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Protocol for Culturing Rat Hippocampal Neurons

 

Hippocampal neural cell cultures are a commonly used model system for not only investigating the physiological properties of learning and memory, but the cellular mechanisms of neurobiology in general. Hippocampal cell cultures contain relatively few interneurons, and these interneurons are morphologically distinguishable from pyramidal neurons, the major cell type in the hippocampus1. Another benefit of hippocampal cell cultures is that they form fully developed dendrites that are covered with spines and make substantial synaptic connections. This protocol provides step-by-step instructions for dissecting and culturing hippocampal neurons from both embryonic (E17–E18) and postnatal (P1–P2) rat pups.
Download PDF of Illustrated Protocol

 

Please read the protocol in its entirety before starting.

Note: In order to yield a healthy neuron population, brain tissue from P1–P2 rat pups needs to be enzymatically digested before trituration. Extra reagents and steps are required.

Note: Aseptic techniques should be used in this protocol to ensure there is no bacterial, fungal, or mycoplasma contamination. The initial dissection and collection of the hippocampi can be completed outside of a laminar flow cell culture hood. However, preparation of reagents and cell culture plates, and all steps following tissue harvest, should be conducted within a hood. Likewise, all reagents and materials used should be sterile.

Supplies Required

Reagents

  • Antibiotic-antimycotic (100x, ThermoFisher Scientific, Catalog # 15240062), or equivalent
  • Cultrex® Mouse Laminin I (R&D Systems, Catalog # 3400-010-02)
  • Cultrex® Poly-D-Lysine (R&D Systems, Catalog # 3439-200-01)
  • Deionized or distilled H2O, sterile (dH2O)
  • DME medium, high glucose, no L-glutamine (Irvine Scientific, Catalog # 9024) or Neurobasal® medium (ThermoFisher Scientific, Catalog # 21103049), or equivalent
  • L-glutamine solution (200 mM, Irvine Scientific, Catalog # 9317), or equivalent
  • N21-MAX Media Supplement (50x, R&D Systems, Catalog # AR008)
  • PBS, sterile (1x): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4
  • Trypan blue (0.4%)

Additional Reagents for P1–P2 Rat Pups

  • DNase I (Worthington Biochemical Corp., Catalog # LK003170), or equivalent
  • EBSS, Ca2+, Mg2+, phenol red (ThermoFisher Scientific, Catalog # 24010043)
  • Ovomucoid protease inhibitor with BSA (Worthington Biochemical Corp., Catalog # LK003182), or equivalent
  • Papain (Worthington Biochemical Corp., Catalog # LK003176), or equivalent

Optional Reagents:

Materials

  • 15 mL conical centrifuge tube
  • Cell culture plates
  • E17–E18 timed pregnant rat or P1–P2 rat pups
  • Ice
  • Parafilm®
  • Pasteur pipette, glass, fire-polished, sterile
  • Petri dishes, 60 × 15 mm
  • Petri dishes, 100 × 20 mm
  • Pipette tips

Note: Locate your desired seeding densities for your hippocampal cell culture in Table 1 (pg. 30) to determine the size of the cell culture plate that should be used.

Equipment

  • 37 °C, 5% CO2 humidified incubator
  • 37 °C water bath
  • Autoclave
  • Centrifuge
  • Dissection microscope
  • Dissection tools
    • Dissecting forceps, curved (e.g. Gillies)
    • Fine forceps, #5, straight
    • Forceps, #7, curved
    • Surgical scissors, small
    • Surgical scissors, large
    • Vannas-Tübingen spring scissors, straight
  • Hemocytometer
  • Inverted microscope
  • Laminar flow cell culture hood
  • Pipettes

 

Reagent Preparation

Note: Prepare all solutions in a laminar flow cell culture hood.

Culture Media

  1. 1x N21-MAX Media Supplement, 1x antibiotic-antimycotic, 0.5 mM L-glutamine in DME (or Neurobasal®) medium

Note: Recombinant Human BDNF Protein and Recombinant Human IGF-I Protein can be added to enhance the hippocampal cell culture.

 

Procedure

Coating and Preparation of Cell Culture Plates

Note: Preparation of the culture plates should be done in a laminar flow cell culture hood.

  1. Dilute the Cultrex® Poly-D-Lysine solution with sterile dH2O to a final concentration of 50 µg/mL.
  2. Cover the wells of the culture plates with 50 µg/mL Cultrex® Poly-D-Lysine solution (e.g., 50 µL/well for 96-well plate). Tilt the plates to ensure even coating of the well surface.
  3. Incubate plates for 1 hour in a 37 °C, 5% CO2 humidified incubator.
  4. Aspirate the poly-D-lysine solution. Wash the wells three times with sterile dH2O. Aspirate the wells to remove all liquids.
  5. Wrap plates with parafilm® to seal. Store plates at 2–8 °C for up to 2 weeks.

    Note: Start these remaining steps the day before collection of the rat hippocampi.

  6. Dilute the Cultrex® Mouse Laminin I solution with sterile PBS to a final concentration of 10 µg/mL.
  7. Cover the wells of the poly-D-Lysine-coated plates with 10 µg/mL Cultrex® Mouse Laminin I (e.g., 50 µL/well for 96-well plate). Tilt the plates to ensure even coating of the well surface.
  8. Incubate the plates overnight at 2–8 °C.
  9. Aspirate the mouse Laminin I solution. Wash the wells two times with sterile dH2O. Aspirate the wells to remove all liquids.

