title: A rapid method for the quantification of mouse hippocampal neurogenesis in vivo by flow cytometry. Validation with conventional and enhanced immunohistochemical methods.
abstract: Neural stem cells reside in the subventricular zone and the dentate gyrus of the hippocampus in adult mammalian brain. In the hippocampus, a number of factors are reported to modulate the rate of neural progenitor proliferation in the hippocampus, such as exercise, corticosteroids, and many pharmacological agents including several classes of antidepressants. It is currently unclear whether this increased proliferation is physiologically relevant, but it provides a potentially useful biomarker to assess novel antidepressant compounds. Changes in neurogenesis are typically quantified by administration of bromodeoxyuridine (BrdU) in vivo, and subsequent quantification of labelled nuclei. A robust and rapid means of quantifying BrdU labelling in adult hippocampus in vivo would allow higher throughput screening of potential antidepressant compounds. In this study we describe a FACS-based method for quantification of BrdU labelled cells in fixed cell suspensions from BrdU-treated adult mouse hippocampus. A variety of experimental conditions known to modulate proliferation were tested, including administration of corticosterone and the antidepressants imipramine and fluoxetine. The robust changes compared to control groups observed in these models were similar to previously reported studies, thus offering a more rapid and streamlined means to quantify effects of compounds on hippocampal proliferation.
authors.affiliation: Department of Molecular and Cellular Neuroscience, Merck, Sharp and Dohme Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, UK. james.bilsland@pfizer.com
authors.forename: James George
authors.initials: JG
authors.lastname: Bilsland
authors.forename: Christine
authors.initials: C
authors.lastname: Haldon
authors.forename: Julie
authors.initials: J
authors.lastname: Goddard
authors.forename: Kevin
authors.initials: K
authors.lastname: Oliver
authors.forename: Fraser
authors.initials: F
authors.lastname: Murray
authors.forename: Alan
authors.initials: A
authors.lastname: Wheeldon
authors.forename: Janine
authors.initials: J
authors.lastname: Cumberbatch
authors.forename: George
authors.initials: G
authors.lastname: McAllister
authors.forename: Ignacio
authors.initials: I
authors.lastname: Munoz-Sanjuan
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000008:administration & dosage / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D003609:Dactinomycin / Q000008:administration & dosage / Q000031:analogs & derivatives; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D006624:Hippocampus / Q000166:cytology*; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D009474:Neurons / Q000502:physiology*; D038081:Organogenesis / Q000502:physiology*; D015203:Reproducibility of Results
chemical_list: D003609:Dactinomycin; C025942:7-aminoactinomycin D; D001973:Bromodeoxyuridine