Supported by NIH grant R01HL105635 to SEJ. The authors would like to thank Diego V. Boh&#243;rquez for technical advice. We also thank R. Ian Cumming for technical assistance and performing the flow cytometry at the Duke Human Vaccine Institute Research Flow Cytometry Shared Resource Facility (Durham, NC). Flow cytometry was performed in the Regional Biocontainment Laboratory at Duke which received partial support for construction from the National Institutes of Health, National Institute of Allergy and Infectious Diseases (UC6-AI058607).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University.
1. Intranasal Administration of Fast Blue
For Fast Blue, administer the dye at least 2 days before euthanizing the mouse. The dye will persist for up to eight weeks.
<ol>
	<li>Anesthetize the mouse with light inhalation anesthesia (2.5% sevoflurane) until breathing starts to slow.</li>
	<li>Use a 200 &#181;l&#160;pipette with filtered tips to slowly instill 40 &#181;l&#160;of dye solution (0.4 mM Fast Blue, 1% dimethyl sulfoxide, DMSO, in phosphate buffered saline, PBS) into the nostril, pausing occasionally to ensure the mouse is aspirating the solution (Figure 1A).</li>
	<li>Hold the mouse vertically, head up, and gently massage the chest to ensure the dye spreads throughout the lungs.</li>
</ol>
2. Preparations on Dissection and Analysis Day
<ol>
	<li>Prepare 10 ml&#160;Ganglia Dissociation Solution (GaDS) by combining the ingredients as specified in Table 1.</li>
	<li>Perform RNA extraction in a dedicated lab area. Before beginning the experiment, clean surfaces with 70% ethanol, 10% bleach, and RNase decontamination reagent. This includes the RNA centrifuge, any tube racks, and pipettes. Ensure there are dedicated filter tip boxes for RNA use only. Do not use this equipment and area for DNA preparation, as this will contaminate the samples.</li>
	<li>Mix fresh 70%, and 80% ethanol with RNase-free water. Prepare 1 ml&#160;per sample to be used in RNA extraction.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Advanced DMEM/f12</td>
			<td>9.5 ml</td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>HEPES (10 mM)</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>B27 (no vitamin A)</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>NGF (50 &#181;g/ml)</td>
			<td>10 &#181;l</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Reagent Mixture for Ganglia Dissociation Solution (GaDS).
3. Dissection Procedure
<ol>
	<li>Fill 1.5 ml&#160;tubes with 500 &#181;l&#160;GaDS, one for each experimental sample, one for a positive sorting control, and one for a negative sorting control. Store tubes on ice throughout dissection.</li>
	<li>Dissect out the nodose ganglion and make two partial cuts to facilitate dissociation, then put it in the experimental sample tube. Please refer to the following JoVE protocol for dissection of the nodose ganglia16 and DRG17.</li>
	<li>Dissect and partially cut non-labeled ganglia, such as the dorsal root ganglia (DRG), for the sorting controls. Use two ganglia for the positive control and two ganglia for the negative control.</li>
	<li>To obtain enough nodose neurons for successful sorting, combine ganglia from 5 mice into one experimental sample tube containing GaDS.</li>
</ol>
4. Sensory Ganglia Dissociation
<ol>
	<li>Pipette out 500 &#181;l&#160;GaDS, leaving ganglia in the tube. Wash ganglia once by adding 1 ml&#160;PBS, wait for ganglia to settle to the bottom of the tube, then pipette out PBS.</li>
	<li>Add 1 ml&#160;GaDS and 22 &#181;l&#160;of digestion enzyme (collagenase I/II and protease mix, 2.5 mg/ml&#160;in H2O) to each tube.</li>
	<li>Add 20 &#181;l&#160;Fast Blue (5.26 mM) to the positive control tube.</li>
	<li>Put the tubes in a water bath or heating block already set at 37 &#176;C. 
	Note: The time of ganglia digestion depends on the age of the digestion enzyme. Within 1 month of dissolving the digestion enzyme, digest for 30 min, within 2 - 6 months digest for 45 min. If the enzyme is over 6 months old, digest ganglia for 60 min.</li>
	<li>Every 15 min&#160;shake tubes briefly and flick them to ensure ganglia are covered by the solution.</li>
	<li>While ganglia are being digested, prepare density gradients using a particle solution (colloidal silica particles coated with polyvinylpyrrolidone).
	<ol>
		<li>Mix 12% particle solution in GaDS, 500 &#181;l&#160;per sample.</li>
		<li>Mix 28% particle solution in GaDS, 500 &#181;l&#160;per sample.</li>
		<li>Slowly and carefully add 400 &#181;l 12% density solution on top of 400 &#181;l&#160;28% density solution in a fresh 1.5 ml&#160;tube for each sample. Do not allow the two layers to mix. Two distinct layers should be visible in the tube; one darker than the other.</li>
	</ol>
	</li>
	<li>While ganglia are being digested, label new 1.5 ml&#160;collection tubes for sorting (one Fast Blue positive and one Fast Blue negative tube per sorting sample). Depending on the sorting facility requirements, these tubes must have detachable lids so as to not interfere with the cell sorter.</li>
