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 &#8211; Funda&#231;&#227;o para a Ci&#234;ncia 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/BD/77789/2011 (J.R.P.) and by Marie Curie Actions - IRG, 7th Framework Programme.

1. Preparation of reagents
NOTE: The materials necessary for this procedure are the following: forceps (n&#186; 5 and n&#186; 55), surgical tweezers, dissection Petri dishes (black bottom), 24-well plates, plastic Pasteur pipette, fire-polished glass Pasteur pipette, 10 mL syringe, 0.22 &#181;m syringe filter.
<ol>
	<li>Prepare and sterilize all the material needed for the protocol including glass coverslips, forceps (n&#186; 5 and n&#186; 55), surgical tweezers, Petri dishes (black bottom), distilled H2O, pipettes and material for surgery.</li>
	<li>Prepare 0.1 mg/mL Poly-D-Lysine (PDL) solution.
	<ol>
		<li>Reconstitute PDL in 0.1 M borate buffer (pH 8.2) to a concentration of 1 mg/mL (10x solution).</li>
		<li>Dilute 1:10 in 166.6 mM borate buffer (pH 8.2) to obtain a final concentration of 0.1 mg/mL.</li>
	</ol>
	</li>
	<li>Prepare 10 &#181;g/mL laminin solution.
	<ol>
		<li>Dilute 1 mg/mL laminin in plain neurobasal medium to a final concentration of 10 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Prepare Hank&#8217;s Balanced Salt Solution (HBSS): 5.36 mM KCl, 0.44 mM KH2PO4, 137 mM NaCl, 4.16 mM NaHCO3, 0.34 mM Na2HPO4&#183;2H2O, 5 mM glucose, 1 mM sodium pyruvate, 10 mM HEPES buffer, 0.001% phenol red. Adjust pH to 7.2.</li>
	<li>Prepare 0.1% trypsin solution.
	<ol>
		<li>Dissolve 5 mg of trypsin 1:250 powder in 5 mL of HBSS for a final concentration of 0.1%.</li>
		<li>Place in a roller mixer at 4 &#176;C until completely dissolved.</li>
		<li>Filter using a 10 mL syringe and a 0.22 &#181;m syringe filter.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Prepare ciliary ganglia complete medium (supplemented with growth factors): to the incomplete medium, add 5 ng/mL GDNF and 5 ng/mL CNTF.</li>
</ol>
2. Preparation of glass coverslips for 24-well plates
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Rinse the coverslips with 75% ethanol twice.</li>
	<li>Carefully separate and place individual coverslips in a metal rack covered with aluminum foil and incubate at 50 &#186;C for 10-15 minutes or until fully dry. 
	NOTE: Do not autoclave glass coverslips as they will stick to each other.</li>
	<li>Sterilize the coverslips under UV light for 10-15 minutes. Maintain coverslips sterile for neuronal tissue culture.</li>
</ol>
3. Coating of glass coverslips for 24-well plates
<ol>
	<li>Using a sterile tweezer, place one coverslip in each well of a 24-well plate.</li>
	<li>Add 500 &#181;L of 0.1 mg/mL PDL and incubate overnight at 37 &#176;C.</li>
	<li>The next day, rinse the coverslips twice with sterile distilled H2O. Then, add 500 &#181;L of distilled water to each coverslip and leave for 30 minutes at room temperature.</li>
	<li>Discard the water and add 350 &#181;L of 10 &#181;g/mL laminin solution in each well.</li>
	<li>Place in a 37 &#176;C incubator for 2 h.</li>
	<li>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.</li>
	<li>Add 300 &#181;L of complete medium and leave in an incubator at 37 &#176;C and 5% CO2 until plating time. Before plating cells, remove this medium.</li>
</ol>
4. Culture of ciliary ganglia from chicken embryo (embryonic day 7)
<ol>
	<li>Dissection of ciliary ganglia (CG)
	<ol>
		<li>Remove eggs from incubator and spray them with 75% ethanol. 
		NOTE: Eggs are stored at ~16 &#176;C before being incubated at 37.7 &#176;C for 7 days (or the desired embryonic stage). Eggs used here are from Ross chicken species.</li>
		<li>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.</li>
		<li>Keep the head of the embryo in ice-cold HBSS.</li>
		<li>Hold the embryo head up and fix it in the beak of the chick with n&#186; 5 forceps. Then with n&#186; 55 forceps, start to remove the thin layer of skin around the eye.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dissociation and culture of ciliary ganglia
	<ol>
		<li>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.</li>
		<li>Centrifuge the ciliary ganglia for 2 minutes at 200 x g.</li>
		<li>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 &#176;C in a water bath, without agitation.</li>
		<li>Centrifuge for 2 minutes at 200 x g.</li>
		<li>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.</li>
		<li>Centrifuge for 2 minutes at 200 x g and remove all medium.</li>
		<li>Add 350-500 &#181;L 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 &#181;L of medium.</li>
		<li>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.</li>
		<li>Determine cellular density using a Trypan blue solution and a standard Neubauer chamber.</li>
		<li>Plate 1 x 104 cells/mL in each well of the 24-well plate by diluting the appropriate volume of cell suspension in 500 &#181;L of complete medium (supplemented with 10 &#181;M 5&#8217;-FDU).</li>
		<li>Incubate cells in a 37 &#176;C, 5% CO2 incubator.</li>
	</ol>
	</li>
</ol>
5. Immunocytochemistry and image analysis of ciliary neurons
<ol>
	<li>Perform the immunocytochemistry assay presented in this paper as previously described16,17.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Mount coverslips using mounting medium with DAPI, for nuclear staining (P36935).</li>
</ol>

