Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

NOTE: This culture method of hippocampal neurons from postnatal 0-day ANK3-E22/23^f/f mice is adapted from Gary Banker’s glia/neuron co-culture system. Therefore, it is critical to perform all steps after dissection in a clean hood using sterilized tools. This protocol takes up to 1 month. The workflow is displayed in Figure 3. The protocol follows the animal guidelines of Duke University.

1. Preparing of coverslips and neuronal plating dishes

1. At least one week before culture day, load coverslips on coverslip rack and soak it in nitric acid (70% W/W) overnight (could be extended for days).
2. Wash nitric acid treated coverslips with distilled water on a low speed shaker in a glass jar 2 times, 1 hour each.
3. Incubate coverslips in saturated KOH dissolved in 100% ethanol overnight. Add KOH into ethanol till it is no longer dissolved.
4. Repeat the wash step with distilled water. Rinse coverslips with 100% ethanol once for 10 minutes.
5. Transfer coverslips from the rack to a glass beaker. Cover the beaker with aluminum foil. Bake coverslips in a 225 °C oven overnight to sterilize the coverslips. (Coverslips could be stored in the beaker for weeks).
6. Place coverslips in a Petri dish and then apply 3-4 wax dots on the coverslip to serve as feet. Use a Pasteur pipette to dip into boiled wax in a glass bottle. Then quickly touch the coverslip to create a dot. A 60 mm Petri dish can hold 4 coverslips. A 10 mm Petri dish can hold ~10 coverslips.
7. 2 days before culture day, coat coverslips (the side with the wax dots) with filter sterilized 1 mg/mL poly-L-lysine in 0.1 M boric acid (pH 8.5) for a minimum of 6 hours and rinse with water 2 times, 1 hour each time. Coverslips remain in the same Petri dish.
8. Add plating medium (MEM supplemented with glucose 0.6% (wt/vol) and 10% (vol/vol) horse serum) to plates slowly without disturbing coverslips. Put plates in the incubator till the culture day to seed neurons.

2. Preparing glia cell feeder dishes (2 weeks before culture day)

1. Dissect the cortex from a postnatal 1-day old mice brain and peel off the meninges.
2. Chop the cortex tissue as finely as possible with a clean scissors in a clean Petri dish in a clean bench.
3. Transfer the chopped tissue into 12 mL of HBSS and add 1.5 mL of 2.5% trypsin and 1% (wt/vol) DNase. Incubate in a 37 °C water bath for 15 min, swing every 5 min. Well digested tissue become sticky and form a big cluster. Triturate 10-15 times with a 10 mL pipette to break the tissue down and get better digestion.
4. Triturate the well digested tissue 10-15 times with a 5 mL pipette till most chunks disappear and the medium turns to cloudy. Pass through a cell strainer to remove remaining chunks and add 15 mL of glia medium (Minimal essential medium (MEM) supplemented with glucose (0.6% wt/vol), 10% (vol/vol) horse serum and Penicillin-Streptomycin (1x) to stop the digestion.
5. Centrifuge the cells at 120 x g for 5 minutes and aspirate the supernatant. Resuspend the cell pellet with fresh glia medium and seed in cell culture dishes (about 10⁵ cells/cm²).
6. Replace medium with fresh glia medium the next day to remove unattached cells.
7. Feed the glia dishes every 3-4 days with fresh glia medium. Slap the flask 5-10 times with a hand to dislodge loosely attached cells before changing medium.
8. After 10 days of culture, glia cells should be nearly confluent. Detach glia cells with 0.25% trypsin-EDTA and seed about 10⁵ cells in a new 60 mm cell culture dish. Remaining cells could be frozen for future use.
9. 3 days before the culture day, change the glia medium to neuronal culture medium (Neurobasal-A Medium with 1x GlutaMAX-I and 1x B27 supplement).

3. Culture hippocampal neurons

NOTE: All steps are performed at room temperature.

