Visualizing Changes in Neuronal Dendritic Morphology in Response to Stress and Pharmacological Challenge

Cara L. Wellman1

1 Department of Psychological and Brain Sciences, Center for the Integrative Study of Animal Behavior, and Program in Neuroscience, Indiana University, Bloomington, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.38
DOI:  10.1002/cpns.18
Online Posting Date:  January, 2017
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Abstract

This unit outlines a protocol for Golgi staining, which has been used extensively to reliably and quantitatively assess alterations in dendritic arborization and spine density as a result of a variety of factors, including chronic administration of glucocorticoids, chronic stress, and pharmacological manipulations. The method stains neurons in their entirety, allowing for sophisticated analyses of branch lengths and numbers as well as patterns of dendritic branching. Advantages of the technique include its usefulness in multisite collaborations and its utility in visualizing neurons in multiple regions within the same brain. Given that it typically labels approximately one in one hundred neurons, many neurons per region of interest can be sampled per animal, greatly increasing the ability to obtain a representative sample of neurons. Limitations include its time‐consuming nature, the hazardous chemicals employed, and the inability to use the stain to identify discrete subpopulations of neurons based on their projections, activation, or protein expression. © 2017 by John Wiley & Sons, Inc.

Keywords: dendrites; dendritic spines; morphology; histology

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparing Golgi‐Cox Solution
  • Basic Protocol 2: Stress Manipulation, Exsanguination, and Chromation
  • Basic Protocol 3: Vibratome Sectioning of Golgi‐Stained Brains
  • Support Protocol 1: 2% (w/v) Pig Gel‐Subbed Slides
  • Basic Protocol 4: Developing, Fixing, Dehydrating, Clearing, and Coverslipping Golgi Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparing Golgi‐Cox Solution

  Materials
  • K 2Cr 2O 7 (potassium dichromate; e.g., Fisher Scientific, cat. no. P188‐500)
  • HgCl 2 (mercury(II) chloride; e.g., Fisher Scientific, cat. no. AC201435000)
  • K 2CrO 4 (potassium chromate; e.g., Fisher Scientific, cat. no. P220‐500)
  • 2‐liter glass beakers (e.g., Fisher Scientific, cat. no. 02‐540R)
  • 4‐liter glass beaker (e.g., Fisher Scientific, cat. no. 02‐540 T) or 5‐liter plastic pitcher (e.g., Fisher Scientific, cat. no. 22‐171132)
  • 4‐liter brown plastic (e.g., B&H Photo, cat. no. DEDG) or glass (e.g., Fisher Scientific, cat. no. 05‐719‐96) bottles
  • Vacuum filtration system including:
    • Buchner funnel (e.g., Fisher Scientific, cat. no. FB966J)
    • Filter flask (e.g., Fisher Scientific, cat. no. 10‐182‐51)
    • Vacuum
CAUTION: The constituents of the Golgi‐Cox solution are toxic; use care and appropriate laboratory hygiene when handling, and dispose of waste properly in consultation with your Environmental Health and Safety office.

Basic Protocol 2: Stress Manipulation, Exsanguination, and Chromation

  Materials
  • Rat or mouse of interest
  • Injectable anesthetic
  • Saline (0.9% [w/v] NaCl)
  • Golgi‐Cox solution (see protocol 1)
  • 30% sucrose in saline (see recipe)
  • Containers for immersing tissue in Golgi‐Cox solution (e.g., 50‐ml plastic centrifuge tubes [Fisher Scientific, cat. no. 05‐539‐8]; 40‐ml plastic specimen cups [Fisher Scientific, cat. no. 13‐711‐94]; or 60‐ml glass jars [Fisher Scientific, cat. no. 13‐756‐734])
  • Wooden applicator sticks or plastic spoons (e.g., Fisher Scientific, cat. nos. 23‐400‐104 or NC9492996, respectively)
  • Additional reagents and equipment for transcardial perfusion and brain removal (unit 1.1)

Basic Protocol 3: Vibratome Sectioning of Golgi‐Stained Brains

  Materials
  • Sucrose‐infiltrated Golgi‐impregnated brains (see protocol 2)
  • Superglue
  • 6% (w/v) sucrose in saline (see recipe)
  • 2% pig gel‐subbed glass slides (see Support Protocol)
  • Vibratome or other vibrating microtome (e.g., Electron Microscopy Sciences 5000 MZ oscillating tissue slicer)
  • Blade for vibrating microtome (e.g., Electron Microscopy Sciences)
  • Small paintbrush
  • Small circular filter papers (e.g., Whatman #1, 32‐mm, Fisher Scientific, cat. no. 09‐805)
  • Small Petri dishes (e.g., Fisher Scientific, cat. no. 07‐201‐980)
  • Forceps (e.g., Fine Science Tools, cat. no. 11251‐20)
  • Bibulous paper (e.g., Fisher Scientific, cat. no. 11‐998)
  • Hard‐rubber brayer (e.g., Speedball Deluxe 4" Brayer; available from art supply stores)
  • Humid chamber (e.g., Fisher Scientific, cat. no. 231974)

Support Protocol 1: 2% (w/v) Pig Gel‐Subbed Slides

  Materials
  • Porcine skin gelatin, 175 bloom (e.g., Sigma‐Aldrich, cat. no. G2625‐100 G)
  • 0.5% (w/v) K 2SO 4 (potassium sulfate; see recipe)
  • Slide racks (e.g., Electron Microscopy Sciences, cat. no. 70312‐54)
  • Slides (e.g., Fisher Scientific, cat. no. 12‐518‐104 C)
  • Vacuum filtration system
  • Filter paper (e.g., Whatman #1)
  • Staining dish (e.g., Electron Microscopy Sciences, cat. no. 70312‐51)
  • 60°C drying oven

Basic Protocol 4: Developing, Fixing, Dehydrating, Clearing, and Coverslipping Golgi Sections

  Materials
  • Ammonium hydroxide (NH 4OH) diluted 2:1 with water (see recipe)
  • Dektol (e.g., Fisher Scientific, cat. no. 50‐267‐64; see recipe)
  • Ilford Rapid Fix concentrate (e.g., Fisher Scientific, cat. no. 50‐313‐78)
  • Slides with Golgi‐impregnated brain slices (see protocol 3)
  • 50%, 70%, 95%, 100% ethanol
  • Xylene (e.g., Fisher Scientific, cat. no. X3P‐1GAL)
  • Mounting medium (e.g., Eukitt, Fisher Scientific, cat. no. 50‐980‐468 or Depex, Fisher Scientific, cat. no. 50‐980‐372)
  • Glass or plastic slide rack (e.g., Fisher Scientific, cat. no. 22‐025587)
  • Glass or plastic staining dishes (e.g., Electron Microscopy Sciences, cat. no. 71423‐DL)
  • Coverslips, #1 thickness (e.g., Fisher Scientific)
  • Light microscope
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Figures

Videos

Literature Cited

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