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Open Access

Peer-reviewed

Lab Protocol

Preparation of acute midbrain slices containing the superior colliculus and periaqueductal Gray for patch-clamp recordings

  • Oriol Pavón Arocas ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    oriol.pavon.16@ucl.ac.uk

    Affiliation Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London, United Kingdom

    ⨯
  • Tiago Branco

    Roles Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing

    Affiliation Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London, United Kingdom

    ⨯

Abstract

This protocol is a practical guide for preparing acute coronal slices from the midbrain of young adult mice for electrophysiology experiments. It describes two different sets of solutions with their respective incubation strategies and two alternative procedures for brain extraction: decapitation under terminal isoflurane anaesthesia and intracardial perfusion with artificial cerebrospinal fluid under terminal isoflurane anaesthesia. Slices can be prepared from wild-type mice as well as from mice that have been genetically modified or transfected with viral constructs to label subsets of cells. The preparation can be used to investigate the electrophysiological properties of midbrain neurons in combination with pharmacology, opto- and chemogenetic manipulations, and calcium imaging; which can be followed by morphological reconstruction, immunohistochemistry, or single-cell transcriptomics. The protocol also provides a detailed list of materials and reagents including the design for a low-cost and easy to assemble 3D printed slice recovery chamber, general advice for troubleshooting common issues leading to suboptimal slice quality, and some suggestions to ensure good maintenance of a patch-clamp rig.

Citation: Pavón Arocas O, Branco T (2022) Preparation of acute midbrain slices containing the superior colliculus and periaqueductal Gray for patch-clamp recordings. PLoS ONE 17(8): e0271832. https://doi.org/10.1371/journal.pone.0271832

Editor: Mark S. Shapiro, University of Texas Health Science Center, UNITED STATES

Received: March 30, 2022; Accepted: July 7, 2022; Published: August 11, 2022

Copyright: © 2022 Pavón Arocas, Branco. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was funded by a Wellcome Senior Research Fellowship (214352/Z/18/Z) and a Sainsbury Wellcome Centre Core Grant from the Gatsby Charitable Foundation and Wellcome (090843/F/09/Z) (T.B.), and by a UCL Wellcome 4-year PhD Programme in Neuroscience Fellowship (203729/Z/16/Z) (O.P.A.). The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The acute brain slice remains an essential preparation for studying the nervous system [1]. For decades, acute brain slices have been combined with the patch-clamp technique [2–4] for investigating the biophysical and electrophysiological properties of different cell types in a multitude of brain areas and animal species, including humans [5–10]. More recently, the refinement of viral and gene-editing approaches and the development of techniques such as optogenetics and single-cell transcriptomics have significantly expanded the range of questions that can be investigated with this preparation [11–27].

Despite the long tradition of patch-clamp experiments and the many adaptations of slicing protocol described in the literature [28–40], the success rate of the method and the quality of the tissue can vary depending on the brain region of interest and the age of the animal. Before starting a set of experiments in a brain area or animal age different from what is normally used in the home institution, it is important to test different combinations of solutions, brain extraction methods, and incubation strategies in order to find the approach that reliably provides the best tissue quality. Here, we describe a step-by-step guide for obtaining acute coronal slices from the midbrain of young adult mice (4–12 weeks old). This includes instructions for preparing solutions and extracting the brain, a detailed list of materials and reagents, and troubleshooting advice.

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io, https://dx.doi.org/10.17504/protocols.io.8epv51pz5l1b/v6 and is included for printing as S1 File with this article.

Expected results

The protocol described here has been routinely used to obtain patch-clamp recordings from midbrain neurons in acute slices containing the superior colliculus (SC) and periaqueductal gray (PAG) for investigating their biophysical properties and their role within the neural circuits controlling instinctive behaviours [18, 22, 25].

This method can be combined with the use of transgenic lines or viral strategies to target genetically labelled subsets of neurons in the midbrain area of interest (Fig 1A) or any of its anatomical subdivisions (Fig 1B). Furthermore, patch-clamp recordings in the cell-attached or whole-cell configuration (Fig 1C) can be obtained while pharmacological, optogenetic, or chemogenetic manipulations are applied to the slice (Fig 1A).

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Fig 1. Targeted patch-clamp recordings in acute midbrain slices.

