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.
