Culture of Mouse Olfactory Sensory Neurons

Qizhi Gong1

1 Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.24
DOI:  10.1002/0471142301.ns0324s58
Online Posting Date:  January, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Olfactory sensory neurons, located in the nasal epithelium, detect and transmit odorant information to the central nervous system. This requires that these neurons form specific neuronal connections within the olfactory bulb and express receptors and signaling molecules specific for these functions. This protocol describes a primary olfactory sensory neuron culture technique that allows in vitro investigation of olfactory sensory neuron differentiation, axon outgrowth, odorant receptor expression, and function. Olfactory epithelium is obtained from the nasal cavity and is enzymatically treated to reduce stroma tissue. Dissociated olfactory sensory neurons are cultured directly on a layer of cortical astrocytes to support their survival. Using this method, cultured olfactory sensory neurons maintain their bipolar morphology and express odorant signal transduction molecules, which are specific for olfactory sensory neurons. Curr. Protoc. Neurosci. 58:3.24.1‐3.24.14. © 2012 by John Wiley & Sons, Inc.

Keywords: olfactory epithelium; bipolar; astrocytes; lentivirus; odorant receptor

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Primary Mouse Olfactory Sensory Neurons
  • Support Protocol 1: Preparation of Glass Coverslips
  • Support Protocol 2: Purification and Culturing Mouse Cortical Astrocytes
  • Support Protocol 3: Immunochemistry of OSN Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Culture of Primary Mouse Olfactory Sensory Neurons

  Materials
  • Cortical astrocytes (see protocol 3)
  • Glia medium (see recipe)
  • 70% ethanol
  • CMF‐HBSS/HEPES (see recipe)
  • OSN medium (see recipe)
  • C57BL/6 embryos at the desired age range (E15‐E20)
  • 2.5% avertin (see recipe) or alternative anesthetics (e.g., sodium pentobarbital)
  • Ice
  • Dispase (Life Technologies, cat. no. 17105041)
  • DNase I
  • Mild trypsinization solution (see recipe)
  • EDTA
  • Fetal bovine serum
  • 12‐mm glass coverslips, culture prepared (see protocol 2)
  • 24‐well plates
  • 37°C, 5% CO 2 incubator
  • Dissection tools:
    • One 5 1/ 2‐ in. scissors
    • One forceps
    • One 4 1/ 2‐ in. fine scissors
    • Two No. 5 forceps
    • One microscissors
  • Horizontal laminar flow culture hood
  • 1‐ml syringe equipped with 28‐G needles
  • 10‐cm petri dishes, sterile
  • Stereo dissecting microscope
  • 35‐mm culture dishes
  • Sterile glass Pasteur pipets (both flame polished and not polished)
  • 15‐ml Falcon tubes
  • Benchtop centrifuge
  • 37°C water bath
  • Microscope and hemacytometer
  • Additional reagents and equipment for counting cells using a hemacytomer (Phelan, )

Support Protocol 1: Preparation of Glass Coverslips

  Materials
  • 10 M NaOH
  • 70% ethanol
  • 1 mg/ml laminin stock (BD Biosciences; dissolved in ddH 2O)
  • 10 mg/ml poly‐D‐lysine stock (BD Biosciences; dissolved in ddH 2O)
  • Appropriate culture medium (e.g., DMEM)
  • No. 1 12‐mm circular glass coverslip
  • Coverslip staining racks
  • Laminar flow culture hood
  • 24‐well plates

Support Protocol 2: Purification and Culturing Mouse Cortical Astrocytes

  Materials
  • 70% ethanol
  • Five to ten neonatal C57 BL/6 mouse pups (P0 to P3)
  • Crushed ice
  • CMF‐HBSS/HEPES (see recipe)
  • Trypsinization solution (see recipe)
  • Fetal bovine serum
  • Glia medium (see recipe)
  • 1 mM AraC (Sigma‐Aldrich)
  • Dimethyl sulfoxide (DMSO)
  • Liquid nitrogen
  • Dissection tools:
    • One 5 1/ 2‐in. scissors
    • One forceps
    • One 4 1/ 2‐in. fine scissors
    • Two no. 5 forceps
    • One micro scissors
    • One micro spatula (Ted Pella, cat. no. 13510)
  • Laminar flow culture hood
  • Latex sleeves fashioned from the fingers of latex gloves
  • 35‐mm dishes
  • Stereomicroscope
  • 50‐ml Falcon tubes
  • Pasteur pipets
  • Benchtop centrifuge
  • 37°C water bath
  • 5‐ml pipets
  • 70‐µm Falcon cell strainer
  • Microscope and hemacytometer
  • 75‐cm2 flasks
  • 37°C, 5% CO 2 incubator
  • Parafilm
  • Rotary platform
  • Cryovials
  • 12‐mm glass coverslips, culture prepared (see protocol 2)
  • 24‐well plates
  • Additional reagents and equipment for counting cells using a hemacytometer (Phelan, )

Support Protocol 3: Immunochemistry of OSN Culture

  Materials
  • OSN culture (see protocol 1)
  • 4% paraformaldehyde
  • Phosphate‐buffered saline (PBS; see recipe) with 0.1% Triton X‐100 (PBS+TX)
  • Normal horse serum
  • Phosphate‐buffered saline (PBS; see recipe)
  • Primary antibodies (see step 4 annotation for details)
  • Secondary antibodies (see the annotation to step 6 for details)
  • Fluoromount G (Southern Biotech, cat. no. 0100‐01)
  • Petri dishes
  • Parafilm
  • Coverslip rack
  • Glass slides
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ayadi, A., Ferrand, G., Cruz, I.G.d., and Warot, X. 2011. Mouse breeding and colony management. Curr. Protoc. Mouse Biol. 1:239‐264.
   Chen, H., Dadsetan, S., Fomina, A.F., and Gong, Q. 2008. Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons. Neural Develop. 3:22.
   Ekberg, J.A., Amaya, D., Chehrehasa, F., Lineburg, K., Claxton, C., Windus, L.C., Key, B., Mackay‐Sim, A., and St John, J.A. 2011. OMP‐ZsGreen fluorescent protein transgenic mice for visualisation of olfactory sensory neurons in vivo and in vitro. J. Neurosci. Methods 196:88‐98.
   Farbman, A.I. 1992. Cell Biology of Olfaction. Cambridge University Press, New York.
   Goslin, K., Asmussen, H., and Banker, G. 1998. Rat hippocampal neurons in low‐density culture. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 339‐369. MIT Press, London.
   Grill, R.J. and Pixley, S.K. 1997. In vitro generation of adult rat olfactory sensory neurons and regulation of maturation by coculture with CNS tissues. J. Neurosci. 17:3120‐3127.
   Mombaerts, P. 2006. Axonal wiring in the mouse olfactory system. Annu. Rev. Cell. Dev. Biol. 22:713‐737.
   Moon, C., Yoo, J.Y., Matarazzo, V., Sung, Y.K., Kim, E.J., and Ronnett, G.V. 2002. Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation. Proc. Natl. Acad. Sci. U.S.A. 99:9015‐9020.
   Packard, A., Schnittke, N., Romano, R.A., Sinha, S., and Schwob, J.E. 2011. DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium. J. Neurosci. 31:8748‐8759.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Weinstein, D.E. 2001. Isolation and purification of primary rodent astrocytes. Curr. Protoc. Neurosci. 1:3.5.1‐3.5.9.
   Zhuang, H. and Matsunami, H. 2007. Synergism of accessory factors in functional expression of mammalian odorant receptors. J. Biol. Chem. 282:15284‐15293.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library