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>
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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 ...

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