This work was supported by the Faculty Development Fund and Summer Research Program grant at Saint Mary&#8217;s College of California. The authors would also like to thank Dr. Pamela Lein at University of California at Davis and Dr. Anthony Iavarone at UC Berkeley Mass spectrometry facility for their advice during the development of these protocols. The authors would also like to thank Haley Nelson in the Office of College Communications at Saint Mary&#8217;s College of California for her help with video production and editing.

All procedures performed in studies involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Saint Mary&#8217;s College of California. The animal care and use guidelines at Saint Mary&#8217;s College were developed based on the guidelines provided by Office of Laboratory Animal Welfare at the National Institute for Health (https://olaw.nih.gov/sites/default/files/PHSPolicyLabAnimals.pdf and https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf).
1. Preparation of culture media (also referred to as control medium)
<ol>
	<li>Add the following ingredients, in the order listed below, to a sterile 500 mL flask: 190 mL of 1x low-glucose DMEM, 10 mL of 20 mg/mL fatty acid free bovine serum albumin(FAF-BSA) in DMEM, 2.8 mL of 200 mM L-glutamine, 4 mL of 100x insulin-selenium-transferrin mixture, 0.4 mL of 125 &#181;g/mL nerve growth factor (in 0.2% inert protein stabilizer in DMEM), and 200 mL of Ham&#8217;s F-12 medium.</li>
	<li>Make sure to coat the pipette with the medium containing FAF-BSA prior to the addition of protein-containing solutions such as insulin-selenium-transferrin and NGF to prevent sticking of proteins to the pipette.</li>
	<li>Swirl the flask to mix the ingredients after each addition. Mix thoroughly with a 25 mL pipette after addition of F-12.</li>
	<li>Aliquot the culture media in 10 mL aliquots and store at -80 &#176;C. The aliquots can be stored for six months without loss of activity.</li>
</ol>
2. Preparation of plates for culturing neurons
<ol>
	<li>Dilute a 1 mg/mL stock of poly-D-lysine (made in 0.5 M Tris buffer, pH 8) to 100 &#181;g/mL with sterile distilled water.</li>
	<li>For proteomic or genomic analysis, one to two days before the dissection, coat 6-well plates with approximately 2 mL of sterile 100 g/mL of poly-D-lysine (PDL). This is necessary to ensure cell adhesion to the well. Wrap the plates with cling film and store the plates overnight at 4 &#176;C.</li>
	<li>For morphological analysis and microscopy, place 12 mm2 pre-treated German glass coverslips (which can be cleaned using nitric acid treatment as described previously18 or purchased) into each of the wells of a 24-well plate.
	<ol>
		<li>Coat the coverslips with 0.3 mL of sterile 100 g/mL of poly-D-lysine (PDL). Store the plates overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>On the day of the dissection, before the start of the dissection, remove the poly-D-lysine solution from the wells and rinse the wells five times with sterile distilled water, followed by once with low glucose DMEM.</li>
	<li>During the enzymatic digestion of the ganglia (approximately an hour before plating the cells), aspirate the DMEM from the plates and replace it with 0.3 mL of control media. Store the plates at 35.5 &#176;C under 5% CO2 in a humidified chamber.</li>
</ol>
3. Dissection setup
<ol>
	<li>Preparation of 200 mL of media for dissection (referred to as Dissection media)
	<ol>
		<li>Add 8 mL of 20 mg/mL fatty acid-free bovine serum albumin (FAF-BSA) in low glucose DMEM (with 1 mg/mL glucose) and 2 mL of 100x penicillin-streptomycin to 193 mL of sterile Leibovitz&#39;s L-15 medium (or any air-buffered medium)</li>
	</ol>
	</li>
	<li>In the hood, set up the following items for dissection: four 50 mL sterile conical tubes with about 20 mL of dissection media in each tube, four sterile 35 mm dishes with 1.5 mL of dissection media, one sterile 50 mL conical tube with 20 mL of dissection media for centrifugation, one sterile 15 mL conical tube for centrifuging the ganglia, and one sterile 10 mL tube for collecting the dissociated cells.</li>
	<li>Use a dry bead sterilizer to sterilize a pair of fine forceps (no.4 or no. 5 forceps) for at least one minute.</li>
</ol>
4. Isolation of the superior cervical ganglia from embryonic rat pups
<ol>
	<li>Removal of E21 embryos from the pregnant rat 
	NOTE: Removal of the uterine horn can be performed outside of the hood if the surrounding area is thoroughly sterilized.
	<ol>
		<li>Euthanize the pregnant rat using CO2 inhalation. Shear the fur from the abdominal region and wipe the skin in the area with 70% alcohol to sterilize it.</li>
		<li>Using a fresh set of sterile scissors and forceps, cut through the skin and then the muscle layer to expose the uterine horns containing the embryos. Remove the uterine horns with the embryos using a new set of scissors and forceps, taking care not to damage the intestines in the process.</li>
		<li>Transfer the uterine horns with the embryos into a 150 mm2 sterile Petri dish and transfer them into the hood.</li>
		<li>Using a new set of forceps and scissors, remove the embryos from the uterine horn and separate the embryos from the amniotic membranes and the placenta.</li>
		<li>To euthanize the embryos, cut the spinal cord of the embryos along the midline under the right arm. This will also reduce the bleeding from the carotid artery during the removal of the SCG.</li>
