Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Recipes

Chick Ringers

Note: Adjust pH to 7.4. Bring final volume to 1000 ml with water.

10X PBS

Note: Adjust pH to 7.4. Bring final volume to 500 ml with water. Working concentration (1X) is made by diluting 1 part 10X stock with 9 parts of tissue-culture grade water, such as that obtained with a Millipore UV irradiation system to generate water with 18 MΩ-cm conductivity.

SAG explant holding medium

• DMEM/F12 with L-glutamine, 10 mM Hepes
• 10% Insulin, Transferring, Selenium (ITS+1)
• 1% pen-strep

Explant culture medium

• SAG explant holding medium
• 10 ng/ml Neurotrophin-3 (NT-3)
• 10 ng/ml ciliary neurotrophic factor (CNTF)

Spinal cord dissection and holding medium

• L-15
• 10% fetal calf serum
• 1% pen-strep

Incubate eggs at 37-38°C. Sterilize dissecting tools and Sylgard dissecting dish in 70% ethanol. Dissecting dishes and tools can also be UV sterilized overnight.

1. Bead preparation

1. Place beads in a microtube with 1 ml sterile PBS, mix, and wait for the beads to settle to the bottom of the tube.
2. Wash the beads several times by removing the supernatant after the beads have settled and resuspend in PBS.
3. Incubate the beads in purified protein (diluted in PBS) or PBS alone (Control) for 1 hour at room temperature.
4. Rinse the beads in PBS and store in a 24-well plate with 1ml PBS until placed in collagen.

2. Statoacoustic ganglion dissection

1. On E4 remove the embryo from the egg and place it in a dish with chick Ringer’s solution to remove embryonic membranes.
2. Place the embryo in a Sylgard© lined petri dish filled with cold HBSS and position the embryo on its side with the otic vesicle facing up. Pin the embryo to the dish using dissecting pins.
3. Using two dissecting pins, make a horizontal cut through the skin immediately ventral to the canal pouches, approximately where it meets the cochlear duct. Make a vertical cut anterior and posterior to the SAG. Use forceps or a dissecting pin to lift and remove the flap of skin bordered by the three cuts.
   
   Note: The SAG is located at the anteroventral edge of the otocyst, about midway between the dorsal pouch which is readily visible with brightfield base illumination, and the ventral cochlear duct which projects ventromedially and is obscured by the pharyngeal arches. The cochlear duct elongates so its dorsoventral dimension will progressively increase on E4 between Hamburger-Hamilton stages 23-25 9.
4. Pull the otocyst in a posterior direction to separate it from the SAG. Displace tissue surrounding the SAG with dissection pins and carefully remove the SAG from the embryo using #55 forceps. Remove large chunks of mesenchymal tissue and protruding nerve bundles from the SAG.
5. Using a wide-mouth pipet tip, transfer the explant to a 24-well plate with 0.5 ml SAG explant holding medium. Keep explants on ice or at room temperature for up to 4 hours.

3. Spinal cord dissection

1. Remove the E6 embryo from the egg and place it in a dish containing chick Ringer’s solution. Remove the head and embryonic membranes.
2. Place the body in a Sylgard© dissecting dish containing cold spinal cord dissection medium. Position and pin the embryo “ventral down” and caudal facing the experimenter. Place dissecting pins through each limb and through the anterior end.
3. Using forceps and/or dissecting pins, carefully remove skin and tissue, moving in a ventral direction, until the dorsal surface of the spinal cord is visible.
4. Cut the dorsal midline of the spinal cord with a dissecting pin, in a posterior to anterior direction, along the entire length of the spinal cord. This creates an “open book” as the left and right sides of the spinal cord separate.
5. Remove surrounding tissue, laterally, to isolate the spinal cord and expose the dorsal root ganglia (DRG). Remove the DRG and meninges by rubbing a dissecting pin or forceps between the spinal cord and DRG.
6. Remove the spinal cord from the body by holding the caudal-most end of the spinal cord with forceps and lifting up and away from the experimenter. Remove remaining DRG and meninges.
7. Hold one end of the the spinal cord with forceps (or pin it to the dish) and use Vannas scissors to cut along the ventral midline, to bisect the spinal cord.
8. Hold one end of the explant with forceps to immobilize the tissue and use Vannas scissors to cut small explants (100-500 um in length) along the length of the tissue. The floor plate can be recognized as a clear thickening of tissue along one explant edge.
9. Use a wide-mouth pipet tip to transfer explants to a 24 well plate with 0.5 ml cold dissection medium. Keep explants on ice to maintain tissue viability, for up to 4 hours.

