Culturing Rat Hippocampal Neurons

Gerald Audesirk1, Teresa Audesirk1, Charles Ferguson1

1 University of Colorado at Denver, Denver, Colorado
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.3
DOI:  10.1002/0471140856.tx1203s04
Online Posting Date:  May, 2001
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Abstract

Cultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.

     
 
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Table of Contents

  • Basic Protocol 1: Isolating Hippocampal Neurons from Rat Embryos
  • Basic Protocol 2: Culturing Hippocampal Neurons
  • Basic Protocol 3: Obtaining Hippocampal Astrocyte Cultures
  • Basic Protocol 4: Coculturing Hippocampal Neurons and Astrocytes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolating Hippocampal Neurons from Rat Embryos

  Materials
  • Timed pregnant female rats (e.g., Sprague‐Dawley) at gestational day 18 or 19 (E18 or E19)
  • 95% (v/v) ethanol
  • HBSS (see recipe), sterile, ice cold
  • CMF‐HBSS (see recipe), sterile, ice cold
  • 2 mg/ml trypsin (Life Technologies) in CMF‐HBSS, prewarmed (37°C)
  • 2 mg/ml trypsin inhibitor (Sigma) in CMF‐HBSS, prewarmed (37°C)
  • Complete EMEM (see recipe; optional) or equivalent
  • Cryopreservation medium (see recipe; optional), cold
  • Propanol (optional)
  • Laminar flow hood
  • Dissecting microscope, total magnification of ∼14×
  • Fiber optic illuminator (e.g., Fiber‐Lite, Dolan Jenner Industries)
  • Large magnifying glass on stand (Edmund Scientific)
  • CO 2 tank and tubing
  • Anesthetizing chamber
  • Dissecting pans of various sizes, all with autoclavable rubber of silicone filling for pinning tissue
    • ∼24 × 32–nm
    • 18 × 20–nm
    • 10‐cm dia., 4‐cm deep
    • 10‐cm tissue culture dish
  • Dissection tools (Roboz Surgical Instruments or Fine Science Tools), sterile:
    • Bone cutters or strong, short‐bladed scissors
    • Rat‐tooth forceps
    • Large and small dissecting scissors (∼4‐ and 2‐cm blades)
    • Microdissecting forceps (Dumont no. 5 or equivalent; old or deliberately blunted forceps work best for pinning tissues, sharpened forceps for actual dissection)
    • Blunt forceps
    • Fine dissecting pins (e.g., Minuten insect pins, Carolina Biological Supply)
    • Large and small spring‐handled microdissecting scissors (10‐ and 3‐mm blades, respectively)
  • 5‐ml disposable plastic tubes (e.g., Falcon), sterile
  • Fire‐polished Pasteur pipet, sterile
  • Hemacytometer and coverslip
  • Upright compound microscope (400× total magnification)
  • Freezing vials (e.g., 2‐ml vials from Nalgene; optional)
  • Vial‐freezing containers (e.g., Nalgene cryo 1° freezing containers; optional)

Basic Protocol 2: Culturing Hippocampal Neurons

  Materials
  • 0.1 mg/ml poly‐D‐lysine (mol. wt. 350,000; Sigma), filter sterilized (cellulose acetate, 0.2‐µm pore size)
  • Dissociated hippocampal cell suspension (see protocol 1)
  • Complete EMEM (see recipe)
  • 10× cytosine arabinoside (typically, 10 to 100 µM) in complete EMEM, filter sterilized (optional)
  • Laminar flow hood
  • 35‐mm plastic cell culture dishes (Corning or Nunc)

Basic Protocol 3: Obtaining Hippocampal Astrocyte Cultures

  Materials
  • Dissociated hippocampal cell suspension (see protocol 1)
  • Complete EMEM (see recipe)
  • CMF‐HBSS (see recipe)
  • 2 mg/ml trypsin (Life Technologies) in recipeCMF‐HBSS
  • Laminar flow hood
  • 35‐mm plastic cell culture dishes, uncoated or poly‐D‐lysine coated (see protocol 2)
  • Rotary shaker (optional)
  • Fire‐polished Pasteur pipet, sterile
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Figures

Videos

Literature Cited

Literature Cited
   Audesirk, G.J. 1997. In vitro systems in neurotoxicological studies. In Comprehensive Toxicology: Nervous System and Behavioral Toxicology (H.E. Lowndes and K.R. Reuhl, eds.) pp. 431‐446. Pergamon Press, New York.
   Banker, G.A. and Cowan, W.M. 1977. Rat hippocampal neurons in dispersed cell culture. Brain Res. 126:397‐425.
   Brewer, G.J., Torricelli, J.R., Evege, E.K., and Price, P.J. 1993. Optimized survival of hippocampal neurons in B27‐supplemented Neurobasal, a new serum‐free medium combination. J. Neurosci. Res. 35:567‐576.
   Goslin, K. and Banker, G. 1991. Rat hippocampal neurons in low‐density culture. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 251‐281. MIT Press, Cambridge,Mass.
   Mattson, M.P. and Kater, S.B. 1988. Isolated hippocampal neurons in cryopreserved long‐term culture. Development of neuroarchitecture and sensitivity to NMDA. Int. J. Dev. Neurosci. 6:439‐452.
Key References
   Goslin and Banker, 1991. See above.
   A superb, illustrated discussion of every step of preparation of hippocampal neurons for culture.
   de Hoop, M.J., Meyn, L., and Dotti, C.G. 1998. Culturing hippocampal neurons and astrocytes from fetal rodent brain. In Cell Biology: A Laboratory Handbook (J.E. Celis, ed.) pp. 154‐163. Academic Press, San Diego.
   Extremely detailed description of hippocampal culture, with emphasis on a specific methodology used in the authors' laboratory.
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