		<li>Transfer these embryos into the prepared 50 mL conical tubes containing dissection media. Make sure the embryos are submerged in the media. Each tube can hold up to 3 embryos.</li>
	</ol>
	</li>
	<li>Isolation of the superior cervical ganglion from the embryo
	<ol>
		<li>Transfer one pup from the dissection media onto a sterile 150 mm2 Petri dish half-filled with solid substrate (either paraffin wax or silicone polymer), with its dorsal surface on the substrate. Using three sterile 23 G needles, pin the pup to the dish with one needle under each arm and a third needle through the mouth to carefully hyperextend the neck.</li>
		<li>Cut through the skin in the neck region using sterile fine forceps (no. 4 or no. 5 forceps) to expose the salivary glands underneath. Remove these glands using fine forceps.</li>
		<li>Locate the sternocleidomastoid and omohyoid muscles near the clavicle and trachea, respectively. First cut the transverse sternocleidomastoid muscles and then carefully cut the thin omohyoid muscle using fine forceps. Once these muscles are removed, the bifurcation in the carotid artery on the anterior end will be visible on either side of the trachea, with the SCG located under this fork in the carotid artery.</li>
		<li>Using closed forceps, gently lift the carotid artery to visualize the SCG. Using one forceps on either side of the SCG, pull out the carotid and transfer it to the prepared sterile 35 mm2 dishes. This tissue will most likely contain the SCG with the carotid artery, the vagus nerve with the nodose ganglia as well as other segments of muscle or fat in the area.</li>
		<li>Repeat the dissection process on the other side. Remove SCGs from all embryos before continuing with the remaining dissection steps outlined below. Distribute the isolated tissues between two of the 35 mm2 dishes to facilitate processing of the tissue samples.</li>
	</ol>
	</li>
	<li>Post-processing of SCG
	<ol>
		<li>To separate the SCG from the dissected tissue, first use fine forceps to remove any extraneous tissue, such as fat or muscle, taking care to avoid the area near the carotid bifurcation.</li>
		<li>Once these tissues have been removed, two ganglia are visible. The nodose ganglion is smaller and circular, while the SCG is almond-shaped. Gently pull on the vagus nerve to separate the vagus nerve and nodose ganglia from the carotid and then separate the SCG from the carotid artery.</li>
		<li>Use fine forceps to remove the capsule that surrounds the SCG. Transfer the SCG to a new 35 mm culture dish. Repeat the process with all the dissected tissue samples.</li>
		<li>Coat a sterilized, cotton plugged glass pipette with dissection media to prevent the tissue from adhering to the pipette walls. Use the pipette to replace the dissection media with sterile 2 mL of Collagenase type II (1 mg/mL)/dispase type II (5 mg/mL) in calcium- and magnesium-free HBSS, and incubate for 50 min at 37 &#176;C to help break down the tissues. 
		NOTE: The incubation time may need to be optimized with different batches of Collagenase/Dispase and usually ranges from 40 min to an hour.</li>
		<li>During the incubation, aspirate the DMEM from the plates and replace it with 0.3 mL of control media. Store the plates at 35.5 &#176;C under 5% CO2 in a humidified chamber.</li>
		<li>Following the incubation, transfer the SCGs in collagenase/dispase to a sterile 15 mL conical tube. Use the dissection media to rinse the plates and transfer the solution to the tube. Add enough dissection media to bring up the volume to approximately 10 mL.</li>
		<li>Centrifuge at 200 x g (1000 - 1200 rpm) for 5 minutes at room temperature to pellet the sample. Aspirate the supernatant, taking care to not dislodge the pellet. Resuspend the pellet with 10 mL of dissection media. Repeat the centrifugation and discard the supernatant.</li>
		<li>Replace with 1 - 2 mL of culture medium. Using a narrow-bore, bent-tip sterile pasteur pipette (pre-coated with culture medium), mechanically dissociate the clumps by gently triturating 5-6 times. Let the larger clumps settle for about one minute. Transfer the supernatant cell suspension to a new 10 mL tube.</li>
		<li>Repeat this process 3 more times, with increasing force of trituration each time to ensure almost complete dissociation of the SCGs. Transfer the supernatant after each round of trituration to a 10 mL tube with the supernatant from the first trituration.</li>
		<li>Add enough culture media to bring up the volume to 8 - 10 mL. Gently mix the cell suspension and quantify the cell density with a hemocytometer.</li>
		<li>Distribute the cell suspension into the wells at the appropriate cell density for the experiments. Mix the cell suspension continually during the plating process to ensure even distribution of cells into the wells. 
		NOTE: For morphological analysis, plate the cells around 8,000 cells/well in a 24 well plates and for genomic and proteomic protocols, plate the cells as high as 30,000 cells/well.</li>
		<li>Transfer the plates to a glass desiccator with sterile water at the bottom to create a humidified chamber. Inject enough CO2 (around 120 mL) to obtain a 5% CO2 environment in the desiccator, prior to sealing. Maintain the plates at 35.5 &#176;C. This is referred to as Day 0 in the protocol. 
		NOTE: These plates can also be maintained in a regular 5% CO2 incubator. The method described above minimizes temperature and pH changes and also helps prevent cross-contamination.</li>
	</ol>
	</li>
</ol>
