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.).

NOTE: This culture method of hippocampal neurons from postnatal 0-day ANK3-E22/23f/f mice is adapted from Gary Banker&#8217;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
<ol>
	<li>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).</li>
	<li>Wash nitric acid treated coverslips with distilled water on a low speed shaker in a glass jar 2 times, 1 hour each.</li>
	<li>Incubate coverslips in saturated KOH dissolved in 100% ethanol overnight. Add KOH into ethanol till it is no longer dissolved.</li>
	<li>Repeat the wash step with distilled water. Rinse coverslips with 100% ethanol once for 10 minutes.</li>
	<li>Transfer coverslips from the rack to a glass beaker. Cover the beaker with aluminum foil. Bake coverslips in a 225 &#176;C oven overnight to sterilize the coverslips. (Coverslips could be stored in the beaker for weeks).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Preparing glia cell feeder dishes (2 weeks before culture day)
<ol>
	<li>Dissect the cortex from a postnatal 1-day old mice brain and peel off the meninges.</li>
	<li>Chop the cortex tissue as finely as possible with a clean scissors in a clean Petri dish in a clean bench.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Replace medium with fresh glia medium the next day to remove unattached cells.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
3. Culture hippocampal neurons
NOTE: All steps are performed at room temperature.
<ol>
	<li>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.</li>
	<li>Wash hippocampi 2x with 5 mL of HBSS in the tube. Leave the hippocampi in 4.5 mL of 1x HBSS after wash.</li>
	<li>Add 0.5 mL of 2.5% trypsin into 4.5 mL of HBSS and incubate in a 37 &#176;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.</li>
	<li>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.</li>
	<li>Add 2 mL of HBSS after the wash and pipette the hippocampi up and down with a Pasteur pipette 15 times.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat step 3.6-3.7 with the remaining chunks till most of the chunks have disappeared.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Optional step: 1 week after seeding, add cytosine arabinoside (1-&#946;-D-arabinofuranosylcytosine) to a final concentration of 5 &#956;M to curb glial proliferation.</li>
</ol>
4. Disruption of AIS by Knockout of Ankyrin-G at earlier stage of neuron development
<ol>
	<li>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.</li>
	<li>Mix 0.25 &#956;g of Cre-BFP DNA with 0.5 &#956;g 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 &#956;L 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.</li>
	<li>Mix 3 &#956;L of transfection reagent (e.g., Lipofectamine 2000) (~ 3 times of DNA) with 100 &#956;L culture medium in a new 1.7 mL tube. Incubate for 5 minutes at RT.</li>
	<li>Mix 100 &#956;L of DNA solution from step 4.2 with 100 &#956;L of transfection reagent from step 4.3. Rest for 5-10 minutes on a rack.</li>
	<li>Add 50 &#956;L 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.</li>
	<li>Slowly bring the dish back to the incubator and incubate for 30-45 minutes.</li>
	<li>Flip the coverslips back to the home glia feeder dish with wax dots side facing down and put the plate back to the incubator.</li>
</ol>
5. Quantification of axon initial segment
<ol>
	<li>Fix neurons on 7-10 div and stain with AIS marker following the standard immunocytochemistry protocol for the protein of interesting.</li>
	<li>Collect fluorescent pictures with desired microscopy.
	<ol>
		<li>Take Z-series sections to collect the signal of the entire AIS. Keep the same Z-depth for all pictures.</li>
		<li>Adjust the laser intensity to reach the best pixel intensity dynamic range.</li>
		<li>Make sure all AIS pictures are taken on the same microscope setup.</li>
		<li>Always check the signal of Cre-BFP.</li>
	</ol>
	</li>
	<li>AIS quantification
	<ol>
		<li>Open picture with Fiji (https://fiji.sc).</li>
		<li>Generate maximum projection of Z-series images.</li>
		<li>Subtract the empty coverslip background signal from the image.</li>
		<li>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.</li>
		<li>Measure the mean pixel intensity alone the line and export to a spreadsheet (~10-15 AISs are needed).</li>
		<li>Generate the average intensity curve of AIS using the MATLAB script adapted from Berger et al.15.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Nelson, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction:+Ankyrin-G+regulates+forebrain+connectivity+and+network+synchronization+via+interaction+with+GABARAP.">Correction: Ankyrin-G regulates forebrain connectivity and network synchronization via interaction with GABARAP.</a> Molecular Psychiatry. , (2019).</li><li>Tseng, W. C., Jenkins, P. M., Tanaka, M., Mooney, R., Bennett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+ankyrin-G+stabilizes+somatodendritic+GABAergic+synapses+through+opposing+endocytosis+of+GABAA+receptors.">Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors.</a> Proceedings of the National Academy of Sciences of the U S A. 112 (4), 1214-1219 (2015).</li><li>Tai, Y., Gallo, N. B., Wang, M., Yu, J. R., Van Aelst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axo-axonic+Innervation+of+Neocortical+Pyramidal+Neurons+by+GABAergic+Chandelier+Cells+Requires+AnkyrinG-Associated+L1CAM.">Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-Associated L1CAM.</a> Neuron. 102 (2), 358-372 (2019).</li><li>Hofflin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+the+Axon+Initial+Segment+in+Interneurons+and+Pyramidal+Cells+of+Rodent+Visual+Cortex.">Heterogeneity of the Axon Initial Segment in Interneurons and Pyramidal Cells of Rodent Visual Cortex.</a> Frontiers in Cellular Neuroscience. 11, 332 (2017).</li><li>Schluter, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Plasticity+of+Synaptopodin+in+the+Axon+Initial+Segment+during+Visual+Cortex+Development.">Structural Plasticity of Synaptopodin in the Axon Initial Segment during Visual Cortex Development.</a> Cerebral Cortex. 27 (9), 4662-4675 (2017).</li><li>Kuba, H., Oichi, Y., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+activity+regulates+Na(+)+channel+distribution+at+the+axon+initial+segment.">Presynaptic activity regulates Na(+) channel distribution at the axon initial segment.</a> Nature. 465 (7301), 1075-1078 (2010).</li><li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+relocation+of+the+axon+initial+segment+fine-tunes+neuronal+excitability.">Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability.</a> Nature. 465 (7301), 1070-1074 (2010).</li><li>Pan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+ankyrin-G-based+mechanism+retains+KCNQ+and+NaV+channels+at+electrically+active+domains+of+the+axon.">A common ankyrin-G-based mechanism retains KCNQ and NaV channels at electrically active domains of the axon.</a> Journal of Neuroscience. 26 (10), 2599-2613 (2006).</li><li>Cooper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Made+for+"anchorin":+Kv7.2/7.3+(KCNQ2/KCNQ3)+channels+and+the+modulation+of+neuronal+excitability+in+vertebrate+axons.">Made for "anchorin": Kv7.2/7.3 (KCNQ2/KCNQ3) channels and the modulation of neuronal excitability in vertebrate axons.</a> Seminars in Cell &amp; Developmental Biology. 22 (2), 185-192 (2011).</li><li>Zhou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AnkyrinG+is+required+for+clustering+of+voltage-gated+Na+channels+at+axon+initial+segments+and+for+normal+action+potential+firing.">AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing.</a> The Journal of Cell Biology. 143 (5), 1295-1304 (1998).</li><li>Kordeli, E., Lambert, S., Bennett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AnkyrinG.+A+new+ankyrin+gene+with+neural-specific+isoforms+localized+at+the+axonal+initial+segment+and+node+of+Ranvier.">AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier.</a> Journal of Biological Chemistry. 270 (5), 2352-2359 (1995).</li><li>Jenkins, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+ankyrin-G:+a+critical+innovation+in+vertebrate+evolution+of+fast+and+integrated+neuronal+signaling.">Giant ankyrin-G: a critical innovation in vertebrate evolution of fast and integrated neuronal signaling.</a> Proceedings of the National Academy of Sciences of the U S A. 112 (4), 957-964 (2015).</li><li>Jenkins, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+polarity+is+determined+by+a+multifunction+motif+mediating+lateral+membrane+retention+through+ankyrin-G+and+apical-lateral+transcytosis+through+clathrin.">E-cadherin polarity is determined by a multifunction motif mediating lateral membrane retention through ankyrin-G and apical-lateral transcytosis through clathrin.</a> Journal of Biological Chemistry. 288 (20), 14018-14031 (2013).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocol. 1 (5), 2406-2415 (2006).</li><li>Berger, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Myosin+II+Activity+Regulates+Assembly+and+Plasticity+of+the+Axon+Initial+Segment.">Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment.</a> Neuron. 97 (3), 555-570 (2018).</li><li>Yang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodevelopmental+mutation+of+giant+ankyrin-G+disrupts+a+core+mechanism+for+axon+initial+segment+assembly.">Neurodevelopmental mutation of giant ankyrin-G disrupts a core mechanism for axon initial segment assembly.</a> Proceedings of the National Academy of Sciences of the U S A. 116 (39), 19717-19726 (2019).</li></ol>