	<li>After the appropriate digestion time, remove GaDS with digestion enzyme and wash ganglia twice with 1 ml&#160;PBS. Add 200 &#181;l&#160;of fresh GaDS to each tube.</li>
	<li>Use a 200 &#181;l&#160;pipette, set to 100 &#181;l&#160;volume, and pipette ganglia up and down several times to break cells apart. Repeat until no intact piece of tissue is seen. Avoid creating bubbles in the solution.</li>
	<li>Pass dissociated cells through a 70 &#181;m cell strainer.</li>
	<li>Add another 100 &#181;l&#160;GaDS to the original dissociation tube to pick up any remaining stray cells left and pass the additional solution through the cell strainer. Using a new pipette tip, collect any remaining cell-containing liquid that has passed through the strainer and clung to the mesh. The final volume of cells suspended in GaDS is 300 &#181;l.</li>
	<li>Carefully layer the 300 &#181;l&#160;of cells on top of the previously made density gradient. Avoid bubbles and mixing of cells with the density layers.</li>
	<li>Centrifuge for 10 min&#160;at 2,900 x g at RT.</li>
	<li>While cells are in the centrifuge, mix lysis buffer (1 ml&#160;per experimental sample) as follows: 10 &#181;l&#160;2-mercaptoethanol in 1 ml&#160;Buffer RLT (from RNA extraction kit).
	<ol>
		<li>Add 300 &#181;l&#160;lysis buffer to Fast Blue positive collection tube and 600 &#181;l&#160;to Fast Blue negative collection tube (tubes labeled from step 4.7). This volume depends on the number of cells expected to be collected. For &#60;2,000 cells, add 300 &#181;l, for &#62;7,000 cells add 600 &#181;l. This will ensure the sorting sheath fluid does not significantly dilute the lysis buffer.</li>
	</ol>
	</li>
	<li>Once centrifugation is complete, carefully remove and discard the top 700 &#181;l&#160;layer. This layer contains the majority of cell debris that will otherwise interfere with cell sorting.</li>
	<li>Add 700 &#181;l&#160;fresh GaDS to the remaining cell containing solution and pipette up and down several times to mix.</li>
	<li>Centrifuge for 15 min&#160;at 2,900 x g to pellet cells.</li>
	<li>While cells are in the centrifuge, mix sorting GaDS (500 &#181;l&#160;per sample) by adding 5 &#181;l&#160;DNase (10 mg/ml) to 1 ml&#160;GaDS.</li>
	<li>Once centrifugation is done, carefully remove and discard supernatant, leaving the cell pellet.</li>
	<li>Re-suspend cell pellet in 200 - 300 &#181;l&#160;sorting GaDS by pipetting up and down with a 1,000 &#181;l pipette set to 200 &#181;l several times. Put samples on ice. Cells are now ready to be sorted.</li>
</ol>
5. Fluorescence Activated Cell (FAC) Sorting
Note: This section requires operational knowledge of a FACS sorter or the assistance of skilled personnel.
<ol>
	<li>Add 1 &#181;l&#160;propidium iodide (PI) stock solution (50 &#181;g/ml) to each sample to test for cell viability. Split negative control into 2 tubes, one with PI and one without. PI binds DNA only in dead cells. If after the first few sorts there are no dead cells to label, the use of PI is not needed.</li>
	<li>Transport cells to flow cytometry instrument, e.g., BD FACS AriaII running Diva 8 software, on ice.</li>
	<li>Since the cell size is variable in this population, use a large nozzle (100 &#181;m) for sorting. To decrease the amount of stress the cells experience and increase cell viability use low pressure (20 psi).</li>
	<li>Use the FACS sorter software to set up the analysis plots, forward scatter, side scatter (SSC), Fast Blue, and PI, if needed.</li>
	<li>To re-suspend cells, gently vortex the sample briefly before loading it into the sorter.</li>
	<li>Begin with the negative control, no Fast Blue or PI, to set the gate threshold (Figure 2A). This will determine the amount of autofluorescence in the cell population. Optional: Run the negative sample with PI to identify whether there is a population of dead cells.</li>
	<li>Sort the positive control sample (incubated with Fast Blue), to set compensation parameters.</li>
	<li>Once the gates and parameters are set, begin sorting the experimental samples. Samples are sorted into the prepared collection tubes containing RNA lysis buffer.</li>
	<li>Keep sorted samples on ice until the cell sorting is complete.</li>
	<li>Begin RNA extraction step immediately after cells are sorted.</li>
</ol>
6. RNA Extraction
<ol>
	<li>Vortex sorted cells in lysis buffer for 1 min&#160;to homogenize.</li>
	<li>Add 1 volume of 70% ethanol and mix by pipetting up and down several times.</li>
	<li>Transfer up to 700 &#181;l&#160;of the sample to a spin column. Centrifuge for 15 sec&#160;at 8,000 x g. Discard the flow-through.
	<ol>
		<li>Repeat until the total volume of the sample is passed through the spin column.</li>
	</ol>
	</li>
	<li>Add 350 &#181;l&#160;Buffer RW1 and continue the RNA purification process, including the DNase treatment step, according to the manufacturer&#39;s protocol for cells.</li>
	<li>Once RNA has been extracted, test the RNA quality using a microfluidic electrophoresis system according to the manufacturer&#39;s specifications.</li>
</ol>

