balsamo & Maria Diamantaki

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  1. Lingjun Ding
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Contributions

L.D. and G.B. set up viral and optogenetic tools and optical stimulation hardware and performed experiments for the establishment of the juxtacellular opto-tagging protocol. L.D. and G.B. acquired and analyzed the data. M.D. contributed to the establishment of juxtacellular recordings in freely moving mice. A.B. and P.P-F. supervised the experiments. A.B. drafted the manuscript. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Patricia Preston-Ferrer or Andrea Burgalossi.

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The authors declare no competing interests.

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Nature Protocols thanks Takashi Kitamura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Diamantaki, M. et al. Cell Rep. 23, 32–38 (2018): https://doi.org/10.1016/j.celrep.2018.03.031

Ding, L. et al. eLife 11, e71720 (2022): https://doi.org/10.7554/eLife.71720

Supplementary information

Supplementary Information

Supplementary Figs. 1–10

Supplementary Data 1

Technical drawing of the micropositioning drive (part 1)

Supplementary Data 2

Technical drawing of the micropositioning drive (part 2)

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Ding, L., Balsamo, G., Diamantaki, M. et al. Opto-juxtacellular interrogation of neural circuits in freely moving mice. Nat Protoc 18, 2415–2440 (2023). https://doi.org/10.1038/s41596-023-00842-7

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