Name: a method to target and isolate airway-innervating sensory neurons in mice
Uploaded: 2016-04-19T15:00:00
Description: Watch this Scientific Journal Video about A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice at JoVE.com

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...

<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>

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 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 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 ...

The overall goal of this experiment is to label and isolate the airway-innervating nodose neurons of the vagus nerve. This method can help answer key questions in the Peripheral Neurobiology field. Such as, what identifying properties do airway-innervating neurons possess?The main advantage of this technique is that it provides a robust and high throughput method for targeting and isolating specific neuronal populations. After harvesting the sensory ganglia, aspirate 500 microliters of ganglia dissociation solution from each tube, without disturbing the ganglia. Next, add one milliliter of PBS to each sample, and wait for the ganglia to settle to the bottom of the tubes.Then, remove the PBS and add one milliliter of ganglia dissociation solution supplemented with digestion enzyme to the neurons, followed by the addition of 20 microliters of Fast Blue to the positive control tube. Incubate the tubes at 37 degrees Celsius for the appropriate time based on the age of the digestion enzyme. Shaking and flicking the tubes briefly every 15 minutes, to ensure the ganglia are covered by the digestion solution.While the nerve cells are being digested, slowly and carefully add 400 microliters of freshly prepared 12%density solution over 400 microliters of 28%density solution in one 1.5 milliliter tube per sample. Two distinct layers should be visible, one darker than the other. Next, label 1.5 milliliter collection tubes for sorting, including one Fast Blue positive and one Fast Blue negative tube per sorting sample.At the end of the digestion period, discard the digestion enzyme and wash the ganglia two times with one milliliter of PBS as just demonstrated. After the second wash add 200 microliters of fresh ganglia dissociation solution to each tube. Then, using a 200 microliter pipette set to a 100 microliter volume, pipette the ganglia up and down several times to dissociate the cells into a single cell suspension, taking care to avoid bubbles.When no intact tissue pieces are visible, holding a 70 micron cell strainer firmly in one hand, pipette the dissociated cell solution into the center of the strainer. Then wash the dissociation tube with 100 microliters of fresh ganglia dissociation solution to collect any additional cells. Filer the wash through the strainer and use a new pipette tip to aspirate any remaining cell containing solution on the underside of the strainer, adding it to the cell suspension.Now carefully layer all 300 microliters of the cells on top of the previously prepared density gradient. And collect the ganglia cells by centrifugation. While the cells are spinning add 300 microliters of freshly prepared lysis buffer to the Fast Blue positive collection tube and 600 microliters of lysis buffer to the negative collection tube to ensure that the sorting sheath fluid does not significantly dilute the lysis buffer.At the end of the centrifugation, carefully remove the top 700 microliters of solution containing the majority of the cell debris. Then mix 700 microliters of fresh ganglia dissociation solution with the remaining cells. Pellet the cells by centrifugation and carefully discard the supernatant.Then re-suspend the cells in 200 to 300 microliters of sorting ganglia dissociation solution with a 1, 000 microliter pipette set to 200 microliters. And place the cells on ice until they are ready for sorting. After purifying the ganglia cells by FACS, homogenize the neurons in lysis buffer for one minute with vortexing.Next, mix the cells several times with one volume of 70%ethanol and transfer up to 700 microliters of the sample to a spin column. Centrifuge the column for 15 seconds at 8, 000 x g in a microcentrifuge and discard the flow through. When the entire sample has been passed through the spin column, extract the RNA according to standard RNA purification protocols.Then use a microfluidic electrophoresis system to test the RNA quality according to the manufacturer's specifications. In these graphs, the negative and positive Fast Blue controls used to set the sorting gates are shown. The dissociated Fast Blue Labeled Nodose cells can then be sorted into Fast Blue positive and Fast Blue negative populations.The sorting efficiency for this method is 73 to 85%with approximately 1, 500 or fewer Fast Blue positive and nearly 60, 000 Fast Blue negative cells collected on average. As observed for this representative sample, the 18S and 28S peaks attained by microfluidic electrophoresis can then be used to calculate the RNA integrity number to determine the quality of the extracted RNA. Once mastered, this technique can be completed in four and a half hours if it's performed properly.While attempting this procedure, it's important to remember to move through the steps quickly and to use freshly prepared solutions for RNA extraction. Following this procedure, other methods like neuronal culturing can be performed to answer additional questions, like what functional properties are unique to airway-innervating neurons? After its development, this technique paved the way for researchers in the field of Peripheral Neurobiology to explore the unique properties of a specific airway-innervating population of neurons in the nodose ganglia of the vagus nerve.After watching this video, you should have a good understanding of how to label and dissociate specific neurons of the nodose ganglia which can then be used for RNA sequencing, or plated for functional studies. Don't forget that working with 2-Mercaptoethanol can be extremely hazardous, and that precautions such as wearing gloves and working in a chemical hood should always be taken when using this compound.

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

Research

JoVE Journal

Neuroscience

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).