5. Maintenance of the cultured SCG neurons and treatments
<ol>
	<li>On Day 1 (24 hours after plating), carefully remove half of the culture media, and replace with 2 &#181;M Ara-C (cytosine &#946;-D-arabinofuranoside, an anti-mitotic agent). Leave the treatment on the cells for 48 h. Usually, this length of treatment is sufficient to eliminate non-neuronal cells in the culture.</li>
	<li>On Day 3, gently aspirate half the medium and replace with control medium.</li>
	<li>On Day 4, the cells are ready for experimental treatments. Feed cultures every other day with the appropriate medium, gently replacing half of the medium in the well with fresh medium.</li>
	<li>Cultured SCG neurons in serum-free medium extend only axons. If experiments require the presence of dendrites, treat cells with either 10% fetal calf serum, 75-100 &#181;g/mL of basement membrane matrix extract or 50 ng/mL of bone morphogenetic protein-7. Dendritic growth is observed within 48 hours of treatment with elaborate dendritic arbor observed within a week of treatment.</li>
</ol>
6. Immunostaining cultured SCG neurons
<ol>
	<li>Gradually replace the cell culture medium in the well with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 in a fume hood.
	<ol>
		<li>Remove half the culture media in the well and replace it gently with 4% paraformaldehyde. Then, repeat the process at least two more times, removing two-thirds of the media in the well each time and replacing with 4% paraformaldehyde. At the end of this process, the color of media in the well should have changed from pink to colorless.</li>
		<li>Once all the medium has been replaced by 4% paraformaldehyde, incubate the wells for 15 - 20 minutes at room temperature.</li>
	</ol>
	</li>
	<li>With a similar process as described above, gradually replace the 4% paraformaldehyde solution with phosphate-buffered saline (PBS), pH 7.2. Leave for 5 minutes. Rinse the wells two more times for 5 minutes each with PBS.
	<ol>
		<li>Once the 4% paraformaldehyde is removed, move the plates to the benchtop for further processing. This step is a good stopping point where plates can be stored at 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Remove all the PBS. Add enough volume of 0.1% Triton X-100 in PBS to cover the cells. Leave it on the cultures for 5 minutes to permeabilize the neurons. The timing of this step is critical to ensure good staining while maintaining the integrity of the cells.</li>
	<li>Carefully remove the Triton X-100 solution and rinse the wells three times for 5 minutes each time with PBS. Remove the PBS solution.</li>
	<li>Add enough 5% BSA in PBS to cover the cells and incubate at room temperature for 20 minutes to reduce non-specific antibody binding.</li>
	<li>Remove the blocking solution and replace it with a mouse monoclonal antibody against MAP-2 protein diluted in 5% BSA in PBS. Leave the primary antibody on the cells overnight at 4 &#176;C.</li>
	<li>Remove and save the primary antibody for reuse. The primary antibody can be reused at least twice without loss of detection intensity.</li>
	<li>Rinse three times with enough PBS to cover the cells. Leave PBS on the cells for 10 minutes per rinse.</li>
	<li>Remove the PBS solution and replace it with fluorescent tag-conjugated Goat anti-Mouse IgG secondary antibody at 1:1000 dilution in 5% BSA in PBS. Incubate for 2 hours in the dark at room temperature.</li>
	<li>Remove the secondary antibody and replace it with PBS. Rinse the wells three times with PBS for 10 minutes per rinse. Rinse the wells one time with water to remove any salts.</li>
	<li>Mount the coverslips onto glass slides containing a drop of aqueous mountant that is appropriate for fluorescently labeled samples. Store the slides at 4 &#176;C, in a slide folder until ready for imaging.</li>
</ol>
7. Sample preparation for analysis of the proteome using liquid chromatography coupled with mass spectrometry
<ol>
	<li>Lysis of cultured neurons
	<ol>
		<li>Gently remove all the culture media in the wells. Replace it with cold, sterile calcium and magnesium-free phosphate-buffered saline (PBS), pH 7.4. Remove quickly and replace with PBS and let it sit for 5 min. This step is done to remove any proteins present in the culture media.</li>
		<li>Carefully remove all the PBS from all the wells for a particular treatment. Repeat the process one more time. Maintain plates over ice when lysing the cells to minimize neuronal damage and proteolysis.</li>
		<li>Add 100 - 150 &#181;L of 50 mM ammonium bicarbonate, pH 7.5 (NH4HCO3) to one of the wells and scrape cells using a sterile cell scraper. Using a micropipette, transfer the liquid and repeat the scraping process with all the wells for a particular treatment. Examine the wells under the microscope after scraping to make sure that most of the cells have been removed.
		<ol>
			<li>With the low number of neurons, use a limited volume to lyse the cells to ensure high enough concentration for proteomic analysis.</li>
		</ol>
		</li>
		<li>Freeze the solution at -80 &#176;C overnight to help with cell lysis. The lysates can be stored at this stage until ready for further processing.</li>
		<li>Once thawed, squirt the samples through a syringe with a 26 G or 28 G needle to mechanically lyse the cells.</li>
		<li>Sonicate the samples in a sonicating water bath two times for 10 minutes. Add ice to the water bath to prevent overheating and denaturation of proteins. Centrifuge for 5 minutes at 4 &#176;C at 12,000 x g.</li>
