Isolation and Culture of Chick Ciliary Ganglion Neurons

Summary

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Abstract

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Introduction

Ciliary ganglion (CG) neurons belong to the parasympathetic nervous system. These neurons are cholinergic, being able to establish muscarinic or nicotinic synapses^1,2,3. Anatomically, the CG is located in the posterior part of the eye between the optic nerve (ON) and the choroid fissure (CF) and consists of around 6000 neurons in early embryonic stages^1,4. For the first week in culture, ciliary ganglion neurons present a multipolar morphology. After one week, they start to transition to a unipolar state, with one neurite extending and forming the axon⁵. In addition, approximately half of CG neurons die between the 8^th and 14^th day of chick embryo development, through a programmed process of cell death. This decrease in the number of neurons results in a total population of the ciliary ganglion of around 3000 neurons^6,7,8. In vitro, there is no reduction in the number of CG neurons when grown with muscle cells⁹ and CG neurons can be cultured for several weeks^1,9.

The ciliary ganglion consists of a homogeneous population of ciliary neurons and choroidal neurons, each representing half of the neuronal population in the CG, innervating the muscle of the eye. These two types of neurons are structurally, anatomically and functionally distinct. Ciliary neurons innervate the striated muscle fibers on the iris and lens, being responsible for pupil contraction. Choroidal neurons innervate the smooth muscle of the choroid^1,10,11,12.

Cultures of chicken ciliary ganglion neurons have been shown to be useful tools for the study of neuromuscular synapses and synapse formation^1,5,9. Considering that neuromuscular synapses are cholinergic¹³, using a neuronal population that is cholinergic – CG neurons – emerged as a potential alternative to previous cell models¹⁴. These models consisted in an heterogenous neuronal population, in which only a small part is cholinergic. Alternatively, ciliary ganglion neurons develop relatively fast in vitro, and after approximately 15 hours already form synapses¹. CG neurons have been used as a model system throughout the years for distinct research studies, due to its relatively ease of isolation and manipulation. These applications include optogenetic studies, synapse development, apoptosis and neuromuscular interactions^14,15.

We describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from embryonic day 7 (E7) chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of cholinergic neurons. We also highlight key steps of the protocol that require special attention and that will improve the quality of the neuronal cultures. These cultures can be maintained in vitro for at least 15 days.

Protocol

1. Preparation of reagents

NOTE: The materials necessary for this procedure are the following: forceps (nº 5 and nº 55), surgical tweezers, dissection Petri dishes (black bottom), 24-well plates, plastic Pasteur pipette, fire-polished glass Pasteur pipette, 10 mL syringe, 0.22 µm syringe filter.

1. Prepare and sterilize all the material needed for the protocol including glass coverslips, forceps (nº 5 and nº 55), surgical tweezers, Petri dishes (black bottom), distilled H₂O, pipettes and material for surgery.
2. Prepare 0.1 mg/mL Poly-D-Lysine (PDL) solution.
   1. Reconstitute PDL in 0.1 M borate buffer (pH 8.2) to a concentration of 1 mg/mL (10x solution).
   2. Dilute 1:10 in 166.6 mM borate buffer (pH 8.2) to obtain a final concentration of 0.1 mg/mL.
3. Prepare 10 µg/mL laminin solution.
   1. Dilute 1 mg/mL laminin in plain neurobasal medium to a final concentration of 10 µg/mL.
4. Prepare Hank’s Balanced Salt Solution (HBSS): 5.36 mM KCl, 0.44 mM KH₂PO₄, 137 mM NaCl, 4.16 mM NaHCO3, 0.34 mM Na₂HPO₄·2H₂O, 5 mM glucose, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.001% phenol red. Adjust pH to 7.2.
5. Prepare 0.1% trypsin solution.
   1. Dissolve 5 mg of trypsin 1:250 powder in 5 mL of HBSS for a final concentration of 0.1%.
   2. Place in a roller mixer at 4 °C until completely dissolved.
   3. Filter using a 10 mL syringe and a 0.22 µm syringe filter.
6. Prepare ciliary ganglia incomplete medium: neurobasal medium without glutamine, 1X B27 (photo-sensitive), 10% heat-inactivated horse serum, 2% heat-inactivated FBS, 12.5 U/mL penicillin/streptomycin (0.25x) and 2 mM glutamine. Use sterile reagents and prepare the medium under sterile conditions.
7. Prepare ciliary ganglia complete medium (supplemented with growth factors): to the incomplete medium, add 5 ng/mL GDNF and 5 ng/mL CNTF.

