Stimulation with EphB-Fc induces spine maturation in cultured rat hippocampal neurons

doi:10.1038/nprot.2007.162

Method Article

Stimulation with EphB-Fc induces spine maturation in cultured rat hippocampal neurons

https://doi.org/10.1038/nprot.2007.162

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Genetics

Biological techniques

Neuroscience

rat hippocampal neurons

transfection

EphB-Fc stimulation

Spine maturation is reflected by decreased spine length and increased size of spine heads leading to a mushroom-like appearance. The following protocol is used to study the involvement of ephrinB reverse signaling in this process. Hippocampal neurons from embryonic rat (E18-19) were isolated and cultured for 14 days. In order to visualize the protrusion the neurons were transfected with Yellow Fluorescent Protein (YFP) 2 days prior to stimulation (12DIV) using Calcium Phosphate method. Activation of ephrinB ligands by soluble EphB receptor ectodomains fused to Fc portions of human IgG (EphB2-Fc) promoted spine maturation in these neurons (14DIV). Spine maturation was analyzed by comparing the average spine length and spine head area of stimulated neurons to that of Fc-stimulated control neurons.

Coverslips (CVS) (Ø 13 mm):

Poly-D-lysine-hydrobromide (Sigma Aldrich)

Borate-Buffer: 3.1mgml-1 Boric acid; 4.75 mg/ml Borax in 1l H₂O, pH 8.5

Laminin (natural mouse laminin; Invitrogen #23017-015)

Sterile PBS

Sterile H₂O

Isolation:

Dissection Medium: HBSS 500ml (Invitrogen #24020-091) ; 5 ml Penecillin/Streptomycin (PAA #P11-026); 5 ml L-Glutamine (PAA #M11-004); 5 ml 1M MgCl₂; 3.5 ml Hepes [pH 7.2] (Invitrogen)

Trypsin: 0.25% Trypsin/1M EDTA 100 ml (Invitrogen #25200-056); 0.7 ml Hepes [pH 7.2]

Serum Medium (SM): D-MEM (Invitrogen #61965-026); 10% Horse serum

Cell culture:

NB medium: Neurobasal Medium (Gibco, Invirtogen #21103-049); 1/50 B27 supplement (Gibco, Invitrogen); 0.5 mM L-Glutamine (PAA #M11-004)

Calcium Phosphate Transfection:

1M CaCl₂

2x BES saline (2x BBS) [pH 7.26]

HBSS (Invitrogen #14025-050)

Stimulation:

Human Fc Fragment (2.2 μg/μl) (Dianova)

Goat anti-human Fc (1.8 μg/μl) (Dianova)

EphB2-Fc (0.2 μg/μl) (recombinant mouse Eph receptor/Fc chimera; R&D)

Ephrinb2-Fc (0.2 μg/μl) (recombinatnt mouse ephrinb2/Fc chimera; R&D)

PFA 4% (4% sucrose)

50mM NH₄Cl chilled

Gel/Mount anti-fading medium (Biomeda)

Dissection tools

CO₂ Incubator (37 ^oC; 5.0% CO₂)

Epifluorescence microscope

Metamorph

Culture dishes (24-well plates)

Vortex

A) CVS Treatment:

CVS are treated with nitric acid o/n, briefly washed with H₂O (5x + 4x 30 min) and spread separately on Whatmann paper to dry. Dry CVS are baked in the oven (165 ^oC; o/n)

B) Coating:

Place CVS into 24-well plates (sterile)
Coat CVS with 400μl/well poly-D-lysine in Borate-Buffer (1mg/ml; sterile filtered) for > 5 hrs into the incubator.
wash CVS 3x with H₂O and let them dry in the hood
coat with 5μg/ml laminin in PBS for > 2 hrs into the incubator (400μl/well)
wash 3x with PBS, replace it with NB medium (400μl/well) and put it into the incubator

C) Isolation of Hippocampal Neurons (adapted from Jenny Lauterbach - ref 1):

put 4ml of SM and 4ml of NB medium each in a dish to pre-warm it in the incubator
pre-warm trypsin at 37 ^oC in the water bath
take E18-19 embryos from rat, cut off the heads and collect them in a dish with chilled dissection medium (DM)
carefully open the skull, remove the brains and transfer them into a new dish with chilled DM
separate the 2 cortices from the rest of the brain, remove their meninges and cut out each hippocampus; collect hippocampi in a 15ml tube filled with 10ml DM on ice
remove DM, replace it with 1-2ml warmed trypsin and incubate 20 min at 37 ^oC in the water bath
flame Pasteur pipette to decrease the diameter of the tip, take off the trypsin and wash hippocampi 2x with 1ml warm SM
add 1.5ml SM and homogenize tissue with the fire-polished Pasteur pipette by pipetting up and down 20-30x
centrifuge 5 min at 80g (650rpm)
remove supernatant, resuspend neurons in 1-3ml pre-warmed NB medium using the fire-polished Pasteur pipette
count cells, and plate them at designated cell density (e.g. high density 25,000 cells/cm² ) on prepared CVS

D) Calcium Phosphate Transfection (general protocol)

Mix for 4 wells (24-well plate):

900μl fresh NB medium

100μl Transfection Mix containing: (37.5 μl H₂O – DNA (4-7μg) + 12.5 μl 1M CaCl₂ + 50 μl BBS)

mix H₂O with CaCl₂
add DNA, mix by slowly pipetting
add BBS to DNA-H₂O-CaCl₂-mix
mix with 900 μl warm NB medium by vortexing
take away the old medium from the cells and quickly apply 250μl of the transfection-medium mix to each of the 4 wells.
incubate for 1.5-2.5 hrs
warm up HBSS for washing
wash cells with pre-warmed HBSS until most of the precipitate is removed
add 300 μl of NB medium per well to the cells
stimulate 2 days later

E) Stimulation/Fixation:

cluster EphB- Fc, ephrinb-Fc or Fc one hour prior stimulation at RT: Mix EphB/ephrin 4 μg/ml + 1/20 anti-hFc (0.2 μg/ml) e.g.: 300μl medium/well: take 6 μl EphB2/ephrinB2 + 0.325 μl anti-hFc (1/10)
stimulate the cells with clustered EphB2, ephrinB2 or Fc by adding the appropriate amount (4μg/ml) into the wells for 8 hrs in the incubator
put cells on ice to stop reaction
remove the medium and fix the cells by adding 200 μl/well 4% PFA (4% sucrose) for 12 min on ice
wash 2x with cold PBS
remove PBS and add 200μl/well cold NH4Cl for 10 min on ice
wash 2x with cold PBS
wash 1x with RT PBS for 5 min
wash with H₂O
drop of H₂O and mount CVS using Gel/Mount anti-fading medium (Biomeda) onto a glass slide
Take pictures using an epifluorescence microscope
Analyze changes in spine length and spine head size using MetaMorph software

Isolation of hippocampal neurons (2-3 hrs)

Cultivation time 14 days

Ca-PO4-transfection: 40 min + 2 hrs incubation

Stimulation: 1hour clustering + 8 hrs incubation

Lauterbach, J. & Klein, R. Release of full-length EphB2 receptors from hippocampal neurons to cocultured glial cells. J Neuroscience 26:11575-81 (2006)

Jenny Lauterbach (The Scripps Research Institute, La Jolla, CA)

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Stimulation with EphB-Fc induces spine maturation in cultured rat hippocampal neurons

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Stimulation with EphB-Fc induces spine maturation in cultured rat hippocampal neurons

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Introduction

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Acknowledgements

Associated Publications

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