		<li>Measure the protein concentration. Typical protein concentrations range from 0.4 - 1 &#181;g/&#181;L</li>
	</ol>
	</li>
	<li>Sample preparation and trypsin digestion for proteome analysis
	<ol>
		<li>Transfer a maximum of 60 &#181;L of the lysate or 50 &#181;g of protein into a DNase-, RNase- and protease-free 1.5 mL tube. If the volume of the lysate is less than 60 &#181;L, add enough 50 mM ammonium bicarbonate to make up the volume.</li>
		<li>Add 25 &#181;L of 0.2% acid labile surfactant and vortex. Incubate the tube in a block heater at 80 &#176;C for 15 minutes. Centrifuge the tube at 12,000 x g for 30 s.</li>
		<li>Add 2.5 &#181;L of 100 mM dithiothreitol and vortex. This makes the protein more accessible for alkylation and digestion. Incubate the tube at 60 &#176;C in a block heater for 30 minutes. Cool to room temperature and centrifuge.</li>
		<li>Add 2.5 &#181;L of 300 mM iodoacetamide to the sample and vortex. This step helps to alkylate the cysteines and prevents them from reforming the disulfide bonds. Incubate the tubes at room temperature in the dark for 30 minutes.</li>
		<li>Add mass spectrometry grade trypsin (0.5 &#181;g/&#181;L) to the tube at a trypsin: protein ratio of 1:10. Digest the samples at 37 &#176;C overnight.</li>
		<li>Add 10 &#181;L of 5% trifluoroacetic acid (TFA) and vortex. Incubate the samples at 37 &#176;C for 90 minutes. This step is necessary to hydrolyze the acid labile surfactant to prevent interference during mass spectrometry.</li>
		<li>Centrifuge the samples at 12,000 x g at 4 &#176;C for 30 minutes and transfer supernatant to a chromatography certified clear glass vial with pre slit Teflon/Silicone septum caps. Samples can be stored at -20 &#176;C prior to mass spectrometry analysis.</li>
		<li>Subject the sample vials to liquid chromatography coupled with high definition mass spectrometry.</li>
	</ol>
	</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP7-induced+dendritic+growth+in+sympathetic+neurons+requires+p75+NTR+signaling.">BMP7-induced dendritic growth in sympathetic neurons requires p75 NTR signaling.</a> Developmental Neurobiology. 76 (9), 1003-1013 (2016).</li><li>Lein, P. J., Fryer, A. D., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Culture:+Autonomic+and+Enteric+Neurons.">Cell Culture: Autonomic and Enteric Neurons.</a> Encyclopedia of Neuroscience. , 625-632 (2009).</li><li>Neto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+Microfluidic+Platforms:+The+Unrivaled+Breakthrough+of+In+vitro+Tools+for+Neurobiological+Research.">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In vitro Tools for Neurobiological Research.</a> Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Research Notes. 9 (1), 82 (2016).</li><li>Conrad, R., Jablonka, S., Sczepan, T., Sendtner, M., Wiese, S., Klausmeyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-based+isolation+and+culture+of+mouse+embryonic+motoneurons.">Lectin-based isolation and culture of mouse embryonic motoneurons.</a> Journal of Visualized Experiments. (55), e3200 (2011).</li><li>Takeuchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+device+for+co-culture+of+sympathetic+neuron+and+iPS-derived+cardiomyocytes.">Microfabricated device for co-culture of sympathetic neuron and iPS-derived cardiomyocytes.</a> Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS. 2013, 3817-3820 (2013).</li><li>Takeuchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+semi-separated+co-culture+system+of+sympathetic+neuron+and+cardiomyocyte.">Development of semi-separated co-culture system of sympathetic neuron and cardiomyocyte.</a> Proceedings of the 31st Annual International Conference of the IEEE Engineering in Medicine and Biology Society: Engineering the Future of Biomedicine, EMBC 2009. , 1832-1835 (2009).</li><li>Chandrasekaran, V., Lea, C., Sosa, J. C., Higgins, D., Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+are+involved+in+BMP-induced+dendritic+growth+in+cultured+rat+sympathetic+neurons.">Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons.</a> Molecular and Cellular Neuroscience. 67, (2015).</li><li>Kim, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statins+decrease+dendritic+arborization+in+rat+sympathetic+neurons+by+blocking+RhoA+activation.">Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation.</a> Journal of Neurochemistry. 108 (4), 1057-1071 (2009).</li><li>Dalby, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+transfection+with+Lipofectamine+2000+reagent:+Primary+neurons,+siRNA,+and+high-throughput+applications.">Advanced transfection with Lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications.</a> Methods. 33 (2), 95-103 (2004).</li><li>Szpara, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+during+neurite+outgrowth+and+regeneration.">Analysis of gene expression during neurite outgrowth and regeneration.</a> BMC Neuroscience. 8 (1), 100 (2007).</li><li>Pop, C., Mogosan, C., Loghin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+rapigest+efficacy+for+the+digestion+of+proteins+from+cell+cultures+and+heart+tissue.">Evaluation of rapigest efficacy for the digestion of proteins from cell cultures and heart tissue.</a> Clujul Medical. 87 (4), 5 (2014).</li><li>Vit, O., Petrak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integral+membrane+proteins+in+proteomics.+How+to+break+open+the+black+box.">Integral membrane proteins in proteomics. How to break open the black box.</a> Journal of Proteomics. 153, 8-20 (2017).</li></ol>