A. Schematics illustrating the use of acute midbrain slices in combination with other techniques to target neurons in a midbrain area of interest (PAG, in yellow). B. Example images from experiments targeting midbrain neurons in different anatomical subdivisions of the PAG in the same slice. Scale bars are 1 mm. Abbreviations indicate the different PAG subdivisions: dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG), and ventrolateral (vlPAG). C. (Left) Spontaneous electrical activity of a PAG neuron during a loose-seal cell-attached recording. (Right) Voltage response to step current injections of a PAG neuron during a whole-cell recording.

https://doi.org/10.1371/journal.pone.0271832.g001

Supporting information

S1 File. Preparation of acute midbrain slices containing the superior colliculus and periaqueductal Gray for patch-clamp recordings.

Step-by-step protocol, also available on protocols.io.

https://doi.org/10.1371/journal.pone.0271832.s001

(PDF)

Acknowledgments

We would like to thank Nicolas Wanaverbecq, Annalisa Scimemi, A. Vanessa Stempel, Yaara Lefler, Yu Lin Tan, Simon Weiler, Sara Mederos, and Hedi Young for their help and advice prior to the development of this protocol. We would also like to thank Robb Barrett from the Fabrications Lab at SWC for designing the 3D printed piece for the recovery chamber.

References

  1. 1. Edwards FA, Konnerth A, Sakmann B, Takahashi T. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflügers Arch Eur J Physiol. 1989;414(5):600–12. Available from: http://link.springer.com/10.1007/BF00580998 pmid:2780225
  2. 2. Neher E, Sakmann B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature. 1976;260(5554):799–802. Available from: https://www.nature.com/articles/260799a0.pdf pmid:1083489
  3. 3. Sakmann B, Neher E. Patch Clamp Techniques for Studying Ionic Channels in Excitable Membranes. Annu Rev Physiol. 1984;46(1):455–72. Available from: https://www.annualreviews.org/doi/10.1146/annurev.ph.46.030184.002323 pmid:6143532
  4. 4. Gibb AJ, Edwards FA. Patch clamp recording from cells in sliced tissues. In: Ogden DC, editor. Microelectrode techniques, The Plymouth workshop handbook. 2nd ed. Cambridge: The Company of Biologists Limited; 1994. p. 255–74.
    • 5. Blanton MG, Lo Turco JJ, Kriegstein AR. Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J Neurosci Methods. 1989;30(3):203–10. Available from: https://linkinghub.elsevier.com/retrieve/pii/0165027089901313 pmid:2607782
    • 6. Madry C, Kyrargyri V, Arancibia-Cárcamo IL, Jolivet R, Kohsaka S, Bryan RM, et al. Microglial Ramification, Surveillance, and Interleukin-1β Release Are Regulated by the Two-Pore Domain K+ Channel THIK-1. Neuron. 2018;97(2):299–312.e6. Available from: https://linkinghub.elsevier.com/retrieve/pii/S089662731731125X pmid:29290552
    • 7. Gouwens NW, Sorensen SA, Berg J, Lee C, Jarsky T, Ting J, et al. Classification of electrophysiological and morphological neuron types in the mouse visual cortex. Nat Neurosci. 2019;22(7):1182–95. Available from: http://dx.doi.org/10.1038/s41593-019-0417-0 pmid:31209381
    • 8. McCauley JP, Petroccione MA, D’Brant LY, Todd GC, Affinnih N, Wisnoski JJ, et al. Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1. Cell Rep. 2020;33(2):108255. Available from: https://doi.org/10.1016/j.celrep.2020.108255 pmid:33053337
    • 9. Dorst MC, Tokarska A, Zhou M, Lee K, Stagkourakis S, Broberger C, et al. Polysynaptic inhibition between striatal cholinergic interneurons shapes their network activity patterns in a dopamine-dependent manner. Nat Commun. 2020;11(1):5113. Available from: pmid:33037215
    • 10. Campagnola L, Seeman SC, Chartrand T, Kim L, Hoggarth A, Gamlin C, et al. Local connectivity and synaptic dynamics in mouse and human neocortex. Science. 2022;375(6585). Available from: https://www.science.org/doi/10.1126/science.abj5861
