The chicken ciliary ganglion, is a structure localized in the posterior part of the eye, adjacent to the optic nerve in the choroid fissure. And the ciliary ganglion neurons, belong to the parasympathetic nervous system, and are cholinergic neurons, and thus they can establish cholinergic synapses. The ciliary ganglion consists of an enormous population of ciliary neurons and choroidal neurons, that are structurally and functionally distinct.
So the ciliary neurons innervates intraocular muscle, and choroidal neurons innervates mood muscle in the eye. And for the first days in culture, the ciliary ganglion neurons present a multipolar morphology, and after these, they start to transition to a unipolar state, where one of the neurites extends and forms the axon. One of the most common studies using ciliary ganglion neurons, is a study of neuromuscular synapses, which are cholinergic synapses, and the use of ciliary ganglion neurons has become a good alternative, compared to previous models, due to the fact that the neuronal population obtained, is homogeneous in the sense that, all the neurons are cholinergic, and thus they can establish functional synapses with the muscle cells, which does not happen when we are working with a neuronal population, that is not entirely cholinergic.
The identification and dissection of the ciliary ganglion can be quite challenging for someone doing it for the first time, so here we provide a step by step protocol, for the appropriate identification and dissection of the ciliary ganglion, as well as the guidelines for successful culture of these neurons. For the preparation of the coverslips, you will need, 13 millimeter glass coverslips, an acid resistant container, 65%nitric acid, tweezers, Milli-Q water, 75%ethanol and a orbital shaker. The preparation of the coverslips for primary cultures, should be done two days before the procedure.
Place the desired number of glass coverslips, inside an acid resistant container, and add 65%nitric acid, until all coverslips are covered. Place the container in an orbital shaker, and incubate overnight at room temperature with agitation. The next day, carefully remove the nitric acid, and star for reusing.
Wash the coverslips, add Milli-Q water to the container. Once again, placing agitation for 30 minutes, discard the washing solution, and repeat this process five times. Rinse the coverslips twice using 75%ethanol.
Carefully separate and place individual coverslips in a metal rack, covered with aluminum foil, and incubate at 50 degrees, for 10 to 15 minutes, or until they are fully dry. Sterilize the coverslips in the UV light, for 10 to 15 minutes. For coating the coverslips, you will need, sterile glass coverslips, a 24-Well plate, sterile tweezers, 0.1 milligrams per milliliter PDL solution, sterile water, a 10 micrograms per milliliter laminin solution, plain neurobasal medium, and ciliary ganglion complete medium.
The coating of the coverslips, should be done the day before the procedure. Using a sterile tweezer, place one coverslip in each well of a 24-Well plate, add 500 microliters of poly dialyzing solution, at a concentration of 0.1 milligrams per milliliter, and incubate overnight at 37 degrees. The next day, wash the coverslips three times with sterile water, discard the water, and add 350 microliters of the laminin solution at a concentration of 10 micrograms per milliliter to each well.
Place in an incubator, at 37 degrees for two hours. After this time, and before cell plating, remove the laminin solution, and wash twice with 300 microliters of plain neurobasal medium. Add 300 microliters of complete medium, and place in an incubator, at 37 degrees and 5%CO2, until you are ready to plate cells.
For the dissection procedure, you will need, chicken eggs at embryonic day seven, 75%ethanol, dissection forceps number 545 and number 55, scissors, a spoon, dissection petri dishes with black bottom and ice-cold HBSS solution. Make sure to sterilize all the dissection tools in 75%ethanol. Eggs should be stored at 16 degrees before being incubated in an egg incubator, at 37.7 degrees, for seven days, or other desired embryonic stage.
In the day of the procedure, remove the eggs from the incubator, spray the eggs with 75%ethanol. Get the top of the egg using a scissor, and carefully take out the embroy using a spoon. Place the embryo, in a petri dish with ice cold HBSS solution, and immediately separate the head from the body, by cutting in the neck region.
Then transfer the head to a new petri dish with clean ice cold HBSS solution. As soon as the embryo is removed from the egg, it is able to produce proteases that are responsible for cell death. So it is important to separate the head from the body as quickly as possible, once the embryo is outside the egg to minimize cell death.
It is also very important to keep the head of the embryo in ice cold HBSS solution. Hold the embryo head up, and fix it in the beak of the chick, and then start to remove the thin layer of skin around the eye. Carefully, remove the eye by gently rotating it away from the head.
