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

Summary

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Abstract

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Introduction

The axon initial segment is located at the proximal axon in most vertebrate neurons. Functionally, AIS is where action potentials are initiated due to the high-density of voltage-gated sodium channels in this region. AIS of some excitatory neurons are also targeted by inhibitory interneurons through forming GABAergic synapses^1,2,3. Therefore, AIS is a critical site to integrate cell signaling and modulate the excitability of neurons. AIS is normally 20-60 μm in length and located within 20 μm of the cell body. The length and position of AIS varies in neurons across brain regions, as well as in different developmental stages of the same neuron^4,5. Accumulated evidence suggested that the composition and position of AIS are dynamic in responding to the change of neuronal activity^4,5,6,7.

480 kDa ankyrin-G is the master organizer of AIS. 480 kDa ankyrin-G is a membrane associated adaptor protein that directly binds to voltage gated sodium channels as well as other major AIS proteins including beta4-spectrin, KCNQ2/3 channels that modulate sodium channel activity^8,9, and 186 kDa neurofascin, a L1CAM that directs GABAergic synapses to the AIS^2,10. 480 kDa ankyrin-G shares canonical ankyrin domains found in the short 190 kDa ankyrin-G isoform (ANK repeats, spectrin binding domain, regulatory domain), but are distinguished by a giant exon that is found only in vertebrates and is specifically expressed in neurons (Figure 1A)^11,12. The 480 kDa ankyrin-G neuron specific domain (NSD) is required for AIS formation¹². The 190 kDa ankyrin-G does not promote AIS assembly or target AIS in ankyrin-G-null neurons¹². However, 190 kDa ankyrin-G is concentrated at the AIS containing 480 kDa ankyrin-G¹². This ability of the 190 kDa ankyrin-G to target pre-assembled AIS of wildtype neurons has been a source of confusion in the literature and has slowed appreciation of the critical specialized functions of the 480 kDa ankyrin-G in AIS assembly. Therefore, it is critical to study AIS assembly in ankyrin-G-null neurons that lack a pre-assembled AIS.

Here, we present a method to study the assembly and structure of the AIS using cultured hippocampal neurons from ANK3-E22/23-flox mice that eliminates all isoforms of ankyrin-G¹³ (Figure 1B). By transfecting neurons with a Cre-BFP construct before AIS is assembled, we generated ankyrin-G-deficient neurons completely lacking an AIS (Figure 1B, Figure 2). The assembly of AIS is fully rescued following co-transfection of 480 kDa ankyrin-G-GFP plasmid with a Cre-BFP plasmid. This method provides a way to study the AIS assembly in a non-pre-assembled AIS environment. We also modified the glia-neuron co-culture system from Gary Banker without using antibiotics, previously designed for embryonic day 18 neurons, for application to postnatal mouse neurons and adapted a AIS quantitation method to average AIS measurements from multiple neurons to normalize the variation of AIS^14,15.

Protocol

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.

Results

A complete set of experiment should include Cre-BFP only transfection as negative control, Cre-BFP plus 480 kDa ankyrin-G co-transfection as positive control and a non-transfected condition as technique control. In Cre-BFP only control, transfected neurons lack the accumulation of AIS markers, including ankyrin-G (ankG), beta4-spectrin (β4), neurofascin (Nf) and voltage gated sodium channels (VSVG) (Figure 4A)¹⁶. In contrast, Cre and 480 kDa ankyrin-G co-transfec...

Discussion

The assembly of AIS is organized by 480 kDa ankyrin-G. However, ankyrin-G has shorter isoforms that can target to the AIS of wildtype neurons, which may lead to difficulty in interpretation of structure-function analyses of AIS assembly. Here we present a method using neurons from ANK3-E22/23-flox mice that allows study of de novo assembly of the AIS. By transfecting with Cre-BFP at 3 div, we eliminate the all endogenous isoforms of ankyrin-G. We could also co-transfect 480 kDa ankyrin-G to rescue the f...

Materials

Name	Company	Catalog Number	Comments
10xHBSS	Thermo Fisher Scientific	14065-056
18mm coverglass (1.5D)	Fisher Scientific	12-545-84-1D
190kDa ankyrin-G-GFP	Addgene	#31059
2.5% Tripsin without phenol red	Thermo Fisher Scientific	14065-056
480kDa ankyrin-G-GFP	lab made	Provide upon request
ANK3-E22/23f/f mice	JAX	Stock No: 029797	B6.129-Ank3tm2.1Bnt/J;
B27 serum-free supplement	Thermo Fisher Scientific	A3582801
Boric acid	Sigma-Aldrich	B6768
Cell strainer with 70-mm mesh	BD Biosciences	352350
Ceramic coverslip-staining rack	Thomas Scientific	8542E40
Cre-BFP	Addgene	#128174
D-Glucose	Sigma-Aldrich	G7021
DMEM	Thermo Fisher Scientific	11995073
GlutaMAX-I supplement	Thermo Fisher Scientific	A1286001
Lipofectamine 2000	Thermo Fisher Scientific	11668030
MEM with Earle’s salts and L-glutamine	Thermo Fisher Scientific	11095-080
Neurobasal Medium	Thermo Fisher Scientific	21103-049
Nitric acid 70%	Sigma-Aldrich	225711
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31985062
Paraformaldehyde	Sigma-Aldrich	P6148
Penicillin-streptomycin	Thermo Fisher Scientific	15140122
Poly-L-lysine hydrochloride	Sigma-Aldrich	26124-78-7
Potassium hydroxide	Sigma-Aldrich	1310-58-3