The authors have nothing to disclose.

This paper describes the protocols for culturing sympathetic neurons from superior cervical ganglia of embryonic rat pups. The advantages of using this model system are that it is possible to obtain a homogeneous population of neurons providing a similar response to growth factors, and since the growth factor requirements for these neurons has been well -characterized, it is possible to grow these neurons in vitro in defined media, under serum-free conditions10. Although the protocol describes the process for isolating SCG from E21 rat pups, this protocol can be used for dissection of SCG from rat pups from E17 to E21 and for dissection of embryonic mouse SCG10,22,23. This dissection protocol can also be used for isolating SCG from postnatal rats with minor modification to the initial steps. These modifications include elimination of need for C-section, euthanasia of the postnatal animals using carbon dioxide, and sterilization of the postnatal pups following euthanasia by immersion of the pups in 70% alcohol, prior to transferring them to dissection media10. This neuronal culturing protocol can also be adapted for studying axonal guidance, axonal transport and synapse formation using Campenot chambers and microfluidic chambers24,25. These neurons can also be co-cultured with neurons from the dorsal root ganglia or spinal motor neurons or with cardiomyocytes to study the neuronal interactions and neuron &#8211; target interactions in the peripheral nervous system26,27,28,29.
The use of cultured SCG neurons for immunocytochemical studies is well documented. The protocol described in the paper provides a good starting point for working with most antibodies. However, there are antibodies, such as those that detect nuclear proteins which may require a modification to the fixation protocol and permeabilization protocol. In the case of phospho-SMAD antibody, treatment with 100% methanol at -20 &#176;C has been used for fixing and permeabilizing the neurons30,31.
The main limitations of working in this model system stem from the small size of SCGs in embryonic rat pups, which results in small number of neurons. This results in a limited amount of nucleic acids or protein that can be obtained from the dissection of one litter of pups. This can be problematic for genomic and proteomic analysis, especially when comparing between multiple treatments in one dissection. Also, it is not possible to obtain SCG neuronal cultures from a single embryo. All the experiments are therefore performed on pooled samples that are obtained from SCG of all rat embryos in a litter. Another limitation of the system is that SCG neurons have lower DNA transfection efficiency (10 - 20%, unpublished observations) and higher toxicity following transfection compared to central neurons32. Although this transfection efficiency is fine for morphological studies with individual neurons, it is difficult to perform molecular or biochemical studies to detect/confirm gene expression changes.
However, in recent years, cultured SCG neurons have been used for studies examining the mechanisms underlying dendritic growth and for transcriptome and miRNome analyses14,15,33. As indicated earlier, due to low protein amounts, cultured neurons from embryonic SCG have not been used previously for proteomic analysis. This protocol and representative results provide evidence that even with low concentration of samples, current high definition mass spectrometry instruments could be used for proteomic studies on these neurons. Although the sample preparation protocol described here is for SCG neurons, this protocol can be used for preparation of any sample with low protein concentration for LC-MS analysis. One of the limitations is that small variations in protein concentration or efficiency of trypsin digestion can result in incomplete fragmentation and a dramatic decrease in the number of proteins detected by LC-MS. Therefore, it is critical to ensure that starting protein concentrations are as close to 50 &#181;g with a 10:1 ratio of protein: trypsin. Also, it is a good practice to aliquot the mass spectrometry-grade trypsin to prevent multiple freeze-thaw cycles.
Another limitation of the protocol is that there were only a limited number of transmembrane proteins detected in the proteome. While the acid labile surfactant has been shown to be an effective surfactant for mass spectrometry, a previous study on cultured cells found that these surfactants can slightly increase the number of cytoplasmic proteins and decrease the membrane proteins in the proteomic profile34. Also, it has been suggested that pre-digestion using a bacterial endopeptidase such as Lys-C, prior to trypsin digestion may improve the efficiency of trypsin digestion of membrane bound proteins and prevent the membrane protein fragments from being retained on the LC columns35.
In summary, this paper provides detailed protocols for growing cultured rat embryonic sympathetic neurons and for performing morphological and proteomic studies on these neurons.

