Assessment of Adult Neurogenesis in Mice

Yung‐Wei Pan1, Wenbin Wang2, Zhengui Xia2

1 Graduate Program, Molecular and Cellular Biology, University of Washington, Seattle, Washington, 2 Toxicology Program, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.20
DOI:  10.1002/0471140856.tx1220s56
Online Posting Date:  May, 2013
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Abstract

Adult neurogenesis is a lifelong developmental process that occurs in two discrete regions in the adult mammalian brain: the subgranular zone of the dentate gyrus (DG) and the subventricular zone (SVZ) along the lateral ventricles. Despite immense interest in the therapeutic potential of adult neural stem cells (aNSCs) residing along these two neurogenic regions, molecular and cellular mechanisms regulating this process are not fully defined. Defining the regulatory mechanisms responsible for the genesis of new neurons in the adult brain is integral to understanding the basic biology of aNSCs. The techniques described here provide a basic blueprint to isolate, culture, and perform experiments using aNSCs in vitro as well as providing methods to perform immunohistochemistry on brain sections. Curr. Protoc. Toxicol. 56:12.20.1‐12.20.16. © 2013 by John Wiley & Sons, Inc.

Keywords: neurogenesis; adult neural stem cells (aNSC); immunohistochemistry; primary culture; primary cell isolation; brain section

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation, Culture, and Expansion of Primary Adult Neural Stem Cells from Both Neurogenic Regions
  • Support Protocol 1: Double Coating Culture Surfaces
  • Basic Protocol 2: In Vivo BrdU Labeling and Immunohistochemical Analysis of Adult Brain Tissue
  • Support Protocol 2: Making Gelatinized Slides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation, Culture, and Expansion of Primary Adult Neural Stem Cells from Both Neurogenic Regions

  Materials
  • 70% ethanol
  • Hank's Balanced Salt Solution (HBSS, Invitrogen #14025‐092)
  • Fresh, whole adult (8‐ to 10‐week‐old) mouse brain
  • Neurosphere dissociation and monolayer culture
    • Solution A (see recipe)
    • Enzyme mix 1 (see recipe)
    • Enzyme mix 2 (see recipe)
    • Advanced DMEM/F‐12 (Invitrogen #12634‐010)
    • Fetal bovine serum (FBS, Gibco #16140)
    • Percoll solution (see recipe)
    • Initial proliferation medium (IPM; see recipe)
    • EGF (EMD Chemicals #324831)
    • bFGF (Millipore #GF003)
    • Serum‐free medium (SFM; see recipe)
    • 10% DMSO (optional)
    • 0.125% trypsin‐EDTA (Invitrogen #25200‐056), diluted from 0.25% in HBSS
    • 0.014% w/v soybean trypsin inhibitor (Invitrogen #17075‐029), diluted in HBSS
  • BrdU labeling and immunocytochemistry
    • BrdU (Sigma #B9285)
    • 4% paraformaldehyde (PFA)/sucrose (see recipe)
    • 1× PBS, prepared from 10× PBS (see recipe)
    • 1 N and 2 N hydrochloric acid
    • 0.1 M borate buffer, pH 8.5 (see recipe)
    • PBST (see recipe)
    • Blocking buffer 1 (5% BSA in PBST; see recipe for PBST)
    • Primary antibodies (see Table 12.20.1)
    • Alexa‐Fluor‐488 or ‐594 secondary antibodies (Invitrogen)
    • 2.5 µg/ml Hoechst 33342 (Invitrogen #H3570), diluted in PBS
    • Aqua‐Poly/Mount medium (Polysciences #18606‐20)
  • Dissecting instruments
    • Small dissecting scissors
    • Forceps
    • Fine‐tip forceps
    • Scalpel
  • 60‐, 100‐, and 35‐mm petri dishes
  • 15‐ml and 50‐ml conical tubes
  • Dissecting microscope
  • Brain matrix (Kent Scientific #RBMA‐200C)
  • Cell strainer (40 µm, Fisher #22‐363‐547)
  • 37°C tissue culture (TC) incubator with 6.5% CO 2 setting
  • Double‐coated culture dishes or Aclar film coverslips (Electron Microscopy Sciences #50425) (see protocol 2)
    Table 2.0.1   MaterialsPrimary Antibodies for Immunolabeling Different Cell Types In Vitro