2. Preparation of glass coverslips for 24-well plates

1. Place the desired number of glass coverslips inside an acid resistant container and add 65% nitric acid until all coverslips are submerged. Place the container in an orbital shaker and incubate overnight at room temperature (RT) at a speed of 1000 rpm.
2. The next day, carefully transfer the nitric acid to a small reservoir and store for further use. Nitric acid can be re-used 2-3x.
3. Carefully, add distilled H₂O to the coverslips to remove the remaining nitric acid. Place in agitation for 30 minutes, discard the washing solution and repeat this 5x.
4. Rinse the coverslips with 75% ethanol twice.
5. Carefully separate and place individual coverslips in a metal rack covered with aluminum foil and incubate at 50 ºC for 10-15 minutes or until fully dry.
   NOTE: Do not autoclave glass coverslips as they will stick to each other.
6. Sterilize the coverslips under UV light for 10-15 minutes. Maintain coverslips sterile for neuronal tissue culture.

3. Coating of glass coverslips for 24-well plates

1. Using a sterile tweezer, place one coverslip in each well of a 24-well plate.
2. Add 500 µL of 0.1 mg/mL PDL and incubate overnight at 37 °C.
3. The next day, rinse the coverslips twice with sterile distilled H₂O. Then, add 500 µL of distilled water to each coverslip and leave for 30 minutes at room temperature.
4. Discard the water and add 350 µL of 10 µg/mL laminin solution in each well.
5. Place in a 37 °C incubator for 2 h.
6. Before cell plating, remove the laminin solution and wash twice with plain neurobasal medium.
   NOTE: It is important that the coverslips do not dry at any time.
7. Add 300 µL of complete medium and leave in an incubator at 37 °C and 5% CO₂ until plating time. Before plating cells, remove this medium.

4. Culture of ciliary ganglia from chicken embryo (embryonic day 7)

1. Dissection of ciliary ganglia (CG)
   1. Remove eggs from incubator and spray them with 75% ethanol.
      NOTE: Eggs are stored at ~16 °C before being incubated at 37.7 °C for 7 days (or the desired embryonic stage). Eggs used here are from Ross chicken species.
   2. Cut the top of the egg with a scissor and take out the embryo using a spoon. Place the embryo in a Petri dish with ice-cold HBSS and separate the head from the body by cutting in the neck region.
      NOTE: As soon as the embryo is removed from the egg, it can produce proteases that are responsible for cell death. It is important to rapidly separate the head from the body once the embryo is outside the shell to minimize cell death.
   3. Keep the head of the embryo in ice-cold HBSS.
   4. Hold the embryo head up and fix it in the beak of the chick with nº 5 forceps. Then with nº 55 forceps, start to remove the thin layer of skin around the eye.
   5. Carefully remove the eye and rotate it to access the posterior part. While separating the eye from the head of the chick, notice the optic nerve being sectioned. This will help to localize the ciliary ganglion.
   6. Once the eye is separated, keep it with the posterior side up and notice the ciliary ganglion adjacent to the sectioned optic nerve and the choroid fissure. The preganglionic nerve might still be attached to the ciliary ganglion, which facilitates its identification.
   7. Dissect the ciliary ganglion from each eye and clean very well by removing the excess tissue around each ganglion.
      NOTE: To have a yield of ~1x10⁶ cells/mL, dissect ~70 CGs. Please note that the cell population obtained contains non-neuronal cells as well. To decrease the number of non-neuronal cells and, consequently, increase the purity of the neuronal population, it is very important to clean the ciliary ganglia as much as possible, removing all the excess tissue.
2. Dissociation and culture of ciliary ganglia
   1. Collect all ciliary ganglia to a 15 mL tube using a sterile plastic Pasteur pipette.
      NOTE: It is important to pre-wet the Pasteur pipette to minimize the attachment of the ganglia to the wall of the pipette.
   2. Centrifuge the ciliary ganglia for 2 minutes at 200 x g.
   3. Carefully, remove all the HBSS medium using a Pasteur pipette and then a P1000 micropipette. Add 1 mL of 0.1% trypsin solution and incubate for 20 minutes at 37 °C in a water bath, without agitation.
   4. Centrifuge for 2 minutes at 200 x g.
   5. Immediately remove the trypsin solution and add 1 mL of incomplete medium.
      NOTE: Incomplete medium contains serum which will immediately stop the effect of trypsin.
   6. Centrifuge for 2 minutes at 200 x g and remove all medium.
   7. Add 350-500 µL of complete medium.
      NOTE: The necessary volume to dissociate cells depends on the number of ciliary ganglia obtained and, thus, on the obtained pellet size. For ~70 CG it is recommended to use 500 µL of medium.
   8. Dissociate CGs by pipetting up and down 10-15x first using a P1000 followed by 10-15x using a fire-polished glass Pasteur pipette. Avoid air bubble formation to minimize cell loss.
      NOTE: Keep the cellular suspension on ice until plating.
   9. Determine cellular density using a Trypan blue solution and a standard Neubauer chamber.
   10. Plate 1 x 10⁴ cells/mL in each well of the 24-well plate by diluting the appropriate volume of cell suspension in 500 µL of complete medium (supplemented with 10 µM 5’-FDU).
   11. Incubate cells in a 37 °C, 5% CO₂ incubator.

