Synaptic Structure Quantification in Cultured Neurons

Pamela J. Roqué1, Marina Guizzetti2, Lucio G. Costa1

1 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, 2 Department of Psychiatry, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, Illinois
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.22
DOI:  10.1002/0471140856.tx1222s60
Online Posting Date:  May, 2014
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Abstract

Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant‐induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre‐ and post‐synaptic markers synaptophysin and PSD‐95, confocal imaging, and three‐dimensional object analysis. Protocols for the long‐term culturing of primary hippocampal neurons and of primary cortical astrocytes, as well as their co‐culture, are included. While the described methods focus on how astrocytes influence synapse formation and how toxicants may interfere in this process, modifications to the experimental plan can easily be implemented. This would allow for the investigation of the effects of toxicants after treating neurons alone, or both astrocytes and neurons in co‐culture. With the common endpoint of synapse structure formation, differences between varying treatment paradigms can expand the understanding of the influence of particular toxicants on these diverse cell types and provide insight into potential mechanisms of effect and the contributions of each to synapse formation. Curr. Protoc. Toxicol. 60:12.22.1‐12.22.32. © 2014 by John Wiley & Sons, Inc.

Keywords: synaptogenesis; astrocytes; sandwich co‐culture; 3‐dimensional analysis; PSD‐95; synaptophysin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Neuronal Preparation and Culture
  • Support Protocol 1: Neuronal Coverslip Preparation
  • Basic Protocol 2: Primary Astrocyte Preparation and Culture
  • Basic Protocol 3: Astrocyte Passage for Experimental Treatment and Co‐Culture
  • Support Protocol 2: 4% Paraformaldehyde (PFA) Preparation
  • Basic Protocol 4: Neuronal Immunostaining for Synaptic Proteins
  • Basic Protocol 5: Synaptic Structure Imaging and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Neuronal Preparation and Culture

  Materials
  • Hank's balanced salt solution with Ca2+ and Mg2+ containing 10 mM HEPES (HBSS plus CM), keep sterile and store at room temperature
  • Papain solution (see recipe)
  • 4 mg/ml deoxyribonuclease‐1 from bovine pancreas (DNase) (see recipe)
  • 1 M magnesium chloride (MgCl 2), sterile
  • E21 Sprague‐Dawley rat fetuses, obtained from timed‐pregnant rat dams (Charles River)
  • Hank's balanced salt solution (Ca2+ and Mg2+‐free) containing 10 mM HEPES (HBSS CM‐free), keep sterile and store at room temperature
  • Neuronal medium (see recipe)
  • Trypan blue
  • 100 µM cytosine β‐D‐arabinofuranoside (ARAC, see recipe)
  • Coverslips in 24‐well plates (see protocol 2)
  • 37°C water bath
  • Sterile surgical tools
  • Sterile filter paper
  • 35‐mm dishes
  • Sterile, disposable 5‐ml syringe and 22‐µm filter
  • 15‐ and 50‐ml tubes
  • Refrigerated centrifuge
  • Aspirator
  • Rubber bulbs
  • Sterile, glass Pasteur pipets (9‐in.)
  • 40‐µm pore nylon mesh filters
  • 5‐ and 10‐ml sterile serological piptets
  • Hemacytometer and microscope
  • Sterile needles
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 1: Neuronal Coverslip Preparation

  Materials
  • Paraffin wax pellets melted
  • 1 M hydrochloric acid (HCl)
  • Sterile water
  • Poly‐L‐ornithine hydrobromide (PLO, 10 mg/ml stock, see recipe)
  • 10‐ml glass beaker
  • Hot plate
  • Aluminum foil
  • 1‐ml disposable syringes
  • Disposable needle (20‐G, 1‐in.)
  • Round, microscope glass coverslips (12‐mm, no. 1)
  • Surgical tweezers
  • 24‐well plates
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 2: Primary Astrocyte Preparation and Culture

  Materials
  • 40 µg/ml poly‐D‐lysine (PDL) working solution (see recipe)
  • Sterile water
  • Phosphate buffered saline (PBS)
  • E21 Sprague‐Dawley rat fetuses obtained from time‐pregnant rat dams (Charles River)
  • HBSS CM‐free
  • 0.25% trypsin (see recipe), 37°C
  • Astrocyte maintenance medium: DMEM‐FBS (see recipe), 37°C
  • 75‐cm2 sterile flasks
  • Sterile surgical tools: tweezers, surgical scissors
  • Sterile filter paper, optional
  • 100‐mm plates
  • 37°C, 5% CO 2 humidified incubator
  • Benchtop centrifuge
  • Vortexer
  • 100‐µm pore nylon mesh filter
  • 50‐ml conical tubes
  • Hemacytometer
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 3: Astrocyte Passage for Experimental Treatment and Co‐Culture

  Materials
  • 0.25% trypsin (see recipe)
  • Astrocyte maintenance medium: DMEM‐FBS (see recipe)
  • 40 µg/ml poly‐D‐lysine (PDL) working solution (see recipe)
  • Sterile water
  • Phosphate buffered saline (PBS), sterile
  • Established primary cortical astrocyte culture grown for 1.5 to 2 weeks (see protocol 3)
  • Astrocyte serum deprivation medium: DMEM‐BSA (see recipe)
  • Compound of interest
  • Neuronal coverslips (see protocol 1)
  • HBSS plus CM
  • 4% paraformaldehyde in HBSS plus CM (see protocol 5)
  • 37°C water bath
  • 24‐well sterile plates
  • 37°C, 5% CO 2 humidified incubator
  • 15‐ or 50‐ml tubes
  • Centrifuge
  • Hemacytometer and microscope
  • Sterile tweezers and needles
  • Platform rocker
  • Parafilm

Support Protocol 2: 4% Paraformaldehyde (PFA) Preparation

  Materials
  • Paraformaldehyde powder
  • HBSS plus CM
  • 1 M sodium hydroxide (NaOH)
  • Fume hood
  • Magnetic heat plate and stirrer
  • Thermometer
  • Covered glass bottle
  • Aluminum foil
  • pH meter

Basic Protocol 4: Neuronal Immunostaining for Synaptic Proteins

  Materials
  • Coverslips in 24‐well plates
  • Phosphate buffered saline (PBS)
  • Bovine serum albumin (BSA)
  • 10% iso‐octylphenoxy‐polyethoxyethanol (Triton‐X) in PBS
  • Rabbit monoclonal antibody to synaptophysin
  • Mouse monoclonal antibody to PSD‐95
  • Day‐2 wash buffer
  • Donkey anti‐rabbit Alexa 488 fluorescent secondary antibody
  • Donkey anti‐mouse Alexa 555 fluorescent secondary antibody
  • 10 mg/ml trihydrochloride, trihydrate (Hoechst 33342)
  • Vectashield mounting medium for fluorescence
  • Nail polish
  • Orbital shakers, room temperature and 4°C
  • 24‐well plates
  • Tweezers and needles
  • Parafilm
  • Centrifuge
  • Aluminum foil
  • Glass microscope slides (75 × 25 × 1–mm)
  • Glass microscope cover glass (22 × 40–mm, no. 1 ½)

Basic Protocol 5: Synaptic Structure Imaging and Analysis

  Materials
  • Scanning confocal microscope (with 488‐nm and 561‐ or 568‐nm lasers)
  • 60× oil immersion objective
  • ImageJ Software (http://rsbweb.nih.gov/ij/index.html)
  • Huygens Professional software (Scientific Volume Imaging; http://www.svi.nl)
  • Huygens Professional Object Analyzer software (Scientific Volume Imaging)
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Figures

Videos

Literature Cited

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