    • 11. Chiang LW. Detection of gene expression in single neurons by patch-clamp and single-cell reverse transcriptase polymerase chain reaction. J Chromatogr A. 1998;806(1):209–18. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0021967398001563 pmid:9639890
    • 12. Carlson GC, Coulter DA. In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording. Nat Protoc. 2008;3(2):249–55. Available from: http://www.nature.com/articles/nprot.2007.539 pmid:18274527
    • 13. Hanemaaijer NAK, Popovic MA, Wilders X, Grasman S, Pavón Arocas O, Kole MHP. Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling. Elife. 2020;9:1–75. Available from: https://elifesciences.org/articles/54566 pmid:32553116
    • 14. Vale R, Campagner D, Iordanidou P, Pavón Arocas O, Tan YL, Vanessa Stempel A, et al. A cortico-collicular circuit for accurate orientation to shelter during escape. bioRxiv. 2020; Available from: https://doi.org/10.1101/2020.05.26.117598
    • 15. Kalmbach BE, Hodge RD, Jorstad NL, Owen S, de Frates R, Yanny AM, et al. Signature morpho-electric, transcriptomic, and dendritic properties of human layer 5 neocortical pyramidal neurons. Neuron. 2021;109(18):2914–2927.e5. Available from: pmid:34534454
    • 16. Bakken TE, Jorstad NL, Hu Q, Lake BB, Tian W, Kalmbach BE, et al. Comparative cellular analysis of motor cortex in human, marmoset and mouse. Nature. 2021;598(7879):111–9. Available from: https://www.nature.com/articles/s41586-021-03465-8 pmid:34616062
    • 17. Fratzl A, Koltchev AM, Vissers N, Tan YL, Marques-Smith A, Stempel AV, et al. Flexible inhibitory control of visually evoked defensive behavior by the ventral lateral geniculate nucleus. Neuron. 2021;109(23):3810–3822.e9. Available from: pmid:34614420
    • 18. Berg J, Sorensen SA, Ting JT, Miller JA, Chartrand T, Buchin A, et al. Human neocortical expansion involves glutamatergic neuron diversification. Nature. 2021;598(7879):151–8. Available from: https://www.nature.com/articles/s41586-021-03813-8 pmid:34616067
    • 19. Callaway EM, Dong H-W, Ecker JR, Hawrylycz MJ, Huang ZJ, Lein ES, et al. A multimodal cell census and atlas of the mammalian primary motor cortex. Nature. 2021;598(7879):86–102. Available from: https://www.nature.com/articles/s41586-021-03950-0 pmid:34616075
    • 20. Fuzik J, Zeisel A, Máté Z, Calvigioni D, Yanagawa Y, Szabó G, et al. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nat Biotechnol. 2016;34(2):175–83. Available from: http://www.nature.com/doifinder/10.1038/nbt.3443 pmid:26689544
    • 21. Cadwell CR, Palasantza A, Jiang X, Berens P, Deng Q, Yilmaz M, et al. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nat Biotechnol. 2016;34(2):199–203. Available from: http://www.nature.com/articles/nbt.3445 pmid:26689543
    • 22. Cadwell CR, Scala F, Li S, Livrizzi G, Shen S, Sandberg R, et al. Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq. Nat Protoc. 2017;12(12):2531–53. Available from: pmid:29189773
    • 23. Muñoz-Manchado AB, Bengtsson Gonzales C, Zeisel A, Munguba H, Bekkouche B, Skene NG, et al. Diversity of Interneurons in the Dorsal Striatum Revealed by Single-Cell RNA Sequencing and PatchSeq. Cell Rep. 2018;24(8):2179–2190.e7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S2211124718311562 pmid:30134177
    • 24. Weiler S, Bauer J, Hübener M, Bonhoeffer T, Rose T, Scheuss V. High-yield in vitro recordings from neurons functionally characterized in vivo. Nat Protoc. 2018;13(6):1275–93. Available from: pmid:29748648
    • 25. Evans DA, Stempel AV, Vale R, Ruehle S, Lefler Y, Branco T. A synaptic threshold mechanism for computing escape decisions. Nature. 2018;558(7711):590–4. Available from: http://www.nature.com/articles/s41586-018-0244-6 pmid:29925954