5. Immunocytochemistry and image analysis of ciliary neurons

1. Perform the immunocytochemistry assay presented in this paper as previously described^16,17.
2. Use the following primary antibodies: mouse monoclonal b-III tubulin (1:1000, T8578), chicken monoclonal neurofilament M (1:1000, AB5735), mouse monoclonal SV2 (1:1000, AB2315387).
3. As secondary antibodies, use Alexa Fluor 568-conjugated goat anti-mouse antibody (1:1000, A11031), Alexa Fluor 568-conjugated goat anti-chicken antibody (1:1000, A11041), Alexa Fluor 647-conjugated goat anti-mouse antibody (1:1000, A21235).
4. Mount coverslips using mounting medium with DAPI, for nuclear staining (P36935).

Results

The estimated duration for this procedure tightly depends on the yield needed for each specific experiment and, thus, on the number of ciliary ganglia that need to be isolated. For an estimated yield of 1 x 10⁶ cells/mL, isolate around 70 ciliary ganglia (35 eggs). For this number of ganglia, it will take 2-3 hours for the dissection procedure and a total of 4-5 hours for the total procedure. A step-by-step illustration of the isolation protocol is shown in Figure 1A. The identifi...

Discussion

In this protocol, we demonstrated how to prepare and culture CG neurons. The identification and dissection of the ciliary ganglion can be difficult for unexperienced users. Therefore, we present a detailed and step-by-step procedure to efficiently dissect E7 chick ciliary ganglia, dissociate the tissue and prepare neuronal cultures that can be maintained for at least 15 days. The ciliary ganglion neurons obtained with this protocol are also suitable for co-culture with muscle cells.

Ciliary ga...

Materials

Name	Company	Catalog Number	Comments
5-fluoro-2’-deoxiuridina (5'-FDU)	Merck (Sigma Aldrich)	F0503
Alexa Fluor 568-conjugated goat anti-chicken antibody	Thermo Fisher Scientific	A11041
Alexa Fluor 568-conjugated goat anti-mouse antibody	Thermo Fisher Scientific	A11031
Alexa Fluor 647-conjugated goat anti-mouse antibody	Thermo Fisher Scientific	A21235
B27 supplement (50x), serum free	Invitrogen (Gibco)	17504-044
Chicken monoclonal neurofilament M	Merck (Sigma Aldrich)	AB5735
D-(+)-Glucose monohydrate	VWR	24371.297
Fetal Bovine Serum (FBS), qualified, Brazil	Invitrogen (Gibco)	10270-106
HEPES, fine white crystals, for molecular biology	Fisher Scientific	10397023
Horse Serum, heat inactivated, New Zealand origin	Invitrogen (Gibco)	26050-070
L-Glutamine (200 mM)	Invitrogen (Gibco)	25030-081
Mouse laminin I	Cultrex (R&D systems)	3400-010-02
Mouse monoclonal b-III tubulin	Merck (Sigma Aldrich)	T8578
Mouse monoclonal SV2	DSHB	AB2315387
Multidishes, cell culture treated, BioLite, MW24 (50x)	Thermo Fisher Scientific	11874235
Neurobasal medium without glutamine	Invitrogen (Gibco)	21103-049
Penicillin/streptomycin (5,000 U/mL)	Invitrogen (Gibco)	15070-063
Phenol red, bioreagent, suitable for cell culture	Merck (Sigma Aldrich)	P3532
Poly-D-Lysine	Merck (Sigma Aldrich)	P7886
Potassium chloride	Fluka (Honeywell Reaarch Chemicals)	31248-1KG
Potassium di-hydrogen phosphate (KH2PO4) for analysis, ACS	Panreac Applichem	131509-1000
Prolong Gold Antifade mounting medium with DAPI	Invitrogen (Gibco)	P36935
Puradisc FP 30mm Syringe Filter, Cellulose Acetate, 0.2µm, sterile 50/pk	Fisher Scientific	10462200
Recombinant human ciliary neurotrophic factor (CNTF)	Peprotech	450-13
Recombinant human glial cell-derived neurotrophic factor (GDNF)	Peprotech	450-10
Sodium chloride for analysis, ACS, ISO	Panreac Applichem	131659-1000
Sodium dihydrogen phosphate 2-hydrate (Na2HPO4·2H2O), pure, pharma grade	Panreac Applichem	141677-1000
Sodium Pyruvate 100 mM (100x)	Thermo Fisher	11360039
Syringe without needle, 10 mL	Thermo Fisher	11587292
Trypsin 1:250 powder	Invitrogen (Gibco)	27250-018