1. Dissect 6-8 hippocampi from postnatal 1-day old pups from ANK3-E22/23^f/f mice with HBSS medium in a Petri dish at room temperate. Chop the hippocampi with dissection scissors to smaller pieces. Transfer hippocampi from the Petri dish to a 15 mL tube.
2. Wash hippocampi 2x with 5 mL of HBSS in the tube. Leave the hippocampi in 4.5 mL of 1x HBSS after wash.
3. Add 0.5 mL of 2.5% trypsin into 4.5 mL of HBSS and incubate in a 37 °C water bath for 15 minutes. Invert the tube every 5 minutes. Well digested hippocampi should become sticky and form a cluster. If needed, extend the digestion for 5 more minutes.
4. Wash hippocampi with HBSS 3 times for 5 minutes each. Do not use a vacuum to remove the HBSS. It is very easy to remove the hippocampi.
5. Add 2 mL of HBSS after the wash and pipette the hippocampi up and down with a Pasteur pipette 15 times.
6. Triturate the tissue with a fire-polished Pasteur pipette (the diameter of the open is narrowed by half) 10 times. Do not go beyond 10 times even if there are still chunks remaining. Overshearing kills neurons.
7. Rest the tube for 5 minutes till all chunks set to the bottom. Gently use a 1 mL pipette tip to transfer the supernatant containing the dissociated neurons to plating dishes (10⁵ cells/60 mm dish). Add it directly to the pre-incubated plating medium and shake the plate gently.
8. Repeat step 3.6-3.7 with the remaining chunks till most of the chunks have disappeared.
9. 2-4 hours after seeding, check the plating dishes with a light microscope. The majority of neurons should have attached to the coverslip. Attached cells are round and bright. Flip coverslips using a fine tip forceps to the glia cell feeder dishes with preconditioned neuronal culture medium with the wax dots side facing downwards.
10. Neurons can grow in the glia cell feeder dishes for up to 1 month. Feed neurons every 7 days with 1 mL of fresh neuronal culture medium.
11. Optional step: 1 week after seeding, add cytosine arabinoside (1-β-D-arabinofuranosylcytosine) to a final concentration of 5 μM to curb glial proliferation.

4. Disruption of AIS by Knockout of Ankyrin-G at earlier stage of neuron development

1. On 3 div (day in vitro), flip the coverslips with wax dots side facing up to a glia cell feeder dish with conditioned neuronal culture medium.
2. Mix 0.25 μg of Cre-BFP DNA with 0.5 μg of ankyrin-G-GFP (WT/mutant) DNA in a 1.7 mL tube to transfect 4 coverslips (~ 2:1 ratio of DNA copy number). Add 100 μL of culture medium (e.g., Opti-MEM), mix and rest on a rack. If only Cre-BFP is transfected, the GFP plasmid backbone is used to match the total amount of DNA.
3. Mix 3 μL of transfection reagent (e.g., Lipofectamine 2000) (~ 3 times of DNA) with 100 μL culture medium in a new 1.7 mL tube. Incubate for 5 minutes at RT.
4. Mix 100 μL of DNA solution from step 4.2 with 100 μL of transfection reagent from step 4.3. Rest for 5-10 minutes on a rack.
5. Add 50 μL of DNA mix from step 4.4 right on top of each coverslip by inserting the tip just below the medium without touching the coverslips. Pipette slowly to avoid spreading of DNA mix.
6. Slowly bring the dish back to the incubator and incubate for 30-45 minutes.
7. Flip the coverslips back to the home glia feeder dish with wax dots side facing down and put the plate back to the incubator.

5. Quantification of axon initial segment

1. Fix neurons on 7-10 div and stain with AIS marker following the standard immunocytochemistry protocol for the protein of interesting.
2. Collect fluorescent pictures with desired microscopy.
   1. Take Z-series sections to collect the signal of the entire AIS. Keep the same Z-depth for all pictures.
   2. Adjust the laser intensity to reach the best pixel intensity dynamic range.
   3. Make sure all AIS pictures are taken on the same microscope setup.
   4. Always check the signal of Cre-BFP.
3. AIS quantification
   1. Open picture with Fiji (https://fiji.sc).
   2. Generate maximum projection of Z-series images.
   3. Subtract the empty coverslip background signal from the image.
   4. Draw a line along the AIS. The width of the line should fully cover the AIS. Start the line before the AIS signal is raised above the background and stop after it drops to the background.
   5. Measure the mean pixel intensity alone the line and export to a spreadsheet (~10-15 AISs are needed).
   6. Generate the average intensity curve of AIS using the MATLAB script adapted from Berger et al.¹⁵.
   7. For each experiment, include Cre only and Cre plus wildtype 480 kDa ankyrin-G transfected neurons as negative and positive controls to make sure that the knockout of AIS is efficiency and the rescue is successful.