Abstract
Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response to brain injury or disease, a process called reactive gliosis. Precisely how astrocytes contribute to healthy brain function and play differential roles in brain pathology and regeneration remain important areas of investigation. To better understand the properties of astrocytes, more sophisticated approaches for probing their rich and complex anatomical and molecular features are needed to fully determine their contribution to brain physiology. Here we present an efficient and straightforward immunolabeling protocol to obtain high-resolution fluorescence-based images from fixed nonhuman primate (common marmoset Callithrix jacchus) and human brain samples. Importantly, the protocol is useful for obtaining images from samples that have been stored in fixative solutions (such as formalin) for years. This approach is especially useful for three-dimensional, multichannel confocal microscopy and can be optimized for super-resolution techniques such as stimulated emission depletion (STED) microscopy. We also present a strategy for using specific combinations of markers to define the phenotypic variations and cellular/subcellular properties of astrocytes to better predict the function of these cells on their surrounding brain microenvironment.
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References
Vasile F, Dossi E, Rouach N (2017) Human astrocytes: structure and functions in the healthy brain. Brain Struct Funct 222(5):2017–2029. https://doi.org/10.1007/s00429-017-1383-5
Farmer WT, Murai K (2017) Resolving astrocyte heterogeneity in the CNS. Front Cell Neurosci 11:300
Oberheim NA, Wang X, Goldman S, Nedergaard M (2006) Astrocytic complexity distinguishes the human brain. Trends Neurosci 29(10):547–553
Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol 119(1):7–35
Bouvier DS, Jones EV, Quesseveur G, Davoli MA, AF T, Quirion R, Mechawar N, Murai KK (2016) High resolution dissection of reactive glial nets in alzheimer's disease. Sci Rep 6:24544
Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K (2013) Structural and molecular interrogation of intact biological systems. Nature 497(7449):332–337
Lai HM, Liu AKL, Ng HHM, Goldfinger MH, Chau TW, DeFelice J, Tilley BS, Wong WM, Wu W, Gentleman SM (2018) Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues. Nat Commun 9(1):1066
Yang Z, Wang KK (2015) Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci 38(6):364–374
Schubert V, Bouvier D, Volterra A (2011) SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis. Glia 59(10):1472–1488
Hubbard JA, Szu JI, Yonan JM, Binder DK (2016) Regulation of astrocyte glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) expression in a model of epilepsy. Exp Neurol 283(Pt A):85–96
Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28(1):264–278
Bittar PG, Charnay Y, Pellerin L, Bouras C, Magistretti PJ (1996) Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain. J Cereb Blood Flow Metab 16(6):1079–1089
Orr AG, Hsiao EC, Wang MM, Ho K, Kim DH, Wang X, Guo W, Kang J, Yu GQ, Adame A, Devidze N, Dubal DB, Masliah E, Conklin BR, Mucke L (2015) Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory. Nat Neurosci 18(3):423–434
Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8(7):571–573
Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780–782
Acknowledgments
This work was supported by the Espoir-en-tête Rotary International award and the Auguste et Simone Prévot foundation (to D.S.B.). This work was supported by the Postdoctoral Training (Applicants living outside of Québec) of the Fonds de Recherche du Québec-Santé (FRQS) Canada (to G.Q.). This work was supported by the Canadian Institutes of Health Research (MOP 111152, PJT148569, PJT156247 to K.K.M.); Natural Sciences and Engineering Research Council of Canada (408044-2011 and 69404 to K.K.M.); Canada Research Chairs Program (K.K.M.); Brain Canada/W. Garfield Weston Foundation (K.K.M.). We thank the Douglas-Bell Canada Brain Bank (Douglas Mental Health University Institute, Montréal, QC, Canada) for providing the human samples and the Molecular Imaging Platform at the Research Institute of the McGill University of Health Centre for instrumentation and support.
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Quesseveur, G., Fouquier d’Hérouël, A., Murai, K.K., Bouvier, D.S. (2019). A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples. In: Di Benedetto, B. (eds) Astrocytes. Methods in Molecular Biology, vol 1938. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9068-9_6
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DOI: https://doi.org/10.1007/978-1-4939-9068-9_6
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