    Antibody Host Dilution Source
    BrdU Rat 1:500 AbD Serotec #MCA2060
    PCNA Mouse 1:1,000 Millipore #MAB424
    Ki67 Rabbit 1:2,000 Leica #NCL‐Ki67p
    Nestin Mouse 1:500 DSHB #Rat‐401
    GFAP Mouse 1:1,000 Millipore #MAB360
    Sox2 Mouse 1:500 Millipore #MAB4343
    DCX Goat 1:500 Santa Cruz #sc‐8067
    PSA‐NCAM Mouse 1:500 DSHB #5A5
    β‐III tubulin Mouse 1:1,000 Promega #G7121
    NeuN Mouse 1:500 Millipore #MAB377

Support Protocol 1: Double Coating Culture Surfaces

  Materials
  • Poly‐L‐ornithine (15 µg/ml)
  • Autoclaved Milli‐Q water for making poly‐L‐ornithine dilution
  • 70% ethanol
  • 1× PBS, prepared from 10× PBS (see recipe)
  • Fibronectin (1 mg/ml) diluted in autoclaved PBS
  • Tissue culture dishes or Aclar film (Electron Microscopy Sciences #50425) and hole punch

Basic Protocol 2: In Vivo BrdU Labeling and Immunohistochemical Analysis of Adult Brain Tissue

  Materials
  • 20 mg/ml BrdU (see recipe)
  • Mice for labeling
  • Cryoprotectant medium (see recipe)
  • 1× PBS, prepared from 10× PBS (see recipe)
  • 1 N and 2 N hydrochloric acid (HCl)
  • 0.1 M borate buffer, pH 8.5 (see recipe)
  • PBST (see recipe)
  • Blocking buffer 2 (see recipe)
  • Primary antibodies (see Table 12.20.2)
  • Alexa‐Fluor Secondary Antibodies (Invitrogen)
  • 2.5 µg/ml Hoechst 33342 (Invitrogen #H3570), diluted in PBS
  • Aqua‐Poly/Mount (Polysciences #18606‐20)
  • SDS, 1% in Milli‐Q water
  • Cryostat, microtome, or other sectioning equipment for frozen tissue
  • Superfrost Plus microscope slides (Fisher #12‐550‐15), not gelatinized, for directly mounted sections
  • 24‐ or 48‐well plates for free‐floating tissue storage in cryoprotectant medium
  • 12‐ or 24‐well plates
  • 12‐ or 24‐well Netwell inserts (Corning)
  • Superfrost Plus microscope slides, gelatinized (see protocol 4), for free‐floating IHC method
  • Size 0 paintbrush
  • Coverslips
  • Pap‐pen liquid blocker (Electron Microscopy #71312)
    Table 2.0.2   MaterialsPrimary Antibodies for Immunolabeling Different Cell Types In Vivo

    Antibody Host Dilution Source
    BrdU Rat 1:500 AbD Serotec #MCA2060
    PCNA Mouse 1:500 Millipore #MAB424
    Ki67 Rabbit 1:1,000 Leica #NCL‐Ki67p
    GFAP Mouse 1:1,000 Millipore #MAB360
    Sox2 Mouse 1:500 R&D Systems #MAB2018
    DCX Goat 1:200 Santa Cruz #sc‐8067
    PSA‐NCAM Mouse 1:500 DSHB #5A5
    NeuroD Goat 1:200 Santa Cruz #sc‐1084
    Calretinin Mouse 1:500 Santa Cruz #sc‐135853
    NeuN Mouse 1:500 Millipore #MAB377
    Calbindin Mouse 1:500 Santa Cruz #sc‐58699

Support Protocol 2: Making Gelatinized Slides

  Materials
  • 2% gelatin, dissolved in Milli‐Q H 2O
  • Slide dipper reservoir
  • Superfrost Plus microscope slides (Fisher #12‐550‐15)
  • Slide box for storage
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Figures