Sympathetic neurons derived from embryonic superior cervical ganglia (SCG) have been widely used as a primary neuronal culture system for studying many aspects of neuronal development including growth factor dependence, neuron-target interactions, neurotransmitter signaling, axonal growth, dendrite development and plasticity, synaptogenesis and signaling mechanisms underlying nerve-target/neuron-glia interactions1,2,3,4,5,6,7,8,9. Despite their small size (around 10000 neurons/ganglia), there are three main reasons for the development and extensive use of this culture system are i) being the first ganglia in the sympathetic chain, they are larger, and therefore easier to isolate, than the rest of the sympathetic ganglia10; ii) unlike central neurons, the neurons in the SCG are fairly homogeneous with all the neurons being derived from the neural crest, having a similar size, dependence on nerve growth factor and being nor-adrenergic. This makes them a valuable model for morphological and genomic studies10,11 and iii) these neurons can be maintained in a defined serum-free medium containing nerve growth factor for over a month10,12. Perinatal SCG neurons have been extensively used for studying the mechanisms underlying the initiation and maintenance of dendrites2. This is mainly because, although SCG neurons have an extensive dendritic arbor in vivo, they do not extend dendrites in vitro in the absence of serum but can be induced to grow dendrites in the presence of certain growth factors such as bone morphogenetic proteins2,12,13.
This paper describes the protocol for isolating and culturing embryonic rat SCG neurons. Over the past 50 years, primary neuronal cultures from the SCG have been mainly used for morphological studies with a limited number of studies examining the large-scale genomic or proteomic changes. This is mainly due to small tissue size resulting in the isolation of low amounts of DNA or protein, which makes it difficult to perform genomic and proteomic analyses on these neurons. However, in recent years, increased detection sensitivity has enabled development of methods to examine the genome, miRNome and proteome in the SCG neurons during dendritic growth development14,15,16,17. This paper will also describe the method for morphological analysis of these neurons using immunocytochemistry and a protocol to obtain neuronal protein extracts for mass spectrometric analysis.