    • 26. Mandelbaum G, Taranda J, Haynes TM, Hochbaum DR, Huang KW, Hyun M, et al. Distinct Cortical-Thalamic-Striatal Circuits through the Parafascicular Nucleus. Neuron. 2019;102(3):636–652.e7. Available from: pmid:30905392
    • 27. Gouwens NW, Sorensen SA, Baftizadeh F, Budzillo A, Lee BR, Jarsky T, et al. Integrated Morphoelectric and Transcriptomic Classification of Cortical GABAergic Cells. Cell. 2020;183(4):935–953.e19. Available from: https://linkinghub.elsevier.com/retrieve/pii/S009286742031254X pmid:33186530
    • 28. Lipton P, Aitken PG, Dudek FE, Eskessen K, Espanol MT, Ferchmin PA, et al. Making the best of brain slices: comparing preparative methods. J Neurosci Methods. 1995;59(1):151–6. Available from: https://linkinghub.elsevier.com/retrieve/pii/016502709400205U pmid:7475245
    • 29. Aitken PG, Breese GR, Dudek FF, Edwards F, Espanol MT, Larkman PM, et al. Preparative methods for brain slices: a discussion. J Neurosci Methods. 1995;59(1):139–49. Available from: https://linkinghub.elsevier.com/retrieve/pii/016502709400204T pmid:7475244
    • 30. Moyer JR, Brown TH. Methods for whole-cell recording from visually preselected neurons of perirhinal cortex in brain slices from young and aging rats. J Neurosci Methods. 1998;86(1):35–54. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0165027098001435 pmid:9894784
    • 31. Brahma B, Forman RE, Stewart EE, Nicholson C, Rice ME. Ascorbate inhibits edema in brain slices. J Neurochem. 2000;74(3):1263–70. Available from: http://doi.wiley.com/10.1046/j.1471-4159.2000.741263.x pmid:10693960
    • 32. MacGregor DG, Chesler M, Rice ME. HEPES prevents edema in rat brain slices. Neurosci Lett. 2001;303(3):141–4. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0304394001016901 pmid:11323105
    • 33. Bischofberger J, Engel D, Li L, Geiger JRP, Jonas P. Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nat Protoc. 2006;1(4):2075–81. Available from: http://www.nature.com/articles/nprot.2006.312 pmid:17487197
    • 34. Tanaka Y, Tanaka Y, Furuta T, Yanagawa Y, Kaneko T. The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice. J Neurosci Methods. 2008;171(1):118–25. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0165027008001386 pmid:18430473
    • 35. Huang S, Uusisaari MY. Physiological temperature during brain slicing enhances the quality of acute slice preparations. Front Cell Neurosci. 2013;7(APR):1–8. Available from: http://journal.frontiersin.org/article/10.3389/fncel.2013.00048/abstract pmid:23630465
    • 36. Ting JT, Daigle TL, Chen Q, Feng G. Acute brain slice methods for adult and aging animals: Application of targeted patch clamp analysis and optogenetics. In: Martina M, Taverna S, editors. Methods in Molecular Biology. New York, NY: Springer New York; 2014. p. 221–42. (Methods in Molecular Biology; vol. 1183). Available from: http://link.springer.com/10.1007/978-1-4939-1096-0_1
      • 37. Buskila Y, Breen PP, Tapson J, van Schaik A, Barton M, Morley JW. Extending the viability of acute brain slices. Sci Rep. 2015;4(1):5309. Available from: http://www.nature.com/articles/srep05309
      • 38. Ting JT, Kalmbach B, Chong P, De Frates R, Keene CDi, Gwinn RP, et al. A robust ex vivo experimental platform for molecular-genetic dissection of adult human neocortical cell types and circuits. Sci Rep. 2018;8(1):8407. Available from: pmid:29849137
      • 39. Ting JT, Lee BR, Chong P, Soler-Llavina G, Cobbs C, Koch C, et al. Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method. J Vis Exp. 2018;(132):1–13. Available from: https://www.jove.com/video/53825/preparation-acute-brain-slices-using-an-optimized-n-methyl-d pmid:29553547
      • 40. Eguchi K, Velicky P, Hollergschwandtner E, Itakura M, Fukazawa Y, Danzl JG, et al. Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions. Front Cell Neurosci. 2020;14(March):35–7. Available from: https://www.frontiersin.org/article/10.3389/fncel.2020.00063/full pmid:32265664