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;and neural networks are identified.
The critical steps in this protocol include rapid progression through the dissociation process and subsequent cell sorting. It is important to schedule the sorting time such that it begins immediately after dissociation. It is also critical that all the RNA extraction reagents are fresh, the 70% and 80% ethanol solutions are made fresh, and all tubes and equipment for RNA extraction are clean and RNase free.
This protocol can be modified such that cells are sorted individually into well plates for single cell RNA extraction and sequencing. It can also be used to isolate cells from other sensory ganglia, such as dorsal root ganglia (DRGs). Dye injection of different body parts for tracing back to DRGs has been previously described17. This protocol can also be modified to collect cells for functional analysis. Instead of sorting cells into lysis buffer, cells are sorted into neuronal culture medium, GaDS. These cells are then cultured and used for functional studies such as calcium imaging, or electrophysiology.
If the Fast Blue positive population is smaller than the expected outcome, purchase new digestion enzyme. The age of the digestion enzyme is correlated to the efficiency of digestion. The digestion enzyme needs to be used within 1 year of purchase. In the case where the cell number is high, RNA is extracted and the quality tested. If the RNA is degraded this is an indication that the reagents are not fresh, or have been contaminated. In this case, be sure to thoroughly clean all RNA extraction areas, pipettes, racks, and anything that will come in contact with the sample tubes. Make fresh RNase and DNase free 70% and 80% ethanol. The water and ethanol used to make these solutions should also be kept separate from other lab supplies and used solely for RNA extraction.
FAC sorting is a fast and efficient method for isolating Fast Blue positive cells, however, one limitation is that the cell density should be 1 to 2 million cells per ml&#160;buffer. This protocol pushes the limit of current cell sorter models. The number of cells in the nodose ganglion is small. To get a cell yield sufficient for the cell sorter, cells from five animals are pooled. The sorting volume described is 200 - 300 &#181;l&#160;and has fewer than 100,000 suspended cells. This translates to a concentration of around 450,000 cells per ml, below the recommended density. One alternative to using a cell sorter is to handpick with a fluorescent microscope and glass pipette24. This technique is slower and has been used to collect 30 - 100 cells at a time. Therefore, this protocol offers a faster high-throughput method compared to existing methods.
The future applications of this protocol include the ability to determine the transcriptional profile of a specific population of airway-innervating nerves. The high quality RNA collected using this protocol can be used as a template for cDNA synthesis and next generation sequencing, and other techniques to characterize the transcriptome of the collected neurons. In this way, it can be determined which genes&#160;or pathways are specific to these neurons. This information will help to elucidate the functional role these neurons play in normal physiological conditions. Once basic expression patterns have been established, the system can be challenged by physical and chemical stimuli or by pathogens to determine their effects on gene regulation in lung-innervating neurons. Finally, by identifying which genes are regulated we can identify new pharmacological targets and start to investigate the effects of therapeutics to interfere with acute and chronic changes of nerve function in airway diseases.