<table><tbody><tr><td>2D nanoACQUITY</td><td>Waters Corporation</td><td></td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr><tr><td>BMP-7</td><td>R&amp;amp;D Systems</td><td>354-BP</td><td></td></tr><tr><td>Bovine Serum Alumin</td><td>Sigma-Aldrich</td><td>5470</td><td></td></tr><tr><td>Cell scraper</td><td>Corning</td><td>CLS-3010</td><td></td></tr><tr><td>Collagenase</td><td>Worthington Biochemical</td><td>4176</td><td></td></tr><tr><td>Corning Costar or Nunc Flat bottomed Cell culture plates</td><td>Fisher Scientific</td><td>07-200, 140675, 142475</td><td></td></tr><tr><td>Cytosine- &amp;beta;- D-arabinofuranoside</td><td>Sigma-Aldrich</td><td>C1768</td><td></td></tr><tr><td>D-phosphate buffered saline (Calcium and magnesium free)</td><td>ATCC</td><td>30-2200</td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>4942078001</td><td></td></tr><tr><td>Distilled Water</td><td>Thermo Fisher Scientific</td><td>15230</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>DMEM - Low glucose + Glutamine, + sodium pyruvate</td><td>Thermo Fisher Scientific</td><td>11885</td><td></td></tr><tr><td>Fatty Acid Free BSA</td><td>Calbiochem</td><td>126609</td><td>20 mg/mL stock in low glucose DMEM</td></tr><tr><td>Fine forceps Dumont no.4 and no.5</td><td>Ted Pella Inc</td><td>5621, 5622</td><td></td></tr><tr><td>Forceps and Scissors for Dissection</td><td>Ted Pella Inc</td><td>1328, 1329, 5002</td><td></td></tr><tr><td>Glass coverlips - 12mm</td><td>Neuvitro Corporation</td><td>GG-12</td><td></td></tr><tr><td>Goat-Anti Mouse IgG Alexa 488 conjugated</td><td>Thermo Fisher Scientific</td><td>A32723</td><td></td></tr><tr><td>Ham&#039;s F-12 Nutrient Mix</td><td>Thermo Fisher Scientific</td><td>11765</td><td></td></tr><tr><td>Hank&#039;s balanced salt soltion (Calcium and Magnesium free)</td><td>Thermo Fisher Scientific</td><td>14185</td><td></td></tr><tr><td>Insulin-Selenium-Transferrin (100X)</td><td>Thermo Fisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>A3221</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030</td><td></td></tr><tr><td>Leibovitz L-15 medium</td><td>Thermo Fisher Scientific</td><td>11415064</td><td></td></tr><tr><td>Mounting media for glass coverslips</td><td>Thermo Fisher Scientific</td><td>P36931, P36934</td><td></td></tr><tr><td>Mouse-anti- MAP2 antibody (SMI-52)</td><td>BioLegend</td><td>SMI 52</td><td></td></tr><tr><td>Nerve growth factor</td><td>Envigo Bioproducts (formerly Harlan Bioproducts)</td><td>BT5017</td><td>Stock 125 &amp;mu;g/mL in 0.2% Prionex in DMEM</td></tr><tr><td>Paraformaldehye</td><td>Spectrum Chemicals</td><td>P1010</td><td></td></tr><tr><td>Penicillin-Streptomycin (100X)</td><td>Thermo Fisher Scientific</td><td>15140</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Prionex</td><td>Millipore</td><td>529600</td><td>10% solution, 100 mL</td></tr><tr><td>RapiGest SF</td><td>Waters Corporation</td><td>186001861</td><td>5 X 1 mg</td></tr><tr><td>Synapt G2 High Definition Mass Spectrometry</td><td>Waters Corporation</td><td></td><td></td></tr><tr><td>Trifluoro acetic acid - Sequencing grade</td><td>Thermo Fisher Scientific</td><td>28904</td><td>10 X 1 mL</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Trypsin</td><td>Promega or NEB</td><td>V511A, P8101S</td><td>100 &amp;mu;g or 5 X 20 mg</td></tr><tr><td>Waters Total recovery vials</td><td>Waters Corporation</td><td>186000385c</td><td></td></tr></tbody></table>

culturing rat sympathetic neurons from embryonic superior cervical ganglia for morphological and proteomic analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