<ol><li>Betz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Formation+of+Synapses+between+Chick+Embryo+Skeletal+Muscle+and+Ciliary+Ganglia+Grown+in+vitro%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">The Formation of Synapses between Chick Embryo Skeletal Muscle and Ciliary Ganglia Grown in vitro.</a> Journal of Physiology. 254, 63-73 (1976).</li>
<li>Fischbach, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synapse+Formation+between+Dissociated+Nerve+and+Muscle+Cells+in+Low+Density+Cell+Cultures%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Synapse Formation between Dissociated Nerve and Muscle Cells in Low Density Cell Cultures.</a> Developmental Biology. 28, 407-429 (1972).</li>
<li>Bernstein, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+and+Culturing+of+Chick+Ciliary+Ganglion+Neurons%3A+A+System+well+Suited+to+Synaptic+Study%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Dissection and Culturing of Chick Ciliary Ganglion Neurons: A System well Suited to Synaptic Study.</a> Methods in Cell Biology. 71, 37-50 (2003).</li>
<li>Marwitt, R., Pilar, G., Weakly, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+Two+Ganglion+Cell+Populations+in+Avian+Ciliary+Ganglia%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Characterization of Two Ganglion Cell Populations in Avian Ciliary Ganglia.</a> Brain Research. 25, 317-334 (1971).</li>
<li>Role, L. W., Fishbach, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+Number+of+Chick+Ciliary+Ganglion.+Neuron+Processes+with+Time+in+Cell+Culture%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Changes in the Number of Chick Ciliary Ganglion. Neuron Processes with Time in Cell Culture.</a> Journal of Cell Biology. 104, 363-370 (1987).</li>
<li>Landmesser, L., Pilar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+Transmission+and+Cell+Death+During+Normal+Ganglionic+Development%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Synaptic Transmission and Cell Death During Normal Ganglionic Development.</a> Journal of Physiology. , 737-749 (1974).</li>
<li>Koszinowski, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bid+Expression+Network+Controls+Neuronal+Cell+Fate+During+Avian+Ciliary+Ganglion+Development%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">Bid Expression Network Controls Neuronal Cell Fate During Avian Ciliary Ganglion Development.</a> Frontiers in Physiology. 9, 1-10 (2018).</li>
<li>Landmesser, L., Pilar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synapse+Formation+During+Embryogenesis+on+Ganglion+Cells+Lacking+a+Periphery%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Synapse Formation During Embryogenesis on Ganglion Cells Lacking a Periphery.</a> Journal of Physiology. 241, 715-736 (1974).</li>
<li>Nishi, R., Berg, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissociated+Ciliary+Ganglion+Neurons+in+vitro%3A+Survival+and+Synapse+Formation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Dissociated Ciliary Ganglion Neurons in vitro: Survival and Synapse Formation.</a> Proceedings of the National Academy of Sciences of the United States of America. 74, 5171-5175 (1977).</li>
<li>Nishi, R., Berg, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+Components+from+Eye+Tissue+that+Differentially+Stimulate+the+Growth+and+Development+of+Ciliary+Ganglion+Neurons+in+Cell+Culture%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Two Components from Eye Tissue that Differentially Stimulate the Growth and Development of Ciliary Ganglion Neurons in Cell Culture.</a> Journal of Neuroscience. 1, 505-513 (1981).</li>
<li>Pilar, G., Vaughan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+Investigations+of+the+Pigeon+iris+Neuromuscular+Junctions%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+B%22%5BJournal%5D)">Electrophysiological Investigations of the Pigeon iris Neuromuscular Junctions.</a> Comparative Biochemistry and Physiology B. 29, 51-72 (1969).</li>
<li>Landmesser, L., Pilar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+Reinnervation+of+Two+Cell+Populations+in+the+Adult+Pigeon+Ciliary+Ganglion%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Selective Reinnervation of Two Cell Populations in the Adult Pigeon Ciliary Ganglion.</a> Journal of Physiology. , 203-216 (1970).</li>
<li>Pinto, M. J., Almeida, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Puzzling+Out+Presynaptic+Differentiation%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Puzzling Out Presynaptic Differentiation.</a> Journal of Neurochemistry. 139, 921-942 (2016).</li>
<li>Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+Development+of+the+Parasympathetic+Neurons+of+the+Avian+Ciliary+Ganglion%3A+A+Classic+Model+System+for+the+Study+of+Neuronal+Differentiation+and+Development%5BTitle%5D)+AND+%22Progress+in+Neurobiology%22%5BJournal%5D)">Functional Development of the Parasympathetic Neurons of the Avian Ciliary Ganglion: A Classic Model System for the Study of Neuronal Differentiation and Development.</a> Progress in Neurobiology. 43, 281-322 (1994).</li>
<li>Egawa, R., Yawo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+Neuro-Neuronal+Synapses+using+Embryonic+Chick+Ciliary+Ganglion+via+Single-Axon+Tracing%2C+Electrophysiology%2C+and+Optogenetic+Techniques%5BTitle%5D)+AND+%22Current+Protocols+in+Neuroscience%22%5BJournal%5D)">Analysis of Neuro-Neuronal Synapses using Embryonic Chick Ciliary Ganglion via Single-Axon Tracing, Electrophysiology, and Optogenetic Techniques.</a> Current Protocols in Neuroscience. 87, 1-22 (2019).</li>
<li>Pinto, M. J., Pedro, J. R., Costa, R. O., Almeida, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+K48+Ubiquitination+during+Presynaptic+Formation+by+Ubiquitination-Induced+Fluorescence+Complementation+%28UiFC%29%5BTitle%5D)+AND+%22Frontiers+in+Molecular+Neuroscience%22%5BJournal%5D)">Visualizing K48 Ubiquitination during Presynaptic Formation by Ubiquitination-Induced Fluorescence Complementation (UiFC).</a> Frontiers in Molecular Neuroscience. 9, 1-19 (2016).</li>
<li>Martins, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+Stem+Cells+Secretome-Induced+Axonal+Outgrowth+is+Mediated+by+BDNF%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Mesenchymal Stem Cells Secretome-Induced Axonal Outgrowth is Mediated by BDNF.</a> Scientific Reports. 7, 1-13 (2017).</li>
<li>Nishi, R. Autonomic and Sensory Neuron. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNishi%2C+R+%22Methods+in+Cell+Biology%22">Methods in Cell Biology</a>. , 249-263 (1996).</li>
<li>Rojo, J. M., De Ojeda, G., Portolés, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitory+Mechanisms+of+5-fluorodeoxyuridine+on+Mitogen-induced+Blastogenesis+of+Lymphocytes%5BTitle%5D)+AND+%22International+Journal+of+Immunopharmacology%22%5BJournal%5D)">Inhibitory Mechanisms of 5-fluorodeoxyuridine on Mitogen-induced Blastogenesis of Lymphocytes.</a> International Journal of Immunopharmacology. 6, 61-65 (1984).</li>
<li>Hui, C. W., Zhang, Y., Herrup, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-Neuronal+Cells+are+Required+to+Mediate+the+Effects+of+Neuroinflammation%3A+Results+from+a+Neuron-Enriched+Culture+System%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Non-Neuronal Cells are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System.</a> PLoS One. 11, 1-17 (2016).</li>
<li>Crain, S. M., Alfei, L., Peterson, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuromuscular+Transmission+in+Cultures+of+Adult+Human+and+Rodent+Skeletal+Muscle+After+Innervation+in+vitro+by+Fetal+Rodent+Spinal+Cord%5BTitle%5D)+AND+%22Journal+of+Neurobiology%22%5BJournal%5D)">Neuromuscular Transmission in Cultures of Adult Human and Rodent Skeletal Muscle After Innervation in vitro by Fetal Rodent Spinal Cord.</a> Journal of Neurobiology. 1, 471-489 (1970).</li>
<li>Kano, M., Shimada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innervation+and+Acetylcholine+Sensitivity+of+Skeletal+Muscle+Cells+Differentiated+in+vitro+from+Chick+Embryo%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">Innervation and Acetylcholine Sensitivity of Skeletal Muscle Cells Differentiated in vitro from Chick Embryo.</a> Journal of Cellular Physiology. 78, 233-242 (1971).</li>
<li>Robbins, N., Yonezawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developing+Neuromuscular+Juctions%3A+First+Sings+of+Chemical+Transmission+during+Formation+in+Tissue+Culture%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Developing Neuromuscular Juctions: First Sings of Chemical Transmission during Formation in Tissue Culture.</a> Science. 80, 395-398 (1971).</li>
<li>Squire, L. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSquire%2C+LR+%22Encyclopedia+of+Neuroscience%22">Encyclopedia of Neuroscience</a>. , (2010).</li>
<li>Hooisma, J., Slaaf, D. W., Meeter, E., Stevens, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Innervation+of+Chick+Striated+Muscle+Fibers+by+the+Chick+Ciliary+Ganglion+in+Tissue+Culture%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">The Innervation of Chick Striated Muscle Fibers by the Chick Ciliary Ganglion in Tissue Culture.</a> Brain Research. 85, 79-85 (1975).</li>
<li>Morrison, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuromuscular+Diseases%5BTitle%5D)+AND+%22Seminars+in+Neurology%22%5BJournal%5D)">Neuromuscular Diseases.</a> Seminars in Neurology. , 409-418 (2016).</li>
<li>Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Trigeminal+System%3A+An+Advantageous+Experimental+Model+for+Studying+Neuronal+Development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The Trigeminal System: An Advantageous Experimental Model for Studying Neuronal Development.</a> Development. 103, 175-183 (1988).</li>
</ol>

The authors declare that they have no competing interests.

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 ganglia at different developmental stages of chick embryonic development can be used as a cell model, depending on the purpose of the study. However, for cultures of CG neurons it is suggested that they be isolated from chick embryo between embryonic days 7 and 818. In the embryonic stage E8, CG neurons have not yet undergone neuronal death processes and the number of non-neuronal cells is reduced comparatively with neuronal cells18. This, in combination with a rigorous dissection procedure and very well cleaned ganglia, will contribute for a highly pure culture of ciliary ganglion neurons, with little contamination by non-neuronal cells, such as fibroblasts or glial cells.
During the isolation of CG neurons, one of the critical points is the identification and the cleaning of the CG. The dissection of such a small structure, as the ciliary ganglion, can be difficult considering the localization, the ability to identify the ganglion as well as the size of the ganglion itself. It is normal that the ganglia might attach to the forceps during dissection. High quality dissection instruments are very important for a successful dissection and will minimize the attachment of the ganglia to the forceps. Cleaning the GC is important to prevent contamination with non-neuronal cells. It is necessary to isolate approximately 70 ganglia to obtain a cellular density of ~1x106 cells/mL, in contrast with other neuronal tissues of the peripheral nervous system that have a 5-15x greater number of ganglia3.
In culture, the addition of 5&#39;-FDU to the complete medium decreases the contamination of the GC culture with non-neuronal cells. 5&#39;-FDU is an anti-mitotic compound that inhibits cell proliferation, namely the proliferation of glial cells and fibroblasts. The concentration of 5&#39;-FDU added to the medium is enough to stop the cell cycle in the S phase but is not detrimental to the normal development of CG neurons3,19,20. The time of treatment with 5&#8217;-FDU can be adjusted. However, since CG neurons establish a dense axonal network in a short time, 5&#39;-FDU should be added to the culture as early as the time of plating.
One of the main limitations of this model is that it is not representative of the normal development of CG neurons under physiological conditions. In ovo, about half of CG neurons die between the 8th and 14th day of chick embryo development. In culture, there is no decrease in the number of CG neurons when the medium is supplemented with neurotrophic factors that allow its survival1,6,14.
The neuronal population obtained from the dissection of the chick ciliary ganglion is a homogenous population of cholinergic neurons, belonging to the autonomic nervous system. It should be noted that the expression of neurotransmitters in the choroid population of the CG is target-driven, which might be hampered depending on the type of muscle used in the co-culture24. If the aim of the study is related to the genetic identity or sub-type of the motor neuron itself, then CG neurons might not be the best suitable neuronal model. Also, the specificity of motor neurons in the innervation of muscle fibers may not be accomplished when using CG neuron co-cultures since, in this case, the muscle fibers can be multiply innervated25. However, this neuronal culture has several advantages, it only requires basic equipment to maintain and incubate the eggs, it is a reasonably inexpensive procedure and, more importantly, provides an excellent model for the study of neuromuscular synapses1, since CG neurons neurotransmission mechanisms are very similar to the ones occurring in spinal motor neurons. The cell models previously used for these type of studies were sensory neurons from the spinal cord12,21,22,23. However, these co-cultures were composed of an heterogeneous population of neurons, not all cholinergic and, thus, only a small part of the neurons were able to establish functional contacts with the muscle cells1. Besides the developmental analysis (immunocytochemistry) demonstrated in this work other assays can be performed in CG cultures like electrophysiology and neuronal survival.
Based on this protocol additional scientific questions can be addressed, for example how subcellular localization of specific mRNAs and proteins regulate synapse formation and function. Moreover, nerve-muscle co-cultures can be easily established and be further used to study neuromuscular diseases when the site of injury is the neuromuscular junction. Neuromuscular diseases are heterogeneous in nature in the sense that the dysfunction might be associated with the muscle itself, the peripheral nerves or the neuromuscular junctions26. Thus, through these co-cultures it would be possible to study the neuromuscular junction alterations that ultimately underlie the development and progression of neuromuscular diseases. Another interesting possibility would be to adapt this protocol to the mouse trigeminal system. These neurons are easily accessible, and their developmental pattern is well-known27. Because mice are amenable to genetic manipulation and the trigeminal system is well characterized in terms of topographic map formation new possibilities arise by using a trigeminal-based protocol to study neuronal development.

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 neurite extending and forming the axon5. In addition, approximately half of CG neurons die between the 8th and 14th 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 neurons6,7,8. 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 weeks1,9.
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 choroid1,10,11,12.
Cultures of chicken ciliary ganglion neurons have been shown to be useful tools for the study of neuromuscular synapses and synapse formation1,5,9. Considering that neuromuscular synapses are cholinergic13, using a neuronal population that is cholinergic &#8211; CG neurons &#8211; 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 interactions14,15.
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.

<table><tbody><tr><td>5-fluoro-2&amp;rsquo;-deoxiuridina (5&#039;-FDU)</td><td>Merck (Sigma Aldrich)</td><td>F0503</td><td></td></tr><tr><td>Alexa Fluor 568-conjugated goat anti-chicken antibody</td><td>Thermo Fisher Scientific</td><td>A11041</td><td></td></tr><tr><td>Alexa Fluor 568-conjugated goat anti-mouse antibody</td><td>Thermo Fisher Scientific</td><td>A11031</td><td></td></tr><tr><td>Alexa Fluor 647-conjugated goat anti-mouse antibody</td><td>Thermo Fisher Scientific</td><td>A21235</td><td></td></tr><tr><td>B27 supplement (50x), serum free</td><td>Invitrogen (Gibco)</td><td>17504-044</td><td></td></tr><tr><td>Chicken monoclonal neurofilament M</td><td>Merck (Sigma Aldrich)</td><td>AB5735</td><td></td></tr><tr><td>D-(+)-Glucose monohydrate</td><td>VWR</td><td>24371.297</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, Brazil</td><td>Invitrogen (Gibco)</td><td>10270-106</td><td></td></tr><tr><td>HEPES, fine white crystals, for molecular biology</td><td>Fisher Scientific</td><td>10397023</td><td></td></tr><tr><td>Horse Serum, heat inactivated, New Zealand origin</td><td>Invitrogen (Gibco)</td><td>26050-070</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Invitrogen (Gibco)</td><td>25030-081</td><td></td></tr><tr><td>Mouse laminin I</td><td>Cultrex (R&amp;amp;D systems)</td><td>3400-010-02</td><td></td></tr><tr><td>Mouse monoclonal b-III tubulin</td><td>Merck (Sigma Aldrich)</td><td>T8578</td><td></td></tr><tr><td>Mouse monoclonal SV2</td><td>DSHB</td><td>AB2315387</td><td></td></tr><tr><td>Multidishes, cell culture treated, BioLite, MW24 (50x)</td><td>Thermo Fisher Scientific</td><td>11874235</td><td></td></tr><tr><td>Neurobasal medium without glutamine</td><td>Invitrogen (Gibco)</td><td>21103-049</td><td></td></tr><tr><td>Penicillin/streptomycin (5,000 U/mL)</td><td>Invitrogen (Gibco)</td><td>15070-063</td><td></td></tr><tr><td>Phenol red, bioreagent, suitable for cell culture</td><td>Merck (Sigma Aldrich)</td><td>P3532</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Merck (Sigma Aldrich)</td><td>P7886</td><td></td></tr><tr><td>Potassium chloride</td><td>Fluka (Honeywell Reaarch Chemicals)</td><td>31248-1KG</td><td></td></tr><tr><td>Potassium di-hydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;) for analysis, ACS</td><td>Panreac Applichem</td><td>131509-1000</td><td></td></tr><tr><td>Prolong Gold Antifade mounting medium with DAPI</td><td>Invitrogen (Gibco)</td><td>P36935</td><td></td></tr><tr><td>Puradisc FP 30mm Syringe Filter, Cellulose Acetate, 0.2&amp;micro;m, sterile 50/pk</td><td>Fisher Scientific</td><td>10462200</td><td></td></tr><tr><td>Recombinant human ciliary neurotrophic factor (CNTF)</td><td>Peprotech</td><td>450-13</td><td></td></tr><tr><td>Recombinant human glial cell-derived neurotrophic factor (GDNF)</td><td>Peprotech</td><td>450-10</td><td></td></tr><tr><td>Sodium chloride for analysis, ACS, ISO</td><td>Panreac Applichem</td><td>131659-1000</td><td></td></tr><tr><td>Sodium dihydrogen phosphate 2-hydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O), pure, pharma grade</td><td>Panreac Applichem</td><td>141677-1000</td><td></td></tr><tr><td>Sodium Pyruvate 100 mM (100x)</td><td>Thermo Fisher</td><td>11360039</td><td></td></tr><tr><td>Syringe without needle, 10 mL</td><td>Thermo Fisher</td><td>11587292</td><td></td></tr><tr><td>Trypsin 1:250 powder</td><td>Invitrogen (Gibco)</td><td>27250-018</td><td></td></tr></tbody></table>

isolation and culture of chick ciliary ganglion neurons

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.

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 identification of the ciliary ganglion can be difficult, especially when performing this protocol for the first time. The ciliary ganglion is localized near the optic nerve and the choroid fissure (Figure 1B). The key steps of the dissection procedure are shown in Figure 2. First, the embryo is removed from the egg and placed in ice-cold HBSS. The head is separated from the body and, once again, placed in ice-cold HBSS in a dissection Petri dish (Figure 2A-2C). Then, the eye is removed from the head of the chick and the ciliary ganglion is isolated (Figure 2D-2H).
The cultures obtained with this protocol are highly pure. However, cleaning the ganglia and removing the excess tissue strongly dictates the success and purity of the culture. The cells develop fast and can be used already in the first days in culture if the overall experiment requires so. Nevertheless, the cultures can be maintained for 15 days, or more. If using the cultures for longer than 7-8 days, make sure to replace a third of the culture medium with fresh medium every 2-3 days. After 1 day in vitro, CG neurons show a multipolar morphology. However, neurite extension occurs rapidly, and a primary neuronal network is already established after 24 hours. After 8 days in vitro, neurons already transitioned to a unipolar state, where one of the neurites extends and forms the axon. The neuronal network is very dense at this stage of development (Figure 3 and Figure 4).
Ciliary ganglion neurons are cholinergic neurons that belong to the parasympathetic nervous system. In vivo, these neurons are responsible for muscle innervation in the eye. These neuronal cultures are very well suited for the study of neuromuscular synapses. For this, CG neurons can be plated on top of muscle cells. The chick pectoral muscle was dissected and allowed to develop and maturate in vitro until DIV 4. CG neurons were then plated on top of the muscle layer and the co-culture allowed to develop for 3 more days. At this time point, muscle fibers are formed and can be easily identified by the presence of multiple nuclei (blue). Synaptic vesicle glycoprotein 2A (SV2) immunostaining, a presynaptic marker shows the presence of synapses that are established between the CG neurons axons and the muscle fibers (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61431/61431fig1.jpg"> 
Figure 1: Scheme of the dissection protocol and the ciliary ganglion. (A) Diagram of the isolation and culture protocol. (B) Scheme of the chick ciliary ganglion localization in the posterior part of the eye. Optic nerve, ciliary ganglion and choroid fissure are indicated by arrows. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61431/61431fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Embryo dissection sequence; optic nerve isolation; labeled optic structures; microscopy analysis." class="xfigimg" src="/files/ftp_upload/61431/61431fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Dissection of E7 chick ciliary ganglion. (A) Cut the top of the egg using scissors. (B) Remove the embryo from the egg with a spoon and place it in a dissection Petri dish with ice-cold HBSS. (C) Separate the head from the body by cutting in the neck region. (D) Fix the head of the embryo in the beak, holding with forcep nº 5. (E) Remove the eye by gentle rotation using forcep nº 55. (F) Posterior view of the eye. Arrows indicate the localization of the optic nerve, choroid fissure and ciliary ganglion. (G) Dissect the ciliary ganglion. (H) Dissected ciliary ganglion. Excess tissue should be removed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61431/61431fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Time-lapse microscopy of neuronal culture development; DIV-1, DIV-3, DIV-8, DIV-15." class="xfigimg" src="/files/ftp_upload/61431/61431fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Ciliary ganglion neurons development in vitro. Phase contrast images of CG neurons at DIV 1, 3, 8 and 15. As CG neurons are plated, they immediately initiate neurite outgrowth. At DIV 15, the axonal network is very dense and at this stage neurites are completely differentiated into dendrites and axons. Phase contrast-images were acquired using a confocal microscope with a plan-Apochromat 20x ph2 objective. Scale bar: 50 µm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61431/61431fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="β-III Tubulin and DAPI staining, neuron fluorescence microscopy, cell structure visualization comparison." class="xfigimg" src="/files/ftp_upload/61431/61431fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Immunocytochemistry of CG neurons at DIV 8. CG neurons show a well-established neuronal network after 8 days in vitro. Nuclei were stained with DAPI (blue) and axons were stained with b-III tubulin (red). Fluorescence imagens were acquired using a confocal microscope with a plan-Apochromat 20x objective. Scale bar: 50 µm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61431/61431fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, DAPI-labeled nuclei, NF and SV2 staining, merged cell structure diagram." class="xfigimg" src="/files/ftp_upload/61431/61431fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Cultured CG neurons establish synapses with muscle fibers. Immunocytochemistry images of CG neurons-pectoral muscle co-cultures. Muscle fibers identified by dashed lines present multiple nuclei, which were stained with DAPI (blue). Axons were labeled against neurofilament (red) and synaptic vesicles were labeled against SV2 (cyan). Images were acquired using a confocal microscope with a plan-Apochromat 63x oil objective. Scale bar: 20 µm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61431/61431fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Culture of Chick Ciliary Ganglion Neurons at JoVE.com

Isolation and Culture of Chick Ciliary Ganglion Neurons

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 ...

isolation-and-culture-of-chick-ciliary-ganglion-neurons

Universidad San Sebastian

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Neuroscience

5.7K Views.  University of Aveiro. 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.

Video: Isolation and Culture of Chick Ciliary Ganglion Neurons

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Developmental Biology

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Biology

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Isolation of Cells from Sensory Ganglia

Source: Kaelberer, M. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53917">A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.</a> J. Vis. Exp. (2016).
This video demonstrates the isolation of cells from the sensory ganglia of mice. Ganglia are subjected to enzymatic digestion to dissolve the extracellular matrix. Single cells are then collected via filtering through a cell strainer followed by density gradient centrifugation for debris removal. Finally, washing and centrifugation are done to collect the isolated cells for future analysis.

All procedures involving sample collection have been performed in accordance with the institute&#8217;s IRB guidelines.
1. Sensory Ganglia Dissociation

	...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Source: Jacques-Fricke, B. T., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63799">Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.</a> J. Vis. Exp. (2022).
This video illustrates a technique for isolating cranial neural folds from the midbrain region of chick embryos. It presents a detailed dissection process of the neural folds, their placement, and attachment on fibronectin-coated coverslips, followed by the observation of neural crest cell migration from the neural folds.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Advanced Neuronal Models

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Begin by harvesting Islet-1 EGF-positive embryos from a pregnant mouse approximately 11.5 days post-fertilization. Spray the abdomen thoroughly with ethanol, and remove the uterus with sterile microdissection scissors and a thumb-dressing forceps. Briefly wash the uterus in sterile PBS. Then, transfer it to the dissection plate filled with pre-chilled sterile PBS.
Under the bright light of the microscope, carefully remove the embryos from the uterus using microdissection scissors, thumb-dressing forceps, and Dumont Number 5 tweezers. Then, use a sterile Moria mini-perforated Spoon to transfer each embryo to a separate well of a 24-well plate filled with pre-chilled Hibernate E low fluorescence medium supplemented with 1X V27.
While keeping the plate on ice, transfer one embryo to a sterile dissection plate, and cover it completely with ice-cold sterile Hank&#39;s Balanced Salt Solution or HBSS. Use tweezers to remove the face of the embryo and the tail without damaging the midbrain. Then, place the embryo prone with limbs straddled and tail pointing toward the front of the microscope.
Use tweezers to slit open the roof of the fourth ventricle in order to generate a small opening. Use this opening to hook tweezers into the space created between the fourth ventricle and its roof. Dissect along the dorsal surface of the embryo rostral to the cortex and lateral to the floor plate and motor column.
Then, open the dissected tissue in an open-book manner to reveal the GFP-positive CN3 and CN4 nuclei. Carefully separate the ventral midbrain from the embryo and remove meningeal tissue with tweezers and a microdissection knife.
Dissect the bilateral GFP-positive CN3 and CN4 nuclei away from the floor plate and other GFP-positive surrounding tissue, taking care to avoid touching or damaging the neurons. If collecting separate CN3 and CN4 nuclei, cut along the midbrain of these two nuclei, and use a P1000 pipette to collect the dissected ventral midbrain tissue with minimal HBSS.
Place it in a labeled 1.7-milliliter microcentrifuge tube filled with dissection medium. Store the tube on ice until dissociation. Continue pooling ventral midbrains from additional embryos in the same tube until the total number meets experimental requirements.
To dissect the ventral spinal cord, keep the embryo prone with the head facing the front of the microscope. Hold it with one pair of tweezers and insert the tip of the other pair into the unopened caudal part of the fourth ventricle. Open the rest of the hindbrain and spinal cord dorsally over the whole rostrocaudal extent of the embryo. Cut the dorsal tissue starting from the fourth ventricle and working toward the central canal of the caudal spinal cord, using the forceps as scissors.
Then, hold the embryo with one set of tweezers and pinch off the flap of the dorsal tissue on each side with the other pair. Remove the ventral spinal cord by using the microdissection knife to pierce directly below the GFP-positive SMN, lifting the ventral spinal cord with saw-like movements on both sides.
Cut the spinal cord transversely at the upper boundary of the lower limb, and remove the cervical lumbar portion. Cut transversely directly above C1 where the first GFP-positive anterior horn projects. Place the ventral spinal cord dorsal side up and hold it by pressing the GFP-negative tissue between the GFP-positive SMN columns with a pair of tweezers.
Remove the remaining attached mesenchyme, DRGs, and dorsal spinal cord by trimming both sides of the GFP-positive SMN column with the microdissection knife. Use a P1000 pipette to collect the dissected ventral spinal cord tissue with minimal HBSS, and place it in a labeled 1.7-milliliter microcentrifuge tube filled with dissection medium. Store it on ice until dissociation and continue pooling ventral spinal cords from additional embryos in the same tube.
Add the appropriate volume of papain solution to each of the microcentrifuge tubes with the dissected tissue samples. Incubate the tubes at 37 degrees Celsius for 30 minutes, agitating the tubes every 10 minutes by finger flicking.
After the incubation, gently triturate each suspension eight times with a P200 pipette. Centrifuge it at 300 times g for five minutes. Resuspend the cell pellets with the appropriate volume of albumin ovomucoid inhibitor solution by gently pipetting up and down. Repeat the centrifugation. Then, carefully remove the supernatant with a P1000 pipette. Resuspend the cells in the appropriate volume of dissection medium. Filter the suspensions through 70-micrometer cell strainers.
Next, use FACS sorting to isolate GFP-positive cells dissected from CN3, CN4, and SMN. Dilute the isolated cell suspensions with motor neuron culture medium pre-warmed to 37 degrees Celsius to the appropriate densities, and add 200 microliters of the suspension to a well of a PDL-laminin-coated 96-well plate. Culture the neurons in a 37 degrees Celsius and 5% carbon dioxide incubator, making sure to refresh the motor neuron culture medium every five days.

Isolation and Culture of Chick Ciliary Ganglion Neurons

Summary

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