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Abstract
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Neuroscience

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

Published: February 12, 2021
doi:
10.3791/61411
,
1School of Basic Medical Sciences,Xi’an Jiaotong University, 2Department of Cell Biology,Duke University, 3Department of Biochemistry,Duke University

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 synapses1,2,3. 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 neuron4,5. Accumulated evidence suggested that the composition and position of AIS are dynamic in responding to the change of neuronal activity4,5,6,7.

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 activity8,9, and 186 kDa neurofascin, a L1CAM that directs GABAergic synapses to the AIS2,10. 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 AIS14,15.

Protocol

NOTE: This culture method of hippocampal neurons from postnatal 0-day ANK3-E22/23f/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-E22/23f/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.

Representative 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-transfected neurons have fully assembled AIS revealed by the present of AIS markers (Figure 4B). It is important to confirm the quality of culture by comparing with the non-transfected dishes. Unhealthy neurons tend to show abnormal AIS structure, like discontinued or ectopic AIS (Figure 4C).

Then we showed an example of evaluating how an ankyrin-G human neurodevelopmental disorder mutation (ankG-K2864N) affects AIS assembly (Figure 5). 3 div ANK3-E22/23f/f neurons were transfected with Cre-BFP and wildtype 480 kDa ankyrin-G (ankG-WT) or 480 kDa ankyrin-G baring human mutation (ankG-K2864). Neurons were fixed at div7 and stained for ankyrin-G. Images were collected from 10-15 transfected neurons and 10-15 control neurons on the same coverslips and processed with maximum intensity projection. Then we draw a line at the AIS as shown and measure the mean intensity across the line. After averaging the AIS intensity, we plot the AIS intensity from the soma to the distal axon. AIS enriched protein normally showed a fast increase of signal from the proximal axon and a slow decrease of signal to the distal axon. AIS assembled by ankyrin-G with human mutant showed an increase and decrease of signal. But when aligned with the non-transfected AIS, the mutant curve is wider, and peak of the curve is lower suggesting a structure change of AIS. The wild type ankyrin-G assembled AIS closely aligned with the non-transfected one.

Figure 1
Figure 1: The genomic editing of ANK3-E22/23-flox. (A) Schematic representation of protein domains for 3 ankyrin-G isoforms. The location of exon 22 and 23 encoded regions in canonical domain is pointed by the dash line. (B) The position of LoxP sites in ANK3-E22/23-flox mice is indicated by triangle. In the present of Cre recombinase, exon 22 and 23 is deleted and causes loss the expression of all 3 isoforms of ankyrin-G. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Loss of AIS in ANK3-E22/23-flox neurons in the present of Cre recombinase. A diagram shows the time frame of ankyrin-G expression and AIS assembly in wild type neurons versus in ANK3-E22/23f/f neurons with Cre transfection at 3 div. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Workflow of protocol. Please click here to view a larger version of this figure.

Figure 4
Figure 4: A full rescue of AIS by 480kDa AnkG in ANK3-E22/23-flox neurons transfected with Cre. 3 div neurons of ANK3-E22/23f/f mice were transfected with Cre-BFP (A) or with a Cre-BFP and wild type 480 kDa ankyrin-G-GFP (B). Neurons were fixed at 7 div and stained for ankyrin-G (ankG), β4-spectrin (β4), neurofascin (Nf) and voltage gated sodium channels (VSVG). White arrow head points to the AIS of a transfected neuron. Scale bar is 20 μm. This figure was adapted from Yang et al16. (C) Two unhealthy neurons transfected with tdTM and 480 kDa ankyrin-G-GFP were shown. The formation of aggregates (circled in white and enlarged) is a sign of unhealthy neurons. Top: 480 kDa ankyrin-G shows up at the non-AIS region (pointed by white arrow heads. Bottom: Neuron formed 3 AIS and ectopic accumulation of ankyrin-G on soma. Scale bar is 20 μm. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Quantification of AIS structural change. div3 neurons of ANK3-E22/23f/f mice were transfected with Cre-BFP and wild type 480 kDa ankyrin-G or 480 kDa ankyrin-G-K2864N. At 7 div, neurons were fixed and stained for ankyrin-G. Representative images show the ankyrin-G signal at the AIS. The green line and yellow line indicate where the line for AIS intensity measurement was drew. White dash line circled the cell body of the transfected neuron. Scale bar is 20 μm. Average AIS intensity for both conditions is plotted alone distance and aligned with non-transfected cells (n=10). Please click here to view a larger version of this figure.

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 formation of AIS. This allows study of AIS formation in a clean system. By further adopting the Banker culture system which improves viability without complications of glial cell over-growth, we could reach a high transfection efficiency, which provide us enough neurons for quantitative measurement of AIS dimensions.

There are several critical steps in this protocol. The first critical step is considering the best time window to do the transfection, which needs to be early enough to prevent the assembly of AIS and late enough to reach the highest transfection efficiency. We tried 0 div electroporation transfection, which gave about 10% transfection efficiency with Cre-BFP only, but we were never able to transfect 480 kDa Ankyrin-G at 0 div. We suspect it is due to the large size of the plasmid (about 20 kb). Primary cultured hippocampal neurons have a narrow window for transfection, which is between 3-5 days. The accumulation of ankyrin-G at the AIS starts from 3 div. When we transfect Cre-BFP at 3 div, no AIS formation was seen in transfected neurons (Figure 4A). We could get 10-20 neurons transfected with 480 kDa ankyrin-G from one 18 mm coverslip. Also, for the co-transfection rescue experiment, all DNA must be generated under the same promoter and the ratio of Cre-BFP and 480 kDa ankyrin-G-GFP must be matched. In this experiment, we used chicken beta-actin promoter.

Another critical step is the modification to the Banker culture. The Banker culture was developed for culturing embryonic rat neurons. To better support the more sensitive mouse postnatal hippocampal neuron, we include a step of chopping hippocampi into smaller pieces to improve the trypsinization efficiency. Adding KOH treatment after the nitric acid treatment further reduced the toxicity from the glass coverslips, which help neurons attach and grow better.

A remaining challenge is how to control the expression level of ankyrin-G. A dosage screen helped to determine the optimal amount of plasmid used for transfection. Going forward, it is better to use a neuron-specific promoter to control the level of expression. The current data analysis did not measure the position of AIS. This function should be included in the future.

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Dr. Gary Banker for suggestion on neuronal culture protocol. This work is supported by the Howard Hughes Medical Institute, a grant from NIH, and a George Barth Geller endowed professorship (V.B.).

Materials

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
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References

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Tags

Primary Cultured Hippocampal NeuronsAxon Initial SegmentsAnkyrin-GCRE BFP DNAAnkyrin-G GFP DNATransfectionDissectionTrypsin DigestionTriturationNeuronal CultureGlial Cell Feeder Dishes

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Yang, R., Bennett, V. Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments. J. Vis. Exp. (168), e61411, doi:10.3791/61411 (2021).
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