Morphological Assessment of Neurite Outgrowth in Hippocampal Neuron‐Astrocyte Co‐Cultures

Gennaro Giordano1, Lucio G. Costa2

1 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, 2 Department of Human Anatomy, Pharmacology and Forensic Science, University of Parma Medical School, Parma, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.16
DOI:  10.1002/0471140856.tx1116s52
Online Posting Date:  May, 2012
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Abstract

Neurite outgrowth is a fundamental event in brain development, as well as in regeneration of damaged neurons. Astrocytes play a major role in neuritogenesis, by expressing and releasing factors that facilitate neurite outgrowth, such as extracellular matrix proteins, and factors that can inhibit neuritogenesis, such as the chondroitin sulfate proteoglycan neurocan. In this unit we describe a noncontact co‐culture system of hippocampal neurons and cortical (or hippocampal) astrocytes for measurement of neurite outgrowth. Hippocampal pyramidal neurons are plated on glass coverslips, which are inverted onto an astrocyte feeder layer, allowing exposure of neurons to astrocyte‐derived factors without direct contact between these two cell types. After co‐culture, neurons are stained and photographed, and processes are assessed morphologically using Metamorph software. This method allows exposing astrocytes to various agents before co‐culture in order to assess how these exposures may influence the ability of astrocytes to foster neurite outgrowth. Curr. Protoc. Toxicol. 52:11.16.1‐11.16.26. © 2012 by John Wiley & Sons, Inc.

Keywords: pyramidal hippocampal neurons; astrocytes; co‐culture; neurite outgrowth

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Astrocyte‐Neuro Co‐Cultures
  • Support Protocol 1: Preparation of Hippocampal Neurons
  • Support Protocol 2: Preparation of Astrocyte Cultures
  • Support Protocol 3: Preparation of Glass Coverslips
  • Support Protocol 4: Staining of Hippocampal Neurons
  • Support Protocol 5: Double‐Staining for Axons and Dendrites in Hippocampal Neurons
  • Support Protocol 6: Florescence Imaging of Hippocampal Neurons
  • Basic Protocol 2: Image Analysis Using Metamorph Software
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Astrocyte‐Neuro Co‐Cultures

  Materials
  • Confluent cortical or hippocampal astrocytes monolayer in 24‐well plates (see protocol 3)
  • Desired agent
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐049)
  • Serum‐free Dulbecco's Modified Eagle's medium (see recipe)
  • Coated glass coverslips seeded with hippocampal neurons (see protocol 2)
  • Hank's balanced salt solution supplemented with calcium and magnesium (HBSS+CM; see recipe)
  • 37°C incubator
  • Microscope
  • 20‐G needles
  • Curved forceps (Dumont), sterile

Support Protocol 1: Preparation of Hippocampal Neurons

  Materials
  • 1.5 mg/ml Papain solution (see recipe)
  • 4 mg/ml DNase I solution (see recipe)
  • Pregnant rat
  • Carbon dioxide
  • 70% (v/v) ethanol
  • Hank's balanced salt solution, calcium‐ and magnesium‐free (HBSS‐CM; see recipe)
  • 1 M MgCl 2 stock solution (see recipe)
  • Complete neurobasal medium (CNM; see recipe)
  • 0.04% (w/v) Trypan blue (see recipe)
  • 37°C water bath
  • 35‐ and 100‐mm petri dishes
  • Scissors
  • Spatula
  • Sterile filter paper
  • Sharp scalpels
  • Fine curved‐tip forceps
  • 15‐ml polypropylene tubes
  • Centrifuge
  • Pasteur pipets
  • 40‐µm nylon mesh filter
  • 50‐ml sterile tubes
  • 200‐µl, 5‐, and 10‐ml pipets
  • 1000‐µl pipet tips
  • 1.5‐ml microcentrifuge tubes
  • Hemacytometer
  • 24‐well plate containing coated coverslips
  • 37°C tissue culture incubator
  • Additional reagents and equipment for euthanizing the rat (Donovan and Brown, ) and opening the skulls (unit 12.3)

Support Protocol 2: Preparation of Astrocyte Cultures

  Materials
  • 40 µg/ml poly‐D‐lysine (PLYS; see recipe)
  • Pregnant Sprague Dawley rats
  • Hank's balanced salt solution, calcium‐ and magnesium‐free (HBBS‐CM; see recipe)
  • 0.25% trypsin solution (see recipe)
  • Dulbecco's modified Eagle's medium, low glucose, with serum (DMEM‐FBS; see recipe)
  • 0.04% (w/v) Trypan blue solution (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐049)
  • 75‐cm2 flasks, sterile
  • Sterile filter paper
  • 100‐mm plates
  • Surgical scissors, sterile
  • 50‐ml conical tubes
  • 37°C water bath
  • Centrifuge
  • Vortex mixer
  • 100‐µm pore nylon mesh filter (Falcon)
  • Hemacytometer