Note: Alternatively, hippocampal neurons can be cultured on poly-L-lysine coated, open m-Slides (chambered #1.5 polymer coverslips; ibidi, Catalog # 80824).

Dissection of Rat Hippocampi

Note: Autoclave dissection tools to sterilize.

  1. Warm an appropriate amount of DME (or Neurobasal®) medium and culture media in a 37 °C water bath. Place sterile PBS on ice.

    Note: If using P1–P2 rat pups, skip to step 4.

  2. Asphyxiate the pregnant rat with CO2. Recover embryos via cesarean section using the large surgical scissors and curved dissecting forceps. Place the embryos in a 100 × 20 mm petri dish containing cold PBS. Keep the dish on ice.
  3. Remove the embryos from their individual placenta sacs and wash with cold PBS.
  4. Place cleaned embryos in a new 100 × 20 mm petri dish containing cold PBS. Decapitate each embryo at the head/neck junction using the small surgical scissors. Decapitate P1–P2 rat pups with the small surgical scissors.
  5. Place the heads in a new 60 × 15 mm petri dish containing cold PBS.
  6. Stabilize the dissociated head using the #7 curved forceps and #5 fine forceps. Moving caudal to rostral, cut through the skull with the small surgical scissors.

    Note: Keep cuts shallow to avoid damaging the brain tissue.

  7. Peel back the two halves of the separated skull.
  8. Remove the whole brain from the head cavity using the #7 curved forceps and place in a 60 × 15 mm petri dish containing cold PBS. Keep the dish on ice. Repeat steps 4–8 for the remaining heads.
  9. Put a brain in a new 60 × 15 mm petri dish containing cold PBS. Under a dissecting microscope, cut the brain with the Vannas-Tübingen spring scissors, following the median longitudinal fissure, to separate the hemispheres. Cut off and discard any brain stem tissue.
  10. With the #5 fine forceps, peel off the meninges that cover each hemisphere. Open up the brain to reveal the mid-sagittal side.
  11. Locate the hippocampus, which is the darker, c-shaped region, and remove it using the Vannas-Tübingen spring scissors. Place the hippocampal tissue in a new 60 × 15 mm petri dish containing cold PBS. Keep the dish on ice. Repeat steps 9–11 for the remaining brains.
  12. Using the Vannas-Tübingen spring scissors, cut the isolated hippocampi into smaller pieces (~2 mm2).

Dissociation and Culture of Rat Hippocampal Neurons

Note: From this point forward, the opening of tubes/plates that contain any tissue, cells, media, or reagents should be done in a laminar flow cell culture hood.

Note: If using embryonic hippocampal tissue, start at step 1. If using P1–P2 tissue, start at step 3.

For embryonic hippocampi:

  1. Transfer the tissue pieces to a 15 mL conical tube. Add 5 mL of DME (or Neurobasal®) medium. Gently triturate the tissue with the fire-polished Pasteur pipette until the solution is homogenous (~10–15 times).
  2. Skip to step 6.
  3. In a 15 mL conical tube, mix 20 U/mL Papain and 100 U/mL DNase I in 5 mL of EBSS. Warm the solution in a 37 °C, 5% CO2 humidified incubator for 10 minutes.
  4. Transfer the tissue to the 15 mL conical tube containing the warmed enzyme solution. Incubate for 20–30 minutes in a 37 °C, 5% CO2 humidified incubator.
  5. Gently triturate the tissue with the fire-polished Pasteur pipette until the solution is homogenous (~10–15 times).
  6. Centrifuge at 200 × g for 5 minutes at room temperature. Decant the solution.
  7. Resuspend the cells in…

    For embryonic hippocampi: 10 mL of DME (or Neurobasal®) medium.

    For postnatal hippocampi: 5 mL of EBSS containing 1 µg/mL of Ovomucoid protease inhibitor with BSA.

  8. Centrifuge at 200 × g for 4–6 minutes at room temperature. Decant the solution.
  9. Wash the cells twice with 10 mL of DME (or Neurobasal®) medium. Centrifuge at 200 × g for 5 minutes at room temperature. Decant the media.
  10. Resuspend the cells in warmed culture media (~ 10 mL). Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan blue. Count the live cells.
  11. Dilute the cell suspension to the desired seeding density (see Table 1, pg. 30) with warmed culture media. Plate the neurons on the prepared culture plates.
  12. Keep cultured neurons in a 37 °C, 5% CO2 humidified incubator until use.

Exchanging Media in Hippocampal Neuron Cultures

Note: Healthy cultures can be maintained for up to 4 weeks.

  1. Warm an appropriate amount of culture media in a 37 °C, 5% CO2 humidified incubator.
  2. Remove half the volume of media from each well of the culture plate (e.g., remove 50 µL from each well of a 96-well plate). Gently add an equal amount of new, warmed, culture media to each well.

    Note: Do not remove all the media from the wells of the plate as this will stress the neurons.

  3. Exchange the culture media every 3–4 days.

References

  1. Kaech, S. and G. Banker (2006) Nat. Protoc. 1:2406.
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