<ol>
	<li>Manteniotis, S., 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=Comprehensive+RNA-Seq+Expression+Analysis+of+Sensory+Ganglia+with+a+Focus+on+Ion+Channels+and+GPCRs+in+Trigeminal+Ganglia.">Comprehensive RNA-Seq Expression Analysis of Sensory Ganglia with a Focus on Ion Channels and GPCRs in Trigeminal Ganglia.</a> PLoS One. 8 (11), 1-30 (2013).</li><li>Vandewauw, I., Owsianik, G., Voets, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+quantitative+mRNA+expression+analysis+of+TRP+channel+genes+at+the+single+trigeminal+and+dorsal+root+ganglion+level+in+mouse.">Systematic and quantitative mRNA expression analysis of TRP channel genes at the single trigeminal and dorsal root ganglion level in mouse.</a> BMC Neurosci. 14 (1), 21 (2013).</li><li>Paintal, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagal+sensory+receptors+and+their+reflex+effects.">Vagal sensory receptors and their reflex effects.</a> Physiol Rev. 53 (1), 159-227 (1973).</li><li>Springall, D. R., Cadieux, A., Oliveira, H., Su, H., Royston, D., Polak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+tracing+shows+that+CGRP-immunoreactive+nerves+of+rat+trachea+and+lung+originate+from+vagal+and+dorsal+root+ganglia.">Retrograde tracing shows that CGRP-immunoreactive nerves of rat trachea and lung originate from vagal and dorsal root ganglia.</a> J Auton Nerv Syst. 20 (2), 155-166 (1987).</li><li>Ricco, M. M., Kummer, W., Biglari, B., Myers, A. C., Undem, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interganglionic+segregation+of+distinct+vagal+afferent+fibre+phenotypes+in+guinea-pig+airways.">Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways.</a> J Physiol. 496 (Pt 2), 521-530 (1996).</li><li>Zhao, H., Sprunger, L. K., Simasko, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+transient+receptor+potential+channels+and+two-pore+potassium+channels+in+subtypes+of+vagal+afferent+neurons+in+rat.">Expression of transient receptor potential channels and two-pore potassium channels in subtypes of vagal afferent neurons in rat.</a> Am J Physiol Gastrointest Liver Physiol. 298 (2), 212-221 (2010).</li><li>Zhuo, H., Ichikawa, H., Helke, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurochemistry+of+the+nodose+ganglion.">Neurochemistry of the nodose ganglion.</a> Prog Neurobiol. 52 (2), 79-107 (1997).</li><li>Chang, R. B., Strochlic, D. E., Williams, E. K., Umans, B. D., Liberles, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagal+Sensory+Neuron+Subtypes+that+Differentially+Control+Breathing.">Vagal Sensory Neuron Subtypes that Differentially Control Breathing.</a> Cell. 161, 1-12 (2015).</li><li>Kaczy&#324;ska, K., Szereda-Przestaszewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodose+ganglia-modulatory+effects+on+respiration.">Nodose ganglia-modulatory effects on respiration.</a> Physiol Res. 62, 227-235 (2013).</li><li>Taylor-Clark, T. E., Undem, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+pulmonary+oxidative+stress+by+lung+vagal+afferents.">Sensing pulmonary oxidative stress by lung vagal afferents.</a> Respir Physiol Neurobiol. 178 (3), 406-413 (2011).</li><li>Bautista, D. M., 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=TRPA1+mediates+the+inflammatory+actions+of+environmental+irritants+and+proalgesic+agents.">TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents.</a> Cell. 124 (6), 1269-1282 (2006).</li><li>Ichikawa, H., De Repentigny, Y., Kothary, R., Sugimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+survival+of+vagal+and+glossopharyngeal+sensory+neurons+is+dependent+upon+dystonin.">The survival of vagal and glossopharyngeal sensory neurons is dependent upon dystonin.</a> Neuroscience. 137 (2), 531-536 (2006).