Culturing of Cerebellar Granule Cells to Study Neuronal Migration: Gradient and Local Perfusion Assays

Patricia Guijarro1, Jian Jiang2, Xiao‐bing Yuan3

1 CAS‐MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, 2 Graduate School of the Chinese Academy of Sciences, Shanghai, China, 3 Bio‐X Center, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, China
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.26
DOI:  10.1002/0471142301.ns0326s60
Online Posting Date:  July, 2012
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Abstract

Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time‐lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed. Curr. Protoc. Neurosci. 60:3.26.1‐3.26.12. © 2012 by John Wiley & Sons, Inc.

Keywords: cerebellum; dissociated granule cells; cell culture; neuronal migration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Low‐Density Dissociated Culture of Cerebellar Neurons
  • Basic Protocol 2: Neuronal Migration Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Low‐Density Dissociated Culture of Cerebellar Neurons

  Materials
  • 100 µg/ml poly‐D‐lysine (PDL; see recipe) solution
  • 25 µg/ml laminin solution (see recipe)
  • Double‐distilled water (ddH 2O)
  • Sprague‐Dawley (SD) rat pups, P0‐P1
  • Hank's balanced salt solution (HBSS), without calcium, magnesium, phenol red; with bicarbonate, glucose (Invitrogen, cat. no. 14175‐129)
  • 0.125% trypsin solution (see recipe)
  • Culture medium (see recipe)
  • Transfection solution (supplied with the Rat Neuron Nucleofector kit; Amaxa cat. no. VPG‐1003)
  • Vertical laminar flow clean bench, with HEPA filtration system (Telstar)
  • Glass coverslips, 18 × 18–mm (Fisher, cat. no. S17521), sterilized by autoclaving
  • Nontreated 35‐mm and 60‐mm plastic dishes
  • CO 2 incubator: 37°C, 5% CO 2, humidified atmosphere (Thermo Scientific)
  • Dissection material including:
    • Scissors
    • Small scissors (straight)
    • Fine curved scissors
    • Iris scissors
    • Tweezers (straight)
    • Curved tweezers
    • Fine tweezers
  • 10‐ml glass tubes
  • 1‐ml automatic pipets
  • 37°C water bath (Thermo Scientific)
  • 5‐ml glass pipets
  • 70‐µm mesh cell strainer (Falcon, cat. no. 35235)
  • Centrifuge, suitable for cell centrifugation (Eppendorf)
  • Neubauer Chamber
  • Electroporator (Nucleofector II, Amaxa)
  • Electroporation cuvettes
NOTE: This protocol must be performed from beginning to end under sterile conditions at room temperature (21°C to 25°C). Unless otherwise stated, all reagents and materials used throughout the whole protocol must be sterile.NOTE: Since the aim of this protocol is to culture cerebellar neurons on coverslips, plastic dishes that are sold as optimal for cell culture are not required: regular, nontreated (and thus cheaper) plastic dishes can be used.NOTE: Figure shows an overview of the dissociated granule cell culture protocol, followed by a typical neuronal migration assay.

Basic Protocol 2: Neuronal Migration Assay

  Materials
  • Cultured coverslips (see protocol 1)
  • L15 medium (without phenol red, with L‐glutamine; Invitrogen, cat. no. 21083‐027)
  • Mineral oil (Sigma, cat. no. M8410)
  • Factor/drug of interest
  • Two‐Stage Puller (Narishige, PC‐10) for preparing micropipets
  • Glass capillaries for preparing micropipets (capillary tubing; AM Systems, cat. no. 6010)
  • Inverted bright‐field microscope with two joystick micromanipulators (Narishige MN‐151) and a charge‐coupled device (CCD) camera (TKC1381; JVC)
  • Fine tweezers
  • Cell imaging chamber
  • Culture dish heater, mounted on the microscope (Warner Instruments)
  • Picospritzer II (Parker)
  • ImageJ software
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Figures

  •   FigureFigure 3.26.1 Schematic representation of a complete neuronal migration assay. (1) Cell dissociation: Cerebella from P0‐P1 rat pups are dissected out, chopped, and dissociated by digestion in a trypsin solution before plating them in dishes containing coated coverslips. Alternatively, dissociated cells may be transfected by electroporation before being plated. (2) Cell culture: Dissociated cells plated in coated coverslips are cultured for 12 to 20 hr in culture medium at 37°C in a humid, 5% CO2 atmosphere. (3) Migration assay: A coverslip with dissociated cells is placed inside an imaging chamber containing warm L15 medium, and a drop of mineral oil is added on top of it to prevent evaporation during the experiment. The imaging chamber is placed on a microscope stage connected to a heater, which keeps temperature constant at around 36°C during the experiment. At either side of the stage there is a micromanipulator holding an injection pipet connected to a Picospritzer, and a suction pipet connected to a vacuum pump. Two different kinds of migration assays can be performed. By means of the Picospritzer, a gradient concentration of the factor of interest can be produced through the injection pipet located near the region of the neuron to be tested (growth cone, soma, rear end). Also, a certain region of the neuron can be perfused locally with a homogeneous concentration of the factor of interest, by applying it through the injection pipet and aspiring it using the suction pipet connected to a vacuum pump.
  •   FigureFigure 3.26.2 Slit2 triggers migration reversal of dissociated cerebellar granule cells in a neuronal migration assay. Concentrated conditioned medium from HEK 293 cells stably expressing Slit2 was continuously applied in a gradient concentration in front of the growth cone of a migrating granule cell. Slit2 caused the neuron to stop, collapse its growth cone, and resume migration in the direction opposite to the Slit2 gradient by generating a new growth cone in the original rear end.

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Literature Cited

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