Advertisement

Detecting Neuronal Differentiation Markers in Newborn Cells of the Adult Brain

  • Sara Bonzano
  • Silvia De MarchisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1560)

Abstract

Adult neurogenesis is the lifelong generation of new neurons that occurs into restricted regions of the adult mammalian brain, namely the dentate gyrus of the hippocampus and the olfactory bulb. In this chapter, we describe the procedures aimed to investigate adult neurogenesis in the murine brain. Specifically, we explain how to inject into animals exogenous markers of proliferation (i.e., BrdU) and prepare brain tissues to perform immunofluorescence reactions for neuronal markers in combination with BrdU staining. As BrdU is incorporated in the DNA during the S-phase of the cell cycle of proliferating cells and is then inherited by daughter cells, by coupling BrdU-immunoreactivity together with the immunolabeling for neuronal markers, we provide the general procedures that can be applied to identify adult-born neurons and to characterize their specific phenotypes in different brain regions, under physiological condition or in pathological states.

Key words

BrdU labeling Neuron birth-dating Adult neurogenesis Multiple immunolabeling Neuronal differentiation 

References

  1. 1.
    Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124:319–336CrossRefPubMedGoogle Scholar
  2. 2.
    Altman J (1969) Autoradiographic and histological studies of postnatal neurogenesis: IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 137:433–458CrossRefPubMedGoogle Scholar
  3. 3.
    Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age related decrease of neuronal progenitor proliferation. J Neurosci 16:2027–2033PubMedGoogle Scholar
  4. 4.
    Cooper-Kuhn CM, Kuhn HG (2002) Is it all DNA repair? Methodological considerations for detecting neurogenesis in the adult brain. Dev Brain Res 134:13–21. doi: 10.1016/S0165-3806(01)00243-7 CrossRefGoogle Scholar
  5. 5.
    Taupin P (2006) BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev 53:198–214. doi: 10.1016/j.brainresrev.2006.08.002 CrossRefPubMedGoogle Scholar
  6. 6.
    Wojtowicz JM, Kee N (2006) BrdU assay for neurogenesis in rodents. Nat Protoc 1:1399–1405. doi: 10.1038/nprot.2006.224 CrossRefPubMedGoogle Scholar
  7. 7.
    Mamber C, Kozareva DA, Kamphuis W et al (2013) Shades of gray: the delineation of marker expression within the adult rodent subventricular zone. Prog Neurobiol 111:1–16. doi: 10.1016/j.pneurobio.2013.07.003 CrossRefPubMedGoogle Scholar
  8. 8.
    Kuhn HG, Eisch AJ, Spalding K et al (2016) Detection and phenotypic characterization of adult neurogenesis. Cold Spring Harb Perspect Biol 8(3):a025981CrossRefPubMedGoogle Scholar
  9. 9.
    Hayes NL, Nowakowski RS (2000) Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering versus leaving the S-phase. Dev Neurosci 22:44–55CrossRefPubMedGoogle Scholar
  10. 10.
    Bonzano S, Bovetti S, Fasolo A et al (2014) Odour enrichment increases adult-born dopaminergic neurons in the mouse olfactory bulb. Eur J Neurosci 40:3450–3457. doi: 10.1111/ejn.12724 CrossRefPubMedGoogle Scholar
  11. 11.
    Ansorg A, Bornkessel K, Witte OW et al (2015) Immunohistochemistry and multiple labeling with antibodies from the same host species to study adult hippocampal neurogenesis. J Vis Exp 98:e52551. doi: 10.3791/52551 Google Scholar
  12. 12.
    Peterson DA (1999) Quantitative histology using confocal microscopy: implementation of unbiased stereology procedures. Methods 18:493–507. doi: 10.1006/meth.1999.0818 CrossRefPubMedGoogle Scholar
  13. 13.
    Gage GJ, Kipke DR, Shain W (2012) Whole animal perfusion fixation for rodents. J Vis Exp 65:e3564. doi: 10.3791/3564 Google Scholar
  14. 14.
    Latt SA, Wohlleb JC (1975) Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes. Chromosoma 52:297–316CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Life Sciences and Systems Biology (DBIOS)University of TurinTurinItaly
  2. 2.Neuroscience Institute Cavalieri Ottolenghi (NICO)Orbassano, TurinItaly

Personalised recommendations