</li><li>Hondoh, 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=Distinct+expression+of+cold+receptors+(TRPM8+and+TRPA1)+in+the+rat+nodose-petrosal+ganglion+complex.">Distinct expression of cold receptors (TRPM8 and TRPA1) in the rat nodose-petrosal ganglion complex.</a> Brain Res. 1319, 60-69 (2010).</li><li>Kummer, W., Fischer, A., Kurkowski, R., Heym, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensory+and+sympathetic+innervation+of+guinea-pig+lung+and+trachea+as+studied+by+retrograde+neuronal+tracing+and+double-labelling+immunohistochemistry.">The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry.</a> Neuroscience. 49 (3), 715-737 (1992).</li><li>Choi, D., Li, D., Raisman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+retrograde+neuronal+tracers+that+label+the+rat+facial+nucleus:+A+comparison+of+Fast+Blue,+Fluoro-ruby,+Fluoro-emerald,+Fluoro-Gold+and+DiI.">Fluorescent retrograde neuronal tracers that label the rat facial nucleus: A comparison of Fast Blue, Fluoro-ruby, Fluoro-emerald, Fluoro-Gold and DiI.</a> J Neurosci Methods. 117 (2), 167-172 (2002).</li><li>Calik, M. W., Radulovacki, M., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+of+Nodose+Ganglia+Injection+in+Sprague-Dawley+Rat.">A Method of Nodose Ganglia Injection in Sprague-Dawley Rat.</a> J Vis Exp. (93), e1-e5 (2014).</li><li>Ramachandra, R., McGrew, S., Elmslie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+specific+sensory+neuron+populations+for+study+of+expressed+ion+channels.">Identification of specific sensory neuron populations for study of expressed ion channels.</a> J Vis Exp. (82), e50782 (2013).</li><li>Yu, X., Hu, Y., Ru, F., Kollarik, M., Undem, B. J., Yu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM8+function+and+expression+in+vagal+sensory+neurons+and+afferent+nerves+innervating+guinea+pig+esophagus.">TRPM8 function and expression in vagal sensory neurons and afferent nerves innervating guinea pig esophagus.</a> Am J Physiol - Gastrointest Liver Physiol. 308 (6), 489-496 (2015).</li><li>Kwong, K., Lee, L. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGE(2)+sensitizes+cultured+pulmonary+vagal+sensory+neurons+to+chemical+and+electrical+stimuli.">PGE(2) sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli.</a> J Appl Physiol. 93 (4), 1419-1428 (2002).</li><li>Joachim, R. 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=Stress+induces+substance+P+in+vagal+sensory+neurons+innervating+the+mouse+airways.">Stress induces substance P in vagal sensory neurons innervating the mouse airways.</a> Clin Exp Allergy. 36 (8), 1001-1010 (2006).</li><li>Kaan, T. K. 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=Systemic+blockade+of+P2X3+and+P2X2/3+receptors+attenuates+bone+cancer+pain+behaviour+in+rats.">Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats.</a> Brain. 133 (9), 2549-2564 (2010).</li><li>Nakatani, T., Minaki, Y., Kumai, M., Ono, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helt+determines+GABAergic+over+glutamatergic+neuronal+fate+by+repressing+Ngn+genes+in+the+developing+mesencephalon.">Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon.</a> Development. 134 (15), 2783-2793 (2007).</li><li>Lobo, M. K., Karsten, S. L., Gray, M., Geschwind, D. H., Yang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-array+profiling+of+striatal+projection+neuron+subtypes+in+juvenile+and+adult+mouse+brains.">FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains.</a> Nat Neurosci. 9 (3), 443-452 (2006).</li><li>Usoskin, D., 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=Unbiased+classification+of+sensory+neuron+types+by+large-scale+single-cell+RNA+sequencing.">Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.</a> Nat Neurosci. 18, 145-153 (2015).</li></ol>

