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 neur....

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.

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