BrdU assay for neurogenesis in rodents

Neurogenesis within the adult central nervous system is demonstrated using an exogenous cell tracer, 5'-bromo-2'-deoxyuridine (BrdU), in combination with endogenous neuronal markers. Specific primary antibodies raised against these markers are widely available and their visualization is possible with the use of fluorescently tagged secondary antibodies. BrdU is a thymidine analog that incorporates into dividing cells during DNA synthesis. Once incorporated into the new DNA, BrdU will remain in place and be passed down to daughter cells following division. Typically, BrdU is injected intraperitoneally. Different survival times required by the desired experimental time-line will yield data on specific phases of neurogenesis: proliferation, differentiation and maturation. One of the drawbacks of using BrdU is the dependence on a stressful injection procedure and uncertain penetration of the targeted cells with a uniform concentration of the compound. Thus, for experiments requiring measurements of cell proliferation, Ki67 can be used as an acceptable alternative. The protocol takes 3–5 d, allowing for sectioning and staining.

[1]  F. Gage,et al.  More hippocampal neurons in adult mice living in an enriched environment , 1997, Nature.

[2]  R. J. Mullen,et al.  NeuN, a neuronal specific nuclear protein in vertebrates. , 1992, Development.

[3]  Johannes Gerdes,et al.  The Ki‐67 protein: From the known and the unknown , 2000, Journal of cellular physiology.

[4]  J. Maruniak,et al.  Neurogenesis persists in the subependymal layer of the adult mouse brain , 1993, Neuroscience Letters.

[5]  J. Gerdes,et al.  The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins , 1993, The Journal of cell biology.

[6]  J. Miller,et al.  Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat , 1982, Brain Research.

[7]  M. Miller,et al.  Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population , 1989, Journal of neurocytology.

[8]  T. Freund,et al.  Loss of Calbindin-D28K immunoreactivity from dentate granule cells in human temporal lobe epilepsy , 1997, Neuroscience.

[9]  Gerd Kempermann,et al.  Milestones of neuronal development in the adult hippocampus , 2004, Trends in Neurosciences.

[10]  T. Dalkara,et al.  Loss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note , 2004, Brain Research.

[11]  J. Martin Wojtowicz,et al.  Dynamics of neurogenesis in the dentate gyrus of adult rats , 2005, Neuroscience Letters.

[12]  M. Celio,et al.  Calbindin D-28k and parvalbumin in the rat nervous system , 1990, Neuroscience.

[13]  R. Boonstra,et al.  The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis , 2002, Journal of Neuroscience Methods.

[14]  H. Gundersen,et al.  Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator , 1991, The Anatomical record.

[15]  E. Preston,et al.  Electrophysiological correlates of neural plasticity compensating for ischemia-induced damage in the hippocampus , 2005, Experimental Brain Research.

[16]  Jürgen Winkler,et al.  Transient expression of doublecortin during adult neurogenesis , 2003, The Journal of comparative neurology.

[17]  T. Seki,et al.  Temporal and spacial relationships between PSA‐NCAM‐expressing, newly generated granule cells, and radial glia‐like cells in the adult dentate gyrus , 1999, The Journal of comparative neurology.

[18]  J. Gerdes,et al.  The Ki-67 protein: fascinating forms and an unknown function. , 2000, Experimental cell research.

[19]  T. Palmer,et al.  Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[20]  V. Anisimov.  The Sole DNA Damage Induced by Bromodeoxyuridine Is Sufficient for Initiation of Both Aging and Carcinogenesis in Vivo , 1994, Annals of the New York Academy of Sciences.

[21]  R. McKay,et al.  Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus , 2001, The Journal of comparative neurology.

[22]  I. Whishaw,et al.  Embryonic and Postnatal Injections of Bromodeoxyuridine Produce Age-Dependent Morphological and Behavioral Abnormalities , 1999, The Journal of Neuroscience.

[23]  S. Hockfield,et al.  A family of proteins implicated in axon guidance and outgrowth. , 1999, Journal of neurobiology.

[24]  S. Mcconnell,et al.  Doublecortin Microtubule Affinity Is Regulated by a Balance of Kinase and Phosphatase Activity at the Leading Edge of Migrating Neurons , 2004, Neuron.

[25]  R. Kerr.  A cautionary note. , 1988, Science.

[26]  P. Bucher,et al.  The rsp5‐domain is shared by proteins of diverse functions , 1995, FEBS letters.

[27]  K. Gatter,et al.  Ki67 protein: the immaculate deception? , 2002, Histopathology.

[28]  H. Gratzner,et al.  Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. , 1982, Science.

[29]  G. Meyer,et al.  Selective expression of doublecortin and LIS1 in developing human cortex suggests unique modes of neuronal movement. , 2002, Cerebral cortex.