A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
Ciliary ganglion (CG) neurons belong to the parasympathetic nervous system. These neurons are cholinergic, being able to establish muscarinic or nicotinic synapses1,2,3. 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 stages1,4. 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....
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.
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.......
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.......
This work was financed by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under projects CENTRO-01-0145-FEDER-000008:BrainHealth 2020, CENTRO2020 CENTRO-01-0145-FEDER-000003:pAGE, CENTRO-01-0246-FEDER-00018:MEDISIS, and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., under projects UIDB/04539/2020, UIDB/04501/2020, POCI-01-0145-FEDER-022122:PPBI, PTDC/SAU-NEU/104100/2008, and the individual grants SFRH/BD/141092/2018 (M.D.), DL57/2016/CP1448/CT0009 (R.O.C.), SFRH/B....
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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved