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>

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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 ga...

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 border="0" cellpadding="0" cellspacing="0" style="width:1132px;" width="1131">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">5-fluoro-2&#8217;-deoxiuridina (5&#39;-FDU)</td>
			<td style="width:258px;">Merck (Sigma Aldrich)</td>
			<td style="width:119px;">F0503</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568-conjugated goat anti-chicken antibody</td>
			<td style="width:258px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A11041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568-conjugated goat anti-mouse antibody</td>
			<td style="width:258px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A11031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647-conjugated goat anti-mouse antibody</td>
			<td style="width:258px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A21235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">B27 supplement (50x), serum free</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chicken monoclonal neurofilament M</td>
			<td style="width:258px;">Merck (Sigma Aldrich)</td>
			<td>AB5735</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">D-(+)-Glucose monohydrate</td>
			<td style="width:258px;">VWR</td>
			<td style="width:119px;">24371.297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Fetal Bovine Serum (FBS), qualified, Brazil</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, fine white crystals, for molecular biology</td>
			<td style="width:258px;">Fisher Scientific</td>
			<td>10397023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Horse Serum, heat inactivated, New Zealand origin</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">26050-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">L-Glutamine (200 mM)</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Mouse laminin I</td>
			<td style="width:258px;">Cultrex (R&#38;D systems)</td>
			<td style="width:119px;">3400-010-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonal b-III tubulin</td>
			<td style="width:258px;">Merck (Sigma Aldrich)</td>
			<td>T8578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonal SV2</td>
			<td style="width:258px;">DSHB</td>
			<td>AB2315387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Multidishes, cell culture treated, BioLite, MW24 (50x)</td>
			<td style="width:258px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11874235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Neurobasal medium without glutamine</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Penicillin/streptomycin (5,000 U/mL)</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red, bioreagent, suitable for cell culture</td>
			<td style="width:258px;">Merck (Sigma Aldrich)</td>
			<td style="width:119px;">P3532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Poly-D-Lysine</td>
			<td style="width:258px;">Merck (Sigma Aldrich)</td>
			<td style="width:119px;">P7886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Potassium chloride</td>
			<td style="width:258px;">Fluka (Honeywell Reaarch Chemicals)</td>
			<td>31248-1KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:24px;">Potassium di-hydrogen phosphate (KH2PO4) for analysis, ACS</td>
			<td style="width:258px;">Panreac Applichem</td>
			<td>131509-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold Antifade mounting medium with DAPI</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puradisc FP 30mm Syringe Filter, Cellulose Acetate, 0.2&#181;m, sterile 50/pk</td>
			<td style="width:258px;">Fisher Scientific</td>
			<td style="width:119px;">10462200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Recombinant human ciliary neurotrophic factor (CNTF)</td>
			<td style="width:258px;">Peprotech</td>
			<td style="width:119px;">450-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Recombinant human glial cell-derived neurotrophic factor (GDNF)</td>
			<td style="width:258px;">Peprotech</td>
			<td style="width:119px;">450-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride for analysis, ACS, ISO</td>
			<td style="width:258px;">Panreac Applichem</td>
			<td>131659-1000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:24px;">Sodium dihydrogen phosphate 2-hydrate (Na2HPO4&#183;2H2O), pure, pharma grade</td>
			<td style="width:258px;">Panreac Applichem</td>
			<td>141677-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate 100 mM (100x)</td>
			<td style="width:258px;">Thermo Fisher</td>
			<td>11360039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Syringe without needle, 10 mL</td>
			<td style="width:258px;">Thermo Fisher</td>
			<td style="width:119px;">11587292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:574px;">Trypsin 1:250 powder</td>
			<td style="width:258px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">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 identifi...

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

Research

JoVE Journal

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...