And while separating the eye from the head of the chick, notice the optic nerve being sectioned. Keep the eye with the posterior side up, and notice the ciliary ganglion, adjacent to the sensor optic nerve and the choroid fissure. The preganglionic nerve might still be attached to the ciliary ganglion, facilitating its identification.
Dissect the ciliary ganglion, from each eye, and clean it very well by removing the excess tissue. Transfer the dissected ganglia to a petri dish with HBSS solution on ice. In order to have a yield of around 1 million cells per milliliter, you should dissect around 70 ganglia, and also keep in mind that the cell population obtained, has non-neuronal cells as well, so in order to decrease the number of non-neuronal cells, and also to increase the purity of your neuranal population, it is important to clean the ciliary ganglia very well, removing all the excess tissue.
For the tissue dissociation, you will need, a 24-Well plate, 0.1%tripsin solution, incomplete ciliary ganglia medium, a fire polished glass pasteur pipette, and a plastic pasteur pipette. Pre-wet a sterile plastic pasteur pipette, and collect all ciliary ganglia to a 15 ML tube, and centrifuge for two minutes at 200 Gs.Carefully, remove all the HBSS medium, using a pasteur pipette to remove a bigger volume, and then, when you are closer to the pellet, use a micro pipette. Add one milliliter of 0.1%tripsin solution, and incubate for 20 minutes, at 37 degrees in a water bath.
Centrifuge for two minutes, at 200 Gs.Immediately, remove the tripsin solution, and add one milliliter of incomplete medium. The serum in the incomplete medium, will immediately stop the activity of trypsin. Centrifuge for two minutes, at 200 Gs, and remove all medium.
Add 500 microliters of complete medium, and start the tissue dissociation using a P1000 micropipette. Start by pipetting up and down, 10 to 15 times, and then, switch to a fire polished glass pasteur pipette, and again, pipette up and down 10 to 15 times. The cell suspension should become blurred, as a sign of successful tissue dissociation.
The necessary volume to dissociate cells depends on the number of ciliary ganglia obtained in this, and the pellet size. When pipetting up and down to dissociate a tissue, it is important to avoid the formation air bubbles, this will minimize cell loss. After resuspending cells, you can leave the cell suspension on ice until plating.
Determine the cellular density using a trypan blue solution, in the neubauer chamber. That lead cell suspension in complete medium, with 5FTU, at the desired density and plate 500 microliters of cells per well. Incubate cells at 37 degrees and 5%CO2.
Make sure to check our culture every day and follow the development of cells. Ciliary ganglia cells develop quite fast in vitro, and after one day, we can already see some neurites extending from the cell body. After seven to eight days in vitro, the neuronal network is very well established, and the cells can be maintained for at least 15 days.
After one day in vitro, ciliary ganglion neurons show a multipolar mythology. However, neurites extension occurs rapidly in the neurons visibly established the neuronal network, already after 24 hours. In vitro, ciliary ganglion neurons are responsible for the innervation of the muscle in the eye.
And so, these neuronal cultures, are very well suited for the study of neuromuscular synapses. For this, ciliary ganglion neurons can be plated on top of muscle cells. Here, we show a successful culture, of the eye vitreous, CG neurons, on top of the eye V7 chick pectral muscle neurons.
Synaptic vesicle marker, SV2, show active synapses that are established between the CG neurons axons, and the muscle fibers, easily identified by the multiple nuclei. Ciliary ganglion neurons obtained with this protocol are suitable for immunocytochemistry, electrophysiology, and survival essays amongst others. This dissection protocol, yields a very low number of neurons, but if done properly, will provide the pure population of cholinergic neurons.
It is very important that each ganglia is very well cleaned and the excess tissue is removed. For that, high quality instruments should be used, so this tissue can be removed in order to prevent contamination by non-neuronal cells. The age of the embryo will determine the success of our protocol.
Ideally, the dissection should be performed between E7 and E8.This time point is very important because at this stage, the developmental cell death that occurs normally in vitro has not occurred yet. In order to minimize the contamination of these non-neuronal cells, we use 5FTU in the culture media to minimize the growth of glial cells and fibroblasts. This cell model provides an excellent alternative for studying your nueromusclar disease.
Based on this protocol, additional scientific questions can be addressed, for example, how the sub cell localization of specific carbonates and specific proteins regulate synapse formation and function. Additionally, it's fairly easy to establish nerve muscle co-cultures to address further questions like the study of neuromuscular diseases.