The authors have nothing to disclose.

This protocol describes a method to target airway-innervating neurons in the nodose ganglia of the vagus nerve. Once labeled, the ganglia are gently dissociated to optimally preserve cell numbers and viability. These neurons are then FAC sorted directly into lysis buffer and RNA is extracted. The significance of this protocol is the ability to target, isolate, and preserve the quality of a specific sensory cell population. Gene expression is described in this small population of neurons, and organ-specific functions&#160...

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent nerves are located in sensory ganglia, such as the dorsal root, trigeminal, or nodose ganglia. Each sensory ganglion innervates specific regions of the body and contains cells that innervate separate organs and tissues within that region. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs, while the trigeminal ganglia are located in the skull, containing neurons that innervate the face, eyes, meninges or upper airways1,2. The nodose ganglia of the vagus nerve is located in the neck below the skull and contains cell bodies that extend nerve fibers throughout the gastrointestinal tract, heart, and lower airways and lungs3. In humans the nodose ganglion stands alone, however, in the mouse it is fused with the jugular ganglion, which also innervates the lungs4. This fused ganglion is often called the jugular/nodose complex, vagal ganglion, or simply nodose ganglion5. Here, it is referred to as the nodose ganglion.
Afferent fibers of the nodose pass information from the viscera to the nucleus of the solitary tract (NTS) in the brainstem. Sensory input to this unique ganglion controls a diverse array of functions, such as gut motility6, heart rate7, respiration8,9, and irritant-activated respiratory responses10,11. With this diversity of functions and innervated organs, it is critical to target and isolate organ-specific subpopulations of the nodose ganglion in order to study individual neuronal pathways. However, given the small size of the nodose and the limited number of neurons it contains this is not a trivial task. Each mouse nodose ganglion contains roughly 5,000 neurons12 in addition to an extensive population of supporting satellite cells. Of the 5,000 nodose neurons, only 3 - 5% innervate the airways. Therefore, any functional, morphological or molecular changes within airway-innervating neurons, due to respiratory stimulation or pathologies, will be lost in the densely packed nodose ganglion.
To solve this problem, a method was developed to identify and isolate neurons that innervate the airways. The airways were exposed to a fluorescent tracer dye to identify the subsequent innervating nodose neurons. Fast Blue was picked up by neurons and travels quickly to their cell bodies where it is retained for up to eight weeks13-15. Once identified, a gentle, yet efficient, dissociation protocol was used to preserve&#160;dye labeling and cell viability for fluorescent activated cell (FAC) sorting. Sorted cells are used to extract high quality ribonucleic acid (RNA) to determine gene expression or for other downstream molecular analysis. This protocol provides a useful and robust technique for isolating sensory neurons that innervate a tissue of interest.