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 border="0" cellpadding="0" cellspacing="0" width="942">
	<tbody>
		<tr height="38">
			<td height="38" style="height:39px;width:405px;">Fast Blue</td>
			<td style="width:157px;">Polysciences, Inc.</td>
			<td style="width:132px;">17740-2</td>
			<td style="width:248px;">stock 2 mg/ml in water</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">NeuroTrace 530/615 red Nissle stain</td>
			<td>Life Technologies</td>
			<td>N21482</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:405px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS) Ca and Mg free</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">glutamine (Glutamax)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">B27 (no vitamin A)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Nerve Growth Factor (NGF)</td>
			<td>Sigma</td>
			<td>N6009</td>
			<td>stock 50 &#181;g/ml in PBS/10% FBS</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">digestion enzyme, Liberase DH Research Grade</td>
			<td>Roche</td>
			<td>5401054001</td>
			<td>stock 2.5 mg/ml in water</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">particle solution (Percoll)</td>
			<td>Sigma</td>
			<td>P1644-25ML</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Heating block</td>
			<td>LabNet</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">70 um cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Absolute Ethanol (200 proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-500</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNase free water</td>
			<td>Fisher Scientific</td>
			<td>BP2484-100</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNase decontamination reagent, RNase AWAY</td>
			<td>invitrogen</td>
			<td>10328-011</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">2-mercaptoethanol</td>
			<td>VWR</td>
			<td>EM-6010</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNA extraction kit, RNeasy Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>74034</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">DNase kit, RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">DNase</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>stock 10 mg/ml in 0.15 M NaCl</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Propidium Iodide</td>
			<td>Sigma</td>
			<td>P4170-10MG</td>
			<td>stock 10 &#181;g/ml in PBS</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">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 DiI labels nodose neurons. However, DiI takes longer to reach the ganglion, 7 - 14 days after instillation18,19, therefore an actively transported dye was used. Fluorogold is an actively transported dye that has also been used to label airway neurons14,20. In our experience, however, once this dye reached the cell bodies in the nodose ganglion it was quickly picked up by satellite cells and not neurons (data not shown). Fast Blue is an alternate actively transported fluorescent dye that successfully, and quickly, labels DRG in rats21 and airway-innervating nodose neurons13,14. The Fast Blue approach, therefore, appears more robust and reproducible.
The dissection of the nodose ganglion has been previously described16 and its location is briefly illustrated in Figure 1B. Digestion begins immediately after the nodose ganglia have been excised. To prevent neurons from clumping together and allow for a cleaner sort, use a gentle digestion enzyme, clear any debris and add RNase to the ganglia sorting solution. Multiple digestion enzyme mixes were tested for effectiveness. A mild digestion enzyme mix22 was tried, however, for adult ganglion digestion it was not strong enough to break apart cells in a timely fashion. Alternatively, a previously used23 combination of papain, protease, and collagenase enzyme mixture, proved to be too aggressive and caused undesirable cell death (data not shown). The outlined digestion protocol, with the specified enzyme mix, was found to be the ideal for a timely digestion with high cell viability.
Previous methods have used manual cell picking with a glass pipette to separate labeled cells from unlabeled cells24. This technique is better suited for single cell analysis of a small number of cells. However, with a maximal rate of 48 cells/hr this technique is impractical when whole populations of hundreds or thousands of cells need to be collected. When the goal is to isolate high quality RNA, time becomes a key variable. It is important to begin RNA extraction as quickly as possible to prevent degradation. FAC sorting allows collection of the entire labeled population in ~45 min&#160;and RNA extraction is started immediately after.
When sorting neurons it is important to clear as much debris from broken neuronal processes. A density gradient was used to clear the debris from the dissociated samples. It is important to centrifuge at a high enough force to ensure that C-fiber neurons, which have a small radius, ~5 &#181;m24, are forced through the gradient.
Figure 2A and B show the negative and positive controls used to set the sorting gates. As can be seen in Figure 2C, the dissociated Fast Blue labeled nodose cells sort into two populations. The cell numbers collected from this sample are listed in Figure 2D. Sorting efficiency for this method is 73 - 85%. When sorting neurons the forward scatter and side scatter parameters did not add specificity. Since neurons are not spherical these parameters were unable to reliably report cell size.
For high quality RNA yield, cells need to be sorted directly into the lysis buffer and RNA should be extracted immediately after sorting. A microfluidic electrophoresis system was used to test the quality of RNA (Figure 3). The 18S and 28S peaks are determined via gel electrophoresis used to calculate an RNA integrity number (RIN). For RNA sequencing the sample RIN should be greater than 7. This RNA sample is now ready to be sequenced.
<img alt="Figure 1" src="/files/ftp_upload/53917/53917fig1.jpg" /> 
Figure 1.&#160;Fast Blue Labeling of Airway Innervating Neurons in the Nodose Ganglia of the Vagus Nerve. A) A representation of Fast Blue intranasal instillation into the lungs. Under light sevoflurane anesthesia, pipette 40 &#181;l&#160;of Fast Blue in PBS with 1% DMSO into the nostrils of a mouse, making sure the mouse is aspirating in the fluid. B) A diagram showing where the nodose ganglion is located. It appears as a thickening of the vagus nerve, which runs along the carotid artery. C) After at least 2 days, Fast Blue appears in the nodose ganglia, marking airway neurons. The nodose is counterstained with red fluorescent Nissle stain, which stains all neurons. Scale bar is 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53917/53917fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53917/53917fig2.jpg" /> 
Figure 2.&#160;Fluorescence Activated Cell (FAC) Sorting Gates for Fast Blue Positive Cells. To set up sorting gates use the negative (A) and positive (B) controls to establish the Fast Blue positive population. The negative control (A) is dissociated DRG cells that do not innervate the lungs. The positive control (B) is dissociated DRG cells that are incubated with Fast Blue in vitro, after they have been dissected. Due to the non-spherical shape of dissociated neurons, the forward scatter and side scatter parameters will not help define the cell population. C) A representative sort of dissociated nodose ganglia cells of mice whose airways have been exposed to Fast Blue. D) The cell count for (C). Over 1,000 Fast Blue (FB) cells were collected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53917/53917fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53917/53917fig3.jpg" /> 
Figure 3.&#160;Assessment of RNA Quality and Assignment of RNA Integrity Number (RIN) Using a Microfluidic Electrophoresis System. Using an electrophoresis system the quality of RNA is calculated from the 18S and 28S peaks on the pictured gel image. The lower peak represents degraded RNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53917/53917fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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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

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

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

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

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

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).