4. Explant culture with purified proteins to test neurite responsiveness

1. Prepare a 1.5 mg/ml collagen solution in a 15 ml conical tube, on ice, according to the manufacturer’s instructions.
2. Verify the collagen solution has a pH of 7-7.4 using pH indicator paper. Adjust the pH by adding NaOH in 1 μl volumes.
3. Transfer explants to a 24-well plate using a wide mouth pipet tip and aspirate excess liquid. Multiple explants can be cultured in one well, but should be placed at least 500 μm apart from each other.
4. Add 0.5 ml of collagen to the well containing the explant. Position the explant on the bottom surface of the well. Be sure to set each culture completely before proceeding with the next well because the collagen will begin to polymerize at room temperature.
5. Place the culture plate on a 37°C slide warmer for 30-45 min to polymerize the collagen. When the collagen has polymerized it will resemble a gel.
6. Add 0.5 ml warm explant culture medium supplemented with either purified proteins (diluted in PBS) or PBS (Control) and incubate at 37°C, 5% CO₂. Effects on neurite outgrowth should be visible by 24 hours.
   
   NOTE: We have used the same protocol to culture SAG explants up to 42 hours.

5. Explant co-culture with protein-coated beads to test directional neurite outgrowth

1. Begin with explant culture steps 4.1- 4.4.
2. Using a pipet, transfer 1-5 beads from PBS to the well containing the collagen solution and explant.
3. Use forceps to position beads 50-500 μm from the edge of the explant.
4. Place the plate on a 37°C slide warmer for 30-45 min to polymerize the collagen. The tissue or bead may become displaced as the collagen polymerizes. Check the cultures and re-position the tissue and beads during the first 5 min.
5. Add 0.5 ml SAG media to each well and incubate for 24 hours.

6. Visualization of neurite outgrowth by immunohistochemistry

1. Rinse cultures in PBS and fix for 1 hour at room temperature in 4% paraformaldehyde in PBS.
2. Release the gels from the walls of the wells by tracing around the edge of the gels with the rounded end of a teflon micro spatula. Perform the immunostain with the gels in the wells. Rinse several times in PBS.
3. Incubate in 0.5 ml blocking solution (10% calf serum, 0.1% Triton X-100, 0.1% sodium azide in PBS) overnight at 4°C.
4. Incubate in 0.5 ml primary antibody (anti-β-Tubulin antibody diluted 1:500 in blocking solution) overnight at 4°C.
5. Rinse several times in PBS. Incubate in 0.5 ml secondary antibody (Alexa fluor 488 goat anti-mouse IgG_2b antibody diluted 1:500) overnight at 4°C. From this step forward, keep plates in dark or wrap in foil, due to light sensitivity of secondary antibodies.
6. Rinse several times in PBS and store at 4°C in PBS for up to one week.

7. Representative Results

Figure 1. Representative results of the neurite-promoting effects of purified NT-3 and NT-3-coated beads on E4 chick SAG explants. SAG neuron cell bodies and neurites were immunostained with β-Tubulin and imaged with a 10x objective on a confocal microscope. After 24 hours in vitro, SAG explants displayed greater neurite outgrowth in the presence of 100 ng/ml purified human NT-3 added to the cell culture medium (1B), compared to controls (1A). SAG neurons were co-cultured with beads soaked in100 ng/ml NT-3 to demonstrate that a point source of NT-3 can locally promote neurite outgrowth. Explants displayed longer and denser neurite outgrowth on the side of the explant facing the bead compared to the opposite side (1D) and compared to control cultures with beads soaked in PBS (1C). Scale bar = 200 μm.

Figure 2. Representative results of the neurite-promoting effects of purified Wnt5a and Wnt5a-coated beads on E6 chick spinal cord. Spinal cord explants were immunostained with β-Tubulin and imaged with a confocal microscope. After 24 hours of growth in the presence of purified mouse Wnt5a (400ng/ml), neurite outgrowth from chick spinal cord explants was increased (2B) compared to controls cultured without Wnt5a (2A). Beads soaked in 500 ng/ml Wnt5a, placed 300-500 μm from the edge of spinal cord explants, also promoted neurite outgrowth (2D) compared to control cultures (2C). Similar co-culture results were previously published with Wnt5a-expressing cells⁸. Scale bar = 200 μm.