<table><tbody><tr><td>Fast Blue</td><td>Polysciences, Inc.</td><td>17740-2</td><td>stock 2 mg/ml in water</td></tr><tr><td>NeuroTrace 530/615 red Nissle stain</td><td>Life Technologies</td><td>N21482</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-500</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS) Ca and Mg free</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>glutamine (Glutamax)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>N2</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>B27 (no vitamin A)</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>Nerve Growth Factor (NGF)</td><td>Sigma</td><td>N6009</td><td>stock 50 &amp;micro;g/ml in PBS/10% FBS</td></tr><tr><td>digestion enzyme, Liberase DH Research Grade</td><td>Roche</td><td>5401054001</td><td>stock 2.5 mg/ml in water</td></tr><tr><td>particle solution (Percoll)</td><td>Sigma</td><td>P1644-25ML</td><td></td></tr><tr><td>Heating block</td><td>LabNet</td><td></td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Absolute Ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818-500</td><td></td></tr><tr><td>RNase free water</td><td>Fisher Scientific</td><td>BP2484-100</td><td></td></tr><tr><td>RNase decontamination reagent, RNase AWAY</td><td>invitrogen</td><td>10328-011</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>VWR</td><td>EM-6010</td><td></td></tr><tr><td>RNA extraction kit, RNeasy Plus Micro Kit</td><td>Qiagen</td><td>74034</td><td></td></tr><tr><td>DNase kit, RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>DNase</td><td>Sigma</td><td>D5025-15KU</td><td>stock 10 mg/ml in 0.15 M NaCl</td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4170-10MG</td><td>stock 10 &amp;micro;g/ml in PBS</td></tr><tr><td>Microfluidic electrophoresis system (TapeStation 2200)</td><td>Agilent</td><td></td><td></td></tr></tbody></table>

a method to target and isolate airway-innervating sensory neurons in mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Using this method, airway-innervating neurons are labeled by intranasally instilling Fast Blue (Figure 1A). After two days, Fast Blue labeled cells appear in the nodose ganglia (Figure 1C). These cells make up 3 - 5% of the total neuronal population of the nodose ganglia. Other retrograde dyes that have been used for this purpose include DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate) and Fluorogold. Lung exposure to D...

Watch this Scientific Journal Video about A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice at JoVE.com

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

a-method-to-target-isolate-airway-innervating-sensory-neurons

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JoVE Journal

Neuroscience

13.1K Views.  Yale University. Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Video: A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biology

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Medicine

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

Targeting Mouse Sympathetic Neurons: A Lumbar Sympathectomy Approach

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Author Spotlight: Exploring Plasticity of Sympathetic Neurons

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

Calcium Imaging of Thoracic Segment T1 DRG for Cardiopulmonary Sensory Neurons

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.

This study presents a surgical manipulation to expose the T-DRG in anesthetized mice for in vivo calcium imaging, along with synchronous ECG recordings. This method represents a cutting-edge tool for studying peripheral electric nerve stimulation and thoracic visceral organ inputs, as well as their interactions at the primary sensory level.

The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. ...

Isolation of Cells from Sensory Ganglia

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

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

	...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

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Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Surgical Lumbar Sympathectomy in Mice

In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation

Izolacja komórek ze zwojów czuciowych