Videos

Literature Cited

Literature Cited
   Altman, J. and Das, G.D. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol. 124:319‐335.
   Amiri, A., Cho, W., Zhou, J., Birnbaum, S.G., Sinton, C.M., McKay, R.M., and Parada, L.F. 2012. Pten deletion in adult hippocampal neural stem/progenitor cells causes cellular abnormalities and alters neurogenesis. J. Neurosci. 32:5880‐5890.
   Bonaguidi, M.A., Peng, C.Y., McGuire, T., Falciglia, G., Gobeske, K.T., Czeisler, C., and Kessler, J.A. 2008. Noggin expands neural stem cells in the adult hippocampus. J. Neurosci. 28:9194‐9204.
   Brown, J., Cooper‐Kuhn, C.M., Kempermann, G., Van Praag, H., Winkler, J., Gage, F.H., and Kuhn, H.G. 2003. Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur. J. Neurosci. 17:2042‐2046.
   Bull, N.D. and Bartlett, P.F. 2005. The adult mouse hippocampal progenitor is neurogenic but not a stem cell. J. Neurosci. 25:10815‐10821.
   Gage, F.H. 2000. Mammalian neural stem cells. Science 287:1433‐1438.
   Gage, F.H., Kempermann, G., Palmer, T.D., Peterson, D.A., and Ray, J. 1998. Multipotent progenitor cells in the adult dentate gyrus. J. Neurobiol. 36:249‐266.
   Gao, Z., Ure, K., Ding, P., Nashaat, M., Yuan, L., Ma, J., Hammer, R.E., and Hsieh, J. 2011. The master negative regulator REST/NRSF controls adult neurogenesis by restraining the neurogenic program in quiescent stem cells. J. Neurosci. 31:9772‐9786.
   Gould, E., Beylin, A., Tanapat, P., Reeves, A., and Shors, T.J. 1999. Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci. 2:260‐265.
   Guo, W., Patzlaff, N.E., Jobe, E.M., and Zhao, X. 2012. Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse. Nat. Protoc. 7:2005‐2012.
   Hodge, R.D., Kowalczyk, T.D., Wolf, S.A., Encinas, J.M., Rippey, C., Enikolopov, G., Kempermann, G., and Hevner, R.F. 2008. Intermediate progenitors in adult hippocampal neurogenesis: Tbr2 expression and coordinate regulation of neuronal output. J. Neurosci. 28:3707‐3717.
   Imayoshi, I., Sakamoto, M., Ohtsuka, T., Takao, K., Miyakawa, T., Yamaguchi, M., Mori, K., Ikeda, T., Itohara, S., and Kageyama, R. 2008. Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat. Neurosci. 11:1153‐1161.
   Luo, Y., Shan, G., Guo, W., Smrt, R.D., Johnson, E.B., Li, X., Pfeiffer, R.L., Szulwach, K.E., Duan, R., Barkho, B.Z., Li, W., Liu, C., Jin, P., and Zhao, X. 2010. Fragile X mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells. PLoS Genet. 6:e100898.
   Ming, G.L. and Song, H. 2005. Adult neurogenesis in the mammalian central nervous system. Annu. Rev. Neurosci. 28:223‐250.
   Mu, Y., Lee, S.W., and Gage, F.H. 2010. Signaling in adult neurogenesis. Curr. Opin. Neurobiol. 20:416‐423.
   Pan, Y.W., Chan, G.C.K., Kuo, C.T., Storm, D.R., and Xia, Z. 2012a. Inhibition of adult neurogenesis by inducible and targeted deletion of ERK5 mitogen‐activated protein kinase specifically in adult neurogenic regions impairs contextual fear extinction and remote fear memory. J. Neurosci. 32:6444‐6455.
   Pan, Y.W., Zou, J., Wang, W., Sakagami, H., Garelick, M.G., Abel, G., Kuo, C.T., Storm, D.R., and Xia, Z. 2012b. Inducible and conditional deletion of extracellular signal‐regulated kinase 5 disrupts adult hippocampal neurogenesis. J. Biol. Chem. 287:23306.
   Pan, Y.W., Kuo, C.T., Storm, D.R., and Xia, Z. 2012c. Inducible and targeted deletion of the ERK5 MAP kinase in adult neurogenic regions impairs adult neurogenesis in the olfactory bulb and several forms of olfactory behavior. PLoS One 7:e49622.
   Pan, Y.W., Storm, D.R., and Xia, Z. 2012d. The maintenance of established remote contextual fear memory requires ERK5 MAP kinase and ongoing adult neurogenesis in the hippocampus. PLoS One 7:e50455.
   Reynolds, B.A. and Weiss, S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707‐1710.
   Suh, H., Deng, W., and Gage, F.H. 2009. Signaling in adult neurogenesis. Annu. Rev. Cell Dev. Biol. 25:253‐275.
   Taupin, P. 2006. Neurogenesis in the adult central nervous system. C.R. Biol. 329:465‐475.
   van Praag, H., Kempermann, G., and Gage, F.H. 1999. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci. 2:266‐270.
   Zou, J., Pan, Y.W., Wang, Z., Chang, S.Y., Wang, W., Wang, X., Tournier, C., Storm, D.R., and Xia, Z. 2012. Targeted deletion of ERK5 MAP kinase in the developing nervous system impairs development of GABAergic interneurons in the main olfactory bulb and behavioral discrimination between structurally similar odorants. J. Neurosci. 32:4118‐4132.
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