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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Altman, J. Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat. Rec. 145, 573–591 (1963).
Kohler, S. J., Williams, N. I., Stanton, G. B., Cameron, J. L. & Greenough, W. T. Maturation time of new granule cells in the dentate gyrus of adult macaque monkeys exceeds six months. Proc. Natl Acad. Sci. USA 108, 10326–10331 (2011).
Eriksson, P. S. et al. Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313–1317 (1998).
Sahay, A. et al. Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature 472, 466–470 (2011).
Zhao, C., Teng, E. M., Summers, R. G. Jr., Ming, G. L. & Gage, F. H. Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J. Neurosci. 26, 3–11 (2006).
Bischofberger, J. Young and excitable: new neurons in memory networks. Nat. Neurosci. 10, 273–275 (2007).
Marin-Burgin, A., Mongiat, L. A., Pardi, M. B. & Schinder, A. F. Unique processing during a period of high excitation/inhibition balance in adult-born neurons. Science 335, 1238–1242 (2012).
Braak, H. & Braak, E. [Morphology of Alzheimer disease]. Fortschr. Med. 108, 621–624 (1990).
Moreno-Jimenez, E. P. et al. Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat. Med. 25, 554–560 (2019).
Knoth, R. et al. Murine features of neurogenesis in the human hippocampus across the lifespan from 0 to 100 years. PLOS One 5, e8809 (2010).
Boldrini, M. et al. Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell 22, 589–599.e5 (2018).
Tobin, M. K. et al. Human hippocampal neurogenesis persists in aged adults and Alzheimer’s disease patients. Cell Stem Cell 24, 974–982.e3 (2019).
Sorrells, S. F. et al. Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature 555, 377–381 (2018).
Cipriani, S. et al. Hippocampal radial glial subtypes and their neurogenic potential in human fetuses and healthy and Alzheimer’s disease adults. Cereb. Cortex 28, 2458–2478 (2018).
Evers, P., Uylings, H. B. & Suurmeijer, A. J. Antigen retrieval in formaldehyde-fixed human brain tissue. Methods 15, 133–140 (1998).
Dowson, J. H. The evaluation of autofluorescence emission spectra derived from neuronal lipopigment. J. Microsc. 128, 261–270 (1982).
Spalding, K. L. et al. Dynamics of hippocampal neurogenesis in adult humans. Cell 153, 1219–1227 (2013).
Tartt, A. N. et al. Considerations for assessing the extent of hippocampal neurogenesis in the adult and aging human brain. Cell Stem Cell 23, 782–783 (2018).
Manganas, L. N. et al. Magnetic resonance spectroscopy identifies neural progenitor cells in the live human brain. Science 318, 980–985 (2007).
Greenberg, D. A. & Jin, K. Neurodegeneration and neurogenesis: focus on Alzheimer’s disease. Curr. Alzheimer Res. 3, 25–28 (2006).
Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H. & Frisen, J. Retrospective birth dating of cells in humans. Cell 122, 133–143 (2005).
Bao, A. M. & Swaab, D. F. The art of matching brain tissue from patients and controls for postmortem research. Handb. Clin. Neurol. 150, 197–217 (2018).
Boekhoorn, K., Joels, M. & Lucassen, P. J. Increased proliferation reflects glial and vascular-associated changes, but not neurogenesis in the presenile Alzheimer hippocampus. Neurobiol. Dis. 24, 1–14 (2006).
Braak, H., Alafuzoff, I., Arzberger, T., Kretzschmar, H. & Del Tredici, K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 112, 389–404 (2006).
Thavarajah, R., Mudimbaimannar, V. K., Elizabeth, J., Rao, U. K. & Ranganathan, K. Chemical and physical basics of routine formaldehyde fixation. J. Oral. Maxillofac. Pathol. 16, 400–405 (2012).
Plumpe, T. et al. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation. BMC Neurosci. 7, 77 (2006).
Gleeson, J. G., Lin, P. T., Flanagan, L. A. & Walsh, C. A. Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 23, 257–271 (1999).
Pikkarainen, M., Martikainen, P. & Alafuzoff, I. The effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers. J. Neuropathol. Exp. Neurol. 69, 40–52 (2010).
Axthelm, M. K. & Krakowka, S. Immunocytochemical methods for demonstrating canine distemper virus antigen in aldehyde-fixed paraffin-embedded tissue. J. Virol. Methods 13, 215–229 (1986).
Guntern, R., Vallet, P. G., Bouras, C. & Constantinidis, J. An improved immunohistostaining procedure for peptides in human brain. Experientia 45, 159–161 (1989).
Leibnitz, L. & Wunscher, W. [The life-long deposition of intraneuronal lipofuscin in various sections of the human brain]. Anat. Anz. 121, 132–140 (1967).
Nishioka, N., Takahata, N. & Iizuka, R. Histochemical studies on the lipo-pigments in the nerve cells. A comparison with lipofuscin and ceroid pigment. Acta Neuropathol. 11, 174–181 (1968).
Llorens-Martin, M., Torres-Aleman, I. & Trejo, J. L. Pronounced individual variation in the response to the stimulatory action of exercise on immature hippocampal neurons. Hippocampus 16, 480–490 (2006).
Gundersen, H. J. Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson. J. Microsc. 143, (3–45 (1986).
Bendtsen, T. F. & Nyengaard, J. R. Unbiased estimation of particle number using sections—an historical perspective with special reference to the stereology of glomeruli. J. Microsc. 153, 93–102 (1989).
Howell, K., Hopkins, N. & McLoughlin, P. Combined confocal microscopy and stereology: a highly efficient and unbiased approach to quantitative structural measurement in tissues. Exp. Physiol. 87, 747–756 (2002).
Pallas-Bazarra, N. et al. Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis. EMBO J. 35, 1417–1436 (2016).
Martinez-Martin, P. & Avila, J. Alzheimer Center Reina Sofia Foundation: fighting the disease and providing overall solutions. J. Alzheimers Dis. 21, 337–348 (2016).
Mai, J., Majtanik, M. & Paxinos, G. Atlas of the Human Brain 4th edn (Academic Press, 2015).
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.
The authors declare no competing interests.
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