Advertisement

Microglia pp 127-147 | Cite as

Replenishment of Organotypic Hippocampal Slice Cultures with Neonatal or Adult Microglia

  • Annette Masuch
  • Knut Biber
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2034)

Abstract

This protocol describes a method to deplete and repopulate organotypic hippocampal slice cultures with ramified microglia. We describe the slice culture preparation from newborn mice, standard culturing of neonatal microglia, and the acute isolation of microglia from adult mouse brain. Furthermore, we outline the technique for the replenishment of microglia-depleted slice cultures with different microglia populations and subsequent morphological analysis. We show that neonatal and adult microglia acquire specific ramified morphologies, which in case of adult microglia are indistinguishable from the in vivo situation. This procedure not only allows the functional investigation of microglia with different degrees of ramification but also enables the construction of chimeric slice cultures with respect to the microglia phenotype. Preparation of slice cultures can be completed in 3.5 h, preparation of mixed-glial cultures in 4 h, isolation of adult microglia can be accomplished in 3.5 h, and replenishment in 30 min.

Key words

Organotypic hippocampal slice culture Microglia Mixed-glial cell culture Acute microglia isolation Mouse brain 

References

  1. 1.
    Ginhoux F, Greter M, Leboeuf M et al (2010) Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330:841–845.  https://doi.org/10.1126/science.1194637CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Prinz M, Mildner A (2011) Microglia in the CNS: immigrants from another world. Glia 59:177–187.  https://doi.org/10.1002/glia.21104CrossRefPubMedGoogle Scholar
  3. 3.
    Kettenmann H, Hanisch U-K, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91:461–553.  https://doi.org/10.1152/physrev.00011.2010CrossRefGoogle Scholar
  4. 4.
    Haynes SE, Hollopeter G, Yang G et al (2006) The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 9:1512–1519.  https://doi.org/10.1038/nn1805CrossRefGoogle Scholar
  5. 5.
    Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314–1318.  https://doi.org/10.1126/science.1110647CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Tremblay M-È, Majewska AK (2011) A role for microglia in synaptic plasticity? Commun Integr Biol 4:220–222.  https://doi.org/10.4161/cib.4.2.14506CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sierra A, Encinas JM, Deudero JJP et al (2010) Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell Stem Cell 7:483–495.  https://doi.org/10.1016/j.stem.2010.08.014CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Paolicelli RC, Bolasco G, Pagani F et al (2011) Synaptic pruning by microglia is necessary for normal brain development. Science 333:1456–1458.  https://doi.org/10.1126/science.1202529CrossRefGoogle Scholar
  9. 9.
    Schafer DP, Lehrman EK, Stevens B (2013) The “quad-partite” synapse: microglia-synapse interactions in the developing and mature CNS. Glia 61:24–36.  https://doi.org/10.1002/glia.22389CrossRefPubMedGoogle Scholar
  10. 10.
    Scheffold A, Holtman IR, Dieni S et al (2016) Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model. Acta Neuropathol Commun 4:87.  https://doi.org/10.1186/s40478-016-0364-xCrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Spiller KJ, Restrepo CR, Khan T et al (2018) Microglia-mediated recovery from ALS-relevant motor neuron degeneration in a mouse model of TDP-43 proteinopathy. Nat Neurosci 21:329–340.  https://doi.org/10.1038/s41593-018-0083-7CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hughes V (2012) Microglia: the constant gardeners. Nature 485:570–572.  https://doi.org/10.1038/485570aCrossRefPubMedGoogle Scholar
  13. 13.
    Hickman SE, Kingery ND, Ohsumi TK et al (2013) The microglial sensome revealed by direct RNA sequencing. Nat Neurosci 16:1896–1905.  https://doi.org/10.1038/nn.3554CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Butovsky O, Jedrychowski MP, Moore CS et al (2014) Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci 17:131–143.  https://doi.org/10.1038/nn.3599CrossRefGoogle Scholar
  15. 15.
    Gosselin D, Skola D, Coufal NG et al (2017) An environment-dependent transcriptional network specifies human microglia identity. Science 356.  https://doi.org/10.1126/science.aal3222CrossRefGoogle Scholar
  16. 16.
    Bohlen CJ, Bennett FC, Tucker AF et al (2017) Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures. Neuron 94:759–773.e8.  https://doi.org/10.1016/j.neuron.2017.04.043CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Bennett FC, Bennett ML, Yaqoob F et al (2018) A Combination of ontogeny and cns environment establishes microglial identity. Neuron 98:1170–1183.e8.  https://doi.org/10.1016/j.neuron.2018.05.014CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Noraberg J, Poulsen FR, Blaabjerg M et al (2005) Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Curr Drug Targets CNS Neurol Disord 4:435–452CrossRefGoogle Scholar