Support Protocol 3: Preparation of Glass Coverslips

  Materials
  • Paraffin wax pellets
  • 1 M HCl solution (see recipe)
  • Double‐distilled sterile water
  • 100 µg/ml poly‐D‐lysine (PLYS) solution (see recipe)
  • Hank's balanced salt solution (HBSS+CM; see recipe)
  • 12‐mm glass coverslips
  • Glass beakers
  • Hot plate
  • 1‐ml sterile syringes equipped with 20‐G needles
  • Forceps
  • 24‐well tissue culture plates
  • 37°C incubator

Support Protocol 4: Staining of Hippocampal Neurons

  Materials
  • Hank's balanced salt solution supplemented with calcium and magnesium (HBSS+CM; see recipe)
  • 4% Paraformaldehyde solution (PFA; see recipe)
  • Quenching solution (50 mM ammonium chloride; see recipe)
  • Blocking solution (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐049), 1×
  • Anti tubulinβ III isoform primary antibody solution (see recipe)
  • Anti‐mouse Alexa 555 (or 488) secondary antibody solution (see recipe)
  • 5 µg/ml Hoechst 44432 solution (see recipe)
  • Vectashield mounting medium
  • Nail polish
  • Shaker
  • Parafilm
  • Kimwipe
  • Glass slides
  • Cover glass

Support Protocol 5: Double‐Staining for Axons and Dendrites in Hippocampal Neurons

  Materials
  • Hippocampal neurons (see protocol 2)
  • Hank's balanced salt solution (HBSS+CM; see recipe)
  • 4% paraformaldehyde solution (PFA; see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐049)
  • Triton X‐100 (TX‐100) solution (see recipe)
  • PBSF solution (see recipe)
  • Tau‐1/MAP 2 primary antibody solution (see recipe)
  • Tau‐1/MAP2 secondary antibody solution (see recipe)
  • Hoechst 44432 solution, 5 µg/ml (see recipe)
  • Antifade fluorescent mounting medium
  • Nail polish

Support Protocol 6: Florescence Imaging of Hippocampal Neurons

  Materials
  • Imaging software (Nuance 3.0.0)
  • Multispectral imaging system (Nuance, CRI)
  • Fluorescence microscope

Basic Protocol 2: Image Analysis Using Metamorph Software

  Materials
  • Acquired images ( protocol 7)
  • Neurite morphology quantification and analysis software (MetaMorph 6.1 used here)
  • Compatible computer terminal with Microsoft Excel
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Figures

  •   FigureFigure 11.16.1 Application of hot, melted paraffin onto glass coverslips. Note placement of paraffin dots at the outer edges of the coverslip, as well as the use of a 20‐G needle and a sterile 1‐ml syringe. As noted in the text, the paraffin in the needle must be sufficiently hot to flow through the needle and adhere to the glass coverslip, yet not so hot as to flatten out. Ideally, a sphere of tightly adhered paraffin will be formed. Please note: coverslip and paraffin dots are to scale relative to each other; syringe and needle are not to scale. Paraffin (dots and in syringe) has been colored gray for clarity only; this does NOT imply that heated paraffin should be gray.
  •   FigureFigure 11.16.2 Placement of fresh paraffin‐dotted glass coverslips in 24‐well plates. Please note that prior to this step, coverslips should be tested (Basic Protocol 3, step 4) to verify tight adherence of paraffin dots to the glass coverslips. Coverslips that do not have at least three tightly bound paraffin dots should be discarded. At this time, coverslips that pass quality testing (Basic Protocol 3, step 4) should be placed with dots facing UP, followed by addition of 1 N HCL for at least 12 hr as noted in the text.
  •   FigureFigure 11.16.3 Hippocampal pyramidal neuron after 24 hr of co‐culture with control cortical astrocytes. After fixing and staining with a β‐tubulin‐III antibody, the neuron was imaged and neurite length was quantified using Metamorph software. The arrow 1 is indicating the longest neurite (the axon, by Tau‐1 staining), and arrows 2 and 3 the minor neurites.
  •   FigureFigure 11.16.4 Hippocampal pyramidal neurons after 24 hour of co‐culture. After fixing and staining with β‐tubulin‐III antibody, the neurons were imaged. As the processes of the neurons intermingled (arrow), this picture was excluded from the neurite outgrowth analysis. Scale bar = 20 µm.

Videos

Literature Cited

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Key References
   Meijering, 2010. See above.
  Detailed and exhaustive paper describing the state of the art of reconstruction and study of neuronal morphology from image data. It also provides a useful list of commercial and freeware software and tools for automatic image segmentation.
   Dotti et al., 1988. See above.
  First report describing in detail how hippocampal neurons acquire their distinctive axonal and dendritic architecture. The paper focuses on the early events leading to the establishment of polarity collecting observations from living neurons. Dotti et al., classified the neuronal development in 5 steps: formation of lamellipodia, outgrowth of the minor processes, formation and growth of the axon, growth of dendrites, maturation.
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