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 dish...

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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...

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-E22/23f/f 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&#8217;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-transfec...

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, ...

This protocol is used to quantitatively study the assembly and structure of the axon initial segments of Hippocampal Neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G. To begin, dissect six to eight hippocampi from postnatal, one day old pups with HBSS medium in a Petri dish. Chop the hippocampi with dissection scissors into smaller pieces, and transfer them from the dish to a 15 milliliter tube.Wash the hippocampi twice with five milliliters of HBSS, and leave them in 4.5 milliliters of HBSS after the wash. Add 0.5 milliliters of 2.5%trypsin into 4.5 milliliters of HBSS, and incubate in a 37 degree Celsius water bath for 15 minutes, inverting the tube every five minutes. Well digested hippocampi should become sticky and form a cluster.If needed, extend the digestion for five more minutes. Wash hippocampi with HBSS three times for five minutes per wash. After the wash, add two milliliters of HBSS and pipette the hippocampi up and down with a Pasteur pipette 15 times.Triturate the tissue with a fire polished Pasteur pipette 10 times, and rest the tube for five minutes until all chunks set to the bottom. Repeat the trituration with the remaining chunks until most of them have disappeared. Then, use a one milliliter pipette tip to gently transfer the supernatant containing the dissociated neurons to plating dishes.Add it directly to the pre incubated plating medium and shake the plate gently. Two to four hours after seeding, check the plating dishes with a light microscope. The majority of the neurons should have attached to the cover slip.Using a fine tip forceps, flip the cover slips to the glial cell feeder dishes containing preconditioned neuronal culture medium with the wax dots side facing downwards. Neurons can grow in the glial cell feeder dishes for up to one month. Feed neurons every seven days with one milliliter of fresh neuronal culture medium.On the third day in vitro, flip the cover slips with the wax dot side facing up into a glial cell feeder dish with conditioned neuronal culture medium. Mix 0.25 micrograms of CRE BFP DNA with 0.5 micrograms of ankyrin-G GFP DNA in a 1.7 milliliter tube to transfect four cover slips. Add 100 microliters optimum.Then mix the tube and rested on a rack. If only CRE BFP is transfected, the GFP plasmic backbone is used to match the total amount of DNA. Mix three microliters of transfection reagent with 100 microliters of optimum in a new 1.7 milliliter tube and incubate the tube for five minutes at room temperature.Then, mix 100 microliters of the previously mixed DNA with 100 microliters of transfection reagent mix, and leave it for five to 10 minutes on a rack. Add 50 microliters of the DNA mix on top of each cover slip by inserting the tip just below the medium without touching the cover slips. Pipette slowly to avoid spreading the DNA mix.Slowly bring the dish back to the incubator and leave it there for 30 to 45 minutes. Flip the cover slips back to the home glial feeder dish with the wax dot side facing down and put the dish back in the incubator. For AIS quantification, open the Z series images in Fiji and generate a maximum projection of images.After subtracting the empty cover slip background signal from the image, draw a line along the AIS, making sure that the width of the line fully covers the AIS. Start the line before the AIS signal is raised above the background and stop after it drops to the background. Measure the mean pixel intensity along the line, and export it to a spreadsheet.Then, run a MATLAB script to generate the final figure. The experiment should include Cre-BFP only transfection as negative control, Cre-BFP plus 480 kilodalton ankyrin-G cotransfection as positive control, and a non-transfected condition as technique control. In the Cre-BFP only control, transfected neurons lack the accumulation of AIS markers, including ankyrin-G, beta 4 spectrin, neurofascin, and voltage gated sodium channels.In contrast, Cre and 480 kilodalton ankyrin-G co-transfected neurons have fully assembled AIS, which is demonstrated by the presence of AIS markers. It is important to confirm the quality of culture via comparison with the non-transfected dishes. Unhealthy neurons tend to show abnormal AIS structure, like discontinued or ectopic AIS.To evaluate how an ankyrin-G human neurodevelopmental disorder mutation affects AIS assembly, the average AIS intensity was plotted from the soma to the distal axon. AIS enriched proteins normally show a fast increase of signal from the proximal axon and a slow decrease of signal to the distal axon. When aligned with the non-transfected AIS, the mutant curve is wider and the 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.

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Neuroscience

4.9K visualizações.  Xi'an Jiaotong University. Este protocolo é usado para estudar quantitativamente a montagem e estrutura dos segmentos iniciais de axônio de neurônios hipocampais que carecem de AIS pré-montada devido à ausência de um anquilo-G gigante. Para começar, dissecar seis a oito hipocampos do pós-natal, um dia filhotes de idade com meio HBSS em uma placa de Petri. Pique o hipocampi com uma tesoura de dissecção em pedaços menores, e transfira-os do prato para um tubo de 15 mililitros.Lave o hipocampi duas vezes...