  19. 19.
    Lossi L, Alasia S, Salio C, Merighi A (2009) Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Prog Neurobiol 88:221–245.  https://doi.org/10.1016/j.pneurobio.2009.01.002CrossRefPubMedGoogle Scholar
  20. 20.
    Harry GJ, Kraft AD (2008) Neuroinflammation and microglia: considerations and approaches for neurotoxicity assessment. Expert Opin Drug Metab Toxicol 4:1265–1277.  https://doi.org/10.1517/17425255.4.10.1265CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    van Weering HRJ, Boddeke HWGM, Vinet J et al (2011) CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus. Hippocampus 21:220–232.  https://doi.org/10.1002/hipo.20742CrossRefPubMedGoogle Scholar
  22. 22.
    Gähwiler BH, Capogna M, Debanne D et al (1997) Organotypic slice cultures: a technique has come of age. Trends Neurosci 20:471–477CrossRefGoogle Scholar
  23. 23.
    Bahr BA (1995) Long-term hippocampal slices: a model system for investigating synaptic mechanisms and pathologic processes. J Neurosci Res 42:294–305.  https://doi.org/10.1002/jnr.490420303CrossRefPubMedGoogle Scholar
  24. 24.
    Kamada M, Li R-Y, Hashimoto M et al (2004) Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus. Eur J Neurosci 20:2499–2508.  https://doi.org/10.1111/j.1460-9568.2004.03721.xCrossRefPubMedGoogle Scholar
  25. 25.
    Raineteau O, Rietschin L, Gradwohl G et al (2004) Neurogenesis in hippocampal slice cultures. Mol Cell Neurosci 26:241–250.  https://doi.org/10.1016/j.mcn.2004.01.003CrossRefPubMedGoogle Scholar
  26. 26.
    Kreutz S, Koch M, Böttger C et al (2009) 2-Arachidonoylglycerol elicits neuroprotective effects on excitotoxically lesioned dentate gyrus granule cells via abnormal-cannabidiol-sensitive receptors on microglial cells. Glia 57:286–294.  https://doi.org/10.1002/glia.20756CrossRefPubMedGoogle Scholar
  27. 27.
    Van Rooijen N, Sanders A (1994) Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 174:83–93CrossRefGoogle Scholar
  28. 28.
    Vinet J, van Weering HRJ, Heinrich A et al (2012) Neuroprotective function for ramified microglia in hippocampal excitotoxicity. J Neuroinflammation 9:27.  https://doi.org/10.1186/1742-2094-9-27CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:173–182CrossRefGoogle Scholar
  30. 30.
    Howe ML, Barres BA (2012) A novel role for microglia in minimizing excitotoxicity. BMC Biol 10:7.  https://doi.org/10.1186/1741-7007-10-7CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bernardino L, Balosso S, Ravizza T et al (2008) Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury: a crucial role of P2X7 receptor-mediated IL-1beta release. J Neurochem 106:271–280.  https://doi.org/10.1111/j.1471-4159.2008.05387.xCrossRefPubMedGoogle Scholar
  32. 32.
    Masuch A, Shieh C-H, van Rooijen N et al (2016) Mechanism of microglia neuroprotection: involvement of P2X7, TNFα, and valproic acid. Glia 64:76–89.  https://doi.org/10.1002/glia.22904CrossRefPubMedGoogle Scholar
  33. 33.
    Masuch A, van der Pijl R, Füner L et al (2016) Microglia replenished OHSC: a culture system to study in vivo like adult microglia. Glia 64:1285–1297.  https://doi.org/10.1002/glia.23002CrossRefPubMedGoogle Scholar
  34. 34.
    Derecki NC, Cronk JC, Lu Z et al (2012) Wild-type microglia arrest pathology in a mouse model of Rett syndrome. Nature 484:105–109.  https://doi.org/10.1038/nature10907CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Mildner A, Mack M, Schmidt H et al (2009) CCR2+Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system. Brain J Neurol 132:2487–2500.  https://doi.org/10.1093/brain/awp144CrossRefGoogle Scholar
  36. 36.
    de Haas AH, Boddeke HWGM, Brouwer N, Biber K (2007) Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions. Glia 55:1374–1384.  https://doi.org/10.1002/glia.20554CrossRefGoogle Scholar
  37. 37.
    Hailer NP, Jarhult JD, Nitsch R (1996) Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 18:319–331CrossRefGoogle Scholar
  38. 38.
    Xiang Z, Hrabetova S, Moskowitz SI et al (2000) Long-term maintenance of mature hippocampal slices in vitro. J Neurosci Methods 98:145–154CrossRefGoogle Scholar
  39. 39.
    Hellwig S, Masuch A, Nestel S et al (2015) Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-β plaque formation in organotypic hippocampal slice cultures. Sci Rep 5:14624.  https://doi.org/10.1038/srep14624CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Annette Masuch
    • 1
  • Knut Biber
    • 2
  1. 1.Institute of Clinical Chemistry and Laboratory MedicineUniversity Medicine GreifswaldGreifswaldGermany
  2. 2.Department of Psychiatry and Psychotherapy, University Hospital FreiburgUniversity of FreiburgFreiburgGermany

Personalised recommendations