The authors have nothing to disclose.

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.

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 &#956;m in length and located within 20 &#956;m 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.

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

use of primary cultured hippocampal neurons to study the assembly of axon initial segments

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.

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 (&#946;4), 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61411/61411fig01.jpg" /> 
Figure 1: The genomic editing of ANK3-E22/23-flox.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61411/61411fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61411/61411fig02.jpg" /> 
Figure 2: Loss of AIS in ANK3-E22/23-flox neurons in the present of Cre recombinase.&#160;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61411/61411fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61411/61411fig03.jpg" /> 
Figure 3: Workflow of protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61411/61411fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61411/61411fig04v3.jpg" /> 
Figure 4: A full rescue of AIS by 480kDa AnkG in ANK3-E22/23-flox neurons transfected with Cre.&#160;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), &#946;4-spectrin (&#946;4), neurofascin (Nf) and voltage gated sodium channels (VSVG). White arrow head points to the AIS of a transfected neuron. Scale bar is 20 &#956;m. 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 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61411/61411fig04v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61411/61411fig05.jpg" /> 
Figure 5: Quantification of AIS structural change.&#160;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 &#956;m. Average AIS intensity for both conditions is plotted alone distance and aligned with non-transfected cells (n=10). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61411/61411fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments at JoVE.com

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

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.

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.0K Views.  Xi'an Jiaotong University. 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.

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and memory. Primary hippocampal cell culturing allows neuroscientists to examine the activity and properties of neurons at the individual cell and single synapse level. In this video, we will demonstrate how to isolate and grow primary hippocampal cells from newborn rats. The hippocampus may be isolated from each newborn animal in as short as 2 to 3 minutes, and the cultures can be maintained for up to two weeks. We will also briefly demonstrate how to use these hippocampal neurons for ratiometric calcium imaging. While this protocol describes the process for the hippocampus, with little to no modification, it can be applied to other regions of the brain.

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

Biology

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

A Technique to Culture Mouse Hippocampal Neurons

Source: Yang, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61411">Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing neurons from murine hippocampi. Initially, the hippocampi undergo enzymatic digestion to break down the extracellular tissue matrix. Subsequently, the digested tissue is mechanically disrupted to isolate individual neurons. Finally, these neurons are cultured on glial feeder cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing glia cell feeder dishes ...