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Unraveling human adult hippocampal neurogenesis

Nature Protocols volume 15, pages 668–693 (2020)Cite this article

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Abstract

Adult neurogenesis occurs in a few selected regions of the mammalian brain. One such region is the hippocampus, the so-called gateway to memory, where adult hippocampal neurogenesis (AHN) occurs. Here, we provide a comprehensive description of the methods used in our laboratory to unambiguously detect a population of immature neurons in the human hippocampus until the 10th decade of life. The criteria used to refine and develop the current protocol include obtaining post-mortem human samples of remarkable quality and under tightly controlled conditions for immunohistochemistry (IHC) studies, optimizing tissue processing and histological procedures, establishing criteria to reliably validate antibody signal and performing unbiased stereological cell counts. Moreover, we provide a detailed description of the parameters that, in our view, should be reported in human AHN studies. The opposing results obtained by introducing slight variations in the methodological conditions should be considered by future studies that seek to increase our knowledge of this fascinating process. By applying simple and inexpensive tissue pre-treatments, this protocol, which can be completed in 7 days, might be applicable to a variety of IHC studies performed on other tissues of human (or animal) origin.

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Fig. 1: Experimental design.
Fig. 2: Experimental setup.
Fig. 3: Effect of fixation on the performance of different antibodies.
Fig. 4: Effects of variations in the histological procedures on staining with different antibodies in samples fixed in 4% PFA for 24 h at 4 °C.
Fig. 5: Effects of incubation with the autofluorescence eliminator reagent on the outcomes of the IHC.
Fig. 6: Image analysis.

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Acknowledgements

The authors would like to thank the patients and families for generously donating brain samples. Moreover, they would like to thank Izaskun Rodal and Laura Saiz for help with human sample extraction and processing, and Esther García, Raquel Cuadros and the confocal microscopy facility of the CBMSO for technical assistance. The authors are grateful to Prof. Gleeson for providing an anti-doublecortin antibody. This study was supported by the following: the Spanish Ministry of Economy and Competitiveness (SAF-2017-82185-R and RYC-2015-171899 (M.L.-M.); SAF-2014-53040-P (J.A.)); The Alzheimer´s Association (2015-NIRG-340709 and AARG-17-528125 (M.L.-M.)); The Association for Frontotemporal Degeneration (2016 Basic Science Pilot Grant Award (M.L.-M.)); the Comunidad de Madrid (PEJD-2017-PRE/BMD-3439 (M.L.-M.)); and the Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED, Spain) (J.A.). Institutional grants from the Fundación Ramón Areces and Banco de Santander to CBMSO are also acknowledged. The salary of E.P.M.-J. was supported by a Comunidad de Madrid researcher contract (PEJD-2017-PRE/BMD-3439). The salary of J.T.-R. was supported by a Universidad Autónoma de Madrid Doctorate fellowship (FPI-UAM 2017 program). The salary of M.F.-G. was supported by a Formación de personal Investigador (FPI) contract, associated with the SAF-2017-82185-R grant (M.L.-M.), supported by the Spanish Ministry for Economy and Competitiveness (PRE2018-085233). The salary of E.P.M.-J. was supported by a Fundación Tatiana Pérez de Guzmán el Bueno Doctorate on Neuroscience Fellowship.

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Author notes

  1. These authors contributed equally to this work as first author: Miguel Flor-García, Julia Terreros-Roncal, Elena P. Moreno-Jiménez.

Authors and Affiliations

  1. Department of Molecular Neuropathology, Centro de Biología Molecular ‘Severo Ochoa’, CBMSO, CSIC-UAM, Madrid, Spain

    Miguel Flor-García, Julia Terreros-Roncal, Elena P. Moreno-Jiménez, Jesús Ávila & María Llorens-Martín

  2. Department of Molecular Biology, Faculty of Sciences, Universidad Autónoma de Madrid, Madrid, Spain

    Miguel Flor-García, Elena P. Moreno-Jiménez & María Llorens-Martín

  3. Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), Madrid, Spain

    Julia Terreros-Roncal, Jesús Ávila & María Llorens-Martín

  4. Neuropathology Department, CIEN Foundation, Madrid, Spain

    Alberto Rábano

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  1. Miguel Flor-García
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Contributions

M.F.-G., J.T.-R., E.P.M.-J. and M.L.-M. designed and conceived the study. A.R. provided materials and performed the autopsies. M.F.-G., J.T.-R., E.P.M.-J. and M.L.-M. performed experiments and analyzed the data. M.L.-M. wrote the manuscript. J.A. and M.L.-M. obtained funding. All authors critically discussed the data and revised the final version of the manuscript.

Corresponding author

Correspondence to María Llorens-Martín.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Josef Bischofberger 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

Moreno-Jiménez, E.P. et al. Nat. Med. 25, 554–560 (2019): https://doi.org/10.1038/s41591-019-0375-9

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Flor-García, M., Terreros-Roncal, J., Moreno-Jiménez, E.P. et al. Unraveling human adult hippocampal neurogenesis. Nat Protoc 15, 668–693 (2020). https://doi.org/10.1038/s41596-019-0267-y

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