Culturing Embryonic Nasal Explants for Developmental and Physiological Study

Ulrike Klenke1, Carol Taylor‐Burds1

1 Cellular and Developmental Neurobiology Section, NIH/NINDS, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.25
DOI:  10.1002/0471142301.ns0325s59
Online Posting Date:  April, 2012
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Abstract

Primary cultures obtained from embryonic nasal placodes can maintain olfactory neurons, olfactory ensheathing cells, and large numbers of gonadotropin releasing hormone‐1 (GnRH) neurons. Depending on the age of the starting material, one can examine cell interactions important for placode formation or neuronal migration and axonal outgrowth. When generated at E11.5 in mouse, neuronal migration and axon outgrowth away from the main tissue mass occurs. This area of the explant, the periphery, is only a few cells thick. This characteristic offers the opportunity to image single cells and axons and allows pharmacological and molecular manipulations as well as physiological recordings to be performed. Here, we describe a system for culturing nasal explants used in our laboratory. This model system provides a method for obtaining physiological cellular responses with post hoc immunohistochemistry, or gene expression studies, on cells arising from the nasal placode. Curr. Protoc. Neurosci. 59:3.25.1‐3.25.16. © 2012 by John Wiley & Sons, Inc.

Keywords: nasal explants; primary organ culture; GnRH neurons; olfactory neurons; olfactory ensheathing cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Culture of Nasal Explants Generated from Mouse Nasal Placodes
  • Support Protocol 1: Perforated Patch‐Clamp Recordings in Nasal Explants
  • Support Protocol 2: Calcium Imaging in Nasal Explants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Primary Culture of Nasal Explants Generated from Mouse Nasal Placodes

  Materials
  • Timed‐pregnant mouse (E11.5)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A), cold
  • Gey's + glucose (G+G) buffer (see recipe)
  • Chicken plasma (see recipe)
  • Thrombin (see recipe)
  • Serum free medium (SFM; see recipe)
  • 5‐fluoro‐2′‐deoxyuridine (FUDR; Sigma, cat. no. F0503)
  • Dissection tools:
    • Blunt‐tipped operating scissors, 6.5 in.
    • Scissors for removal of uteri 5.5 in.
    • Large grip forceps 5 in.
    • Small forceps
    • Dissecting microscope with at least a 2× magnification, and light source
    • Narrow spatula
    • #5 forceps (2)
    • 4–in. Iris scissors
    • Plastic Pasteur pipets
    • Scalpel and sterile scalpel micro blades (size 15°)
  • Tissue culture dishes (100 × 20 mm; Becton Dickinson Labware, cat no. 35 3003)
  • Tissue culture dishes (150 × 25 mm; Becton Dickinson Labware, cat. no. 35 3025)
  • Tissue culture dishes (35 × 10 mm; Becton Dickinson Labware, cat. no. 35 3001)
  • Spatula
  • Glass coverslips (12 × 24 mm), sterilized in 100% ethanol for 24 hr and dried at 37 C for 60 min
  • Plastic coverslips: e.g., Permanox cell culture slides (Nalge Nunc, cat. no. 160005) cut into ∼1‐cm wide pieces with sterile scissors
  • Additional reagents and equipment for euthanasia of mice ( appendix 4H)

Support Protocol 1: Perforated Patch‐Clamp Recordings in Nasal Explants

  Materials
  • 100% Chlorox bleach
  • Intracellular solution (see recipe)
  • Gramicidin (Sigma, cat. no. G5002)
  • Dimethyl sulfoxide (DMSO; Sigma, cat. no. D8418)
  • Nasal explants ( protocol 1)
  • Extracellular solution (see recipe)
  • Borosilicate glass (1.5 mm OD/0.86 mm ID) (Harvard Apparatus, cat. no. 30‐0058)
  • P‐97 Flaming/Brown micropipet puller (Sutter Instruments)
  • 0.22‐µm sterile filters for syringe (Millipore, cat. no. SLGS0250S)
  • Gravity‐fed perfusion system
  • 3‐ to 5‐ml syringe to apply suction for filling electrode tip
  • 1‐ml syringe and >4‐cm needle for backfilling electrodes
  • Digidata 1312A acquisition system (Axon Instruments)
  • MWO‐3 three‐axis oil hydraulic micromanipulator (Narishige)
  • Axopatch amplifier (Axon Instruments)
  • Nikon 2000E inverted microscope
  • pClamp 9.2 Acquisition and Analysis Software (Axon Instruments)
  • Additional reagents and equipment for pulling pipets (unit 6.3) and preparing a standard electrophysiological setup meeting the requirements and specifications described in The Axon Guide (Sherman‐Gold, )

Support Protocol 2: Calcium Imaging in Nasal Explants

  Materials
  • Nasal explants ( protocol 1)
  • Calcium Green–1 working solution (see recipe)
  • Serum free medium (SFM, see recipe)
  • 40 mM KCl in SFM (see recipe for SFM)
  • Tissue culture dishes (35 × 10 mm; Becton Dickinson Labware, cat. no. 35 3001)
  • Recording/perfusion chamber (Warner Instruments, RC‐22, cat. no. 640228)
  • Peristaltic pump (Spectra Hardware)
  • Inverted imaging microscope (Nikon TE2000) with 20× fluorescence objective
  • Gravity‐fed perfusion system
  • CCD camera (Retiga, Qimaging)
  • Computer
  • Imaging software (iVision 4.5, BioVision; http://www.biovis.com/)
  • MATLAB software (MathWorks; http://www.mathworks.com/)
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Figures

Videos

Literature Cited

Literature Cited
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