Isolating and maintaining neuronal cultures of embryonic SCG neurons 
Dissociated cells from the rat embryonic SCG were plated in a poly-D-lysine coated plate or coverslip and maintained in serum free culture media containing b-nerve growth factor. The dissociated cells containing a mixture of neurons and glial cells look circular upon plating (Figure 1A). Within 24 hours of plating, the neurons extend small axonal processes with glial cells flattening and appearing phase-dark under phase contrast microscopy (Figure 1B). Following the treatment with Ara-C, 99% of the glial cells are eliminated and the cultures contain predominantly neuronal cells with an oval cell body, extensive axonal growth and no dendrites (Figure 1C).
Immunocytochemical staining of cultured embryonic rat superior cervical ganglia neurons 
Previous studies have shown that cultured sympathetic neurons grown in serum-free medium extend only axons and only extend an elaborate dendritic arbor in the presence of basement membrane extract, 10% serum or 50 ng/mL bone morphogenetic protein-7 (BMP-7)2,12. In agreement with these observations, phase contrast microscopy shows that neurons grown in the presence of BMP-7 (50 ng/mL) for 5 days have a flattened cell body with multiple thick tapered processes when compared to neurons grown in serum-free control media (Figure 2). The identity of these processes as dendrites is confirmed by the presence of MAP-2, a cytoskeletal protein predominantly found in the cell body and dendrites19,20. Under control conditions, fluorescent staining for MAP-2 is observed within the cytoplasm and proximal axons and excluded from the nucleus (as evidenced by co-labeling with a nuclear stain to visualize the nucleus (Figure 3A-3D). Following treatment with BMP-7 at 50 ng/mL for 5 days, immunoreactivity for MAP-2 is observed not only in the cytoplasm but also in dendrites (Figure 3E-3H).
Sample proteomic analysis of cultured SCG neurons grown in control media 
E21 rat sympathetic neurons were cultured in control media for 6 days in vitro, lysed and subjected to LC-MS analysis. The sample was run in three replicates on the mass spectrometer and the proteins were identified based on the number of fragmented peptides observed for each sample. The abundance of the proteins (ranging from 0 to over 300000 arbitrary units) and confidence score for protein ID based on the number of fragmented peptides observed for each protein (ranging from 5 - 500) was calculated. A sample data set from the mass spectrometric analysis of the proteome of 30 &#181;g protein from SCG neurons cultured in serum-free control media is shown in Figure 4.
There were 13, 134 peptides detected in the sample which corresponded to 1287 proteins. Of these, 1100 of which were present in all three technical replicates with normalized abundance values greater than 500. Of these 1100 proteins, 90 proteins were identified by the presence of a single peptide hit, which only covered a small portion of the protein. Therefore, it was difficult to determine if the identification was correct and these proteins had a low protein confidence score (score &#60;10).These proteins were eliminated from the analysis and the remaining 1010 proteins were further analyzed using Gene ontology to determine whether the protocol was successful in detecting proteins with different localization and function.
Gene ontology analysis of this dataset was performed using the Panther classification database (http://www.pantherdb.org/) to determine if this data set included proteins with different cellular localization or molecular and biological functions and to examine whether there were the connections between the identified proteins and known pathways21. The analysis showed that although over 700 of the proteins were cytoplasmic, that data set contained proteins that were localized to other regions of the cell including the nucleus, the membrane, several cellular organelles, and cell junctions (Figure 4A). The analysis also revealed that over 400 proteins had catalytic activity, and around 300 proteins were involved in signaling pathways (Figure 4B and Figure 4C). In addition, the dataset contained proteins involved in various signaling pathways. Table 1 shows five of the proteins in G-protein signaling, cell adhesion, growth factor signaling pathway, and synaptic vesicle trafficking pathways, respectively, that were identified in the dataset.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61283/61283fig01.jpg" /> 
Figure 1: Changes in the morphology of cultured E21 rat embryonic SCG neurons over time.&#160;Representative phase contrast microscopy images at 10x magnification of dissociated cells from E21 rat SCG 20 min after plating (A), one day after plating (B) and after 48 hours of Ara-C treatment (5 days after plating) (C). Note the presence of glial cells (arrows) and axonal extension (arrow heads) in (B), and absence of glial cells and neurons showing extensive axonal growth in (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61283/61283fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61283/61283fig02.jpg" /> 
Figure 2: Changes in neuronal morphology of E21 rat SCG neurons following treatment with BMP-7.&#160;Following the elimination of non-neuronal cells, cultured SCG neurons were treated with control media (A) or BMP-7 at 50 ng/mL (B) for 5 days. Representative phase contrast micrographs (10x magnification) show the circular neuronal cell body with axons in control neurons (A) and neurons with multiple short dendrite-like processes when treated with BMP-7 (arrows, B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61283/61283fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61283/61283fig03.jpg" /> 
Figure 3: Immunostaining for MAP-2 protein in cultured embryonic rat sympathetic neurons.&#160;Following the elimination of non-neuronal cells, cultured SCG neurons were treated with control media (A-D) or BMP-7 at 50 ng/mL (E-H) for 5 days and immunostained using a mouse monoclonal antibody against MAP-2 and co-labeled with a nuclear marker. Representative micrographs showing immunocytochemical staining of the neurons with microtubule associated protein-2 (MAP-2) (C,G), a nuclear stain (D,H) with phase contrast micrographs (B,F) and a merge of the three channels (A,E). Immunocytochemical staining for microtubule associated protein-2 is present in the cell body and proximal axons of control neurons (C) and staining for MAP-2 protein in the cell body and dendrites in BMP-7 treated neurons (G). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61283/61283fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61283/61283fig04v2.jpg" /> 
Figure 4: Classification and distribution of proteins identified in the proteome analysis of cultured E21 rat SCG neurons.&#160;Protein IDs for the 1100 proteins were subjected to gene ontology analysis on Panther classification system to examine the distribution of proteins in the data set with respect to cellular localization (A), cellular function and biological processes they were involved in (B) and molecular function class based on protein structure (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61283/61283fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">A) Proteins involved in biological adhesion</td>
		</tr>
		<tr>
			<td>UniProt Accession ID</td>
			<td>Gene name</td>
			<td>Gene symbol</td>
		</tr>
		<tr>
			<td>Q9Z1Y3</td>
			<td>Cadherin-2</td>
			<td>Cdh2</td>
		</tr>
		<tr>
			<td>O35112</td>
			<td>CD166 antigen</td>
			<td>Alcam</td>
		</tr>
		<tr>
			<td>P49134</td>
			<td>Integrin beta-1</td>
			<td>Itgb1</td>
		</tr>
		<tr>
			<td>D3ZES7</td>
			<td>Plexin A4</td>
			<td>Plxna4</td>
		</tr>
		<tr>
			<td>F1LP44</td>
			<td>Integrin subunit alpha L</td>
			<td>Itgal</td>
		</tr>
		<tr>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="3">B) Proteins involved in growth factor signaling</td>
		</tr>
		<tr>
			<td>P35213</td>
			<td>14-3-3 protein beta/alpha</td>
			<td>Ywhab</td>
		</tr>
		<tr>
			<td>Q5BJ92</td>
			<td>Serine/Threonine-protein phosphatase 4 catalytic subunit</td>
			<td>Ppp4c</td>
		</tr>
		<tr>
			<td>P47196</td>
			<td>Rac &#8211; alpha serine/threonine protein kinase</td>
			<td>Akt1</td>
		</tr>
		<tr>
			<td>P62994</td>
			<td>Growth factor receptor bound protein 2</td>
			<td>Grb2</td>
		</tr>
		<tr>
			<td>P21708</td>
			<td>Mitogen-activated protein kinase 3</td>
			<td>Mapk3</td>
		</tr>
		<tr>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="3">C) Proteins involved in G-protein coupled signaling pathway</td>
		</tr>
		<tr>
			<td>P08753</td>
			<td>Guanine nucleotide-binding protein G(k) subunit alpha</td>
			<td>Gnai3</td>
		</tr>
		<tr>
			<td>D4ABV5</td>
			<td>Calmodulin-2</td>
			<td>Calm2</td>
		</tr>
		<tr>
			<td>P18266</td>
			<td>Glycogen synthase kinase-3 beta</td>
			<td>Gsk3b</td>
		</tr>
		<tr>
			<td>P09456</td>
			<td>cAMP-dependent protein kinase type I-alpha regulatory subunit</td>
			<td>Prkar1a</td>
		</tr>
		<tr>
			<td>P53534</td>
			<td>Glycogen phosphorylase</td>
			<td>Pygb (brain form)</td>
		</tr>
		<tr>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="3">D) Proteins involved in synaptic vesicle trafficking</td>
		</tr>
		<tr>
			<td>P47861</td>
			<td>Synaptotagmin-5</td>
			<td>Syt5</td>
		</tr>
		<tr>
			<td>P63045</td>
			<td>Vesicle-associated membrane protein 2</td>
			<td>Vamp2</td>
		</tr>
		<tr>
			<td>P61265</td>
			<td>Syntaxin-1B</td>
			<td>Stx1b</td>
		</tr>
		<tr>
			<td>Q63537</td>
			<td>Synapsin-2</td>
			<td>Syn2</td>
		</tr>
		<tr>
			<td>P60881</td>
			<td>Synaptosomal-associated protein 25</td>
			<td>Snap25</td>
		</tr>
	</tbody>
</table>
Table 1: Representative proteins identified in the proteome data from cultured rat embryonic SCG neurons, classified by their biological functions. The proteome data with the 1100 proteins was subjected to gene ontology analysis using the Panther classification system. Listed below are the top five proteins identified for four biological pathways.

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Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

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Anatomy and Physiology

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Unit 1

The Autonomic Nervous System

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7.5K Views.  Saint Mary's College of California. In deze video zullen we de culturing van de superieure cervicale ganglia van embryonale ratten demonstreren voor morfologische en proteomische analyse. Bereid de controle- en ontledingsmedia voor voordat u met de dissectie begint. Controlemedium bevat F-12 en lage glucose DMEM aangevuld met een insuline-selenium-transferrine mengsel, glutamine, zenuwgroeifactor, en BSA.Dissectiemedium bevat L-15 aangevuld met pen-strep en BSA. Raadpleeg het manuscript voor gedetailleerde protocollen vo...

Video: Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Immunology and Infection

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

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

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...

Cancer Research

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

Medicine

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

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