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

Summary

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.

Abstract

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.

Introduction

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 airways^1,2. 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, respiration^8,9, and irritant-activated respiratory responses^10,11. 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 weeks^13-15. Once identified, a gentle, yet efficient, dissociation protocol was used to preserve 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.

Protocol

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.

1. Anesthetize the mouse with light inhalation anesthesia (2.5% sevoflurane) until breathing starts to slow.
2. Use a 200 µl pipette with filtered tips to slowly instill 40 µl 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).
3. Hold the mouse vertically, head up, and gently massage the chest to ensure the dye spreads throughout the lungs.

2. Preparations on Dissection and Analysis Day

1. Prepare 10 ml Ganglia Dissociation Solution (GaDS) by combining the ingredients as specified in Table 1.
2. 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.
3. Mix fresh 70%, and 80% ethanol with RNase-free water. Prepare 1 ml per sample to be used in RNA extraction.

Ingredient	Amount
Advanced DMEM/f12	9.5 ml
glutamine	100 µl
HEPES (10 mM)	100 µl
N2 supplement	100 µl
B27 (no vitamin A)	200 µl
NGF (50 µg/ml)	10 µl

Table 1. Reagent Mixture for Ganglia Dissociation Solution (GaDS).

3. Dissection Procedure

1. Fill 1.5 ml tubes with 500 µl 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.
2. 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.
3. 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.
4. To obtain enough nodose neurons for successful sorting, combine ganglia from 5 mice into one experimental sample tube containing GaDS.

4. Sensory Ganglia Dissociation

1. Pipette out 500 µl GaDS, leaving ganglia in the tube. Wash ganglia once by adding 1 ml PBS, wait for ganglia to settle to the bottom of the tube, then pipette out PBS.
2. Add 1 ml GaDS and 22 µl of digestion enzyme (collagenase I/II and protease mix, 2.5 mg/ml in H₂O) to each tube.
3. Add 20 µl Fast Blue (5.26 mM) to the positive control tube.
4. Put the tubes in a water bath or heating block already set at 37 °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.
5. Every 15 min shake tubes briefly and flick them to ensure ganglia are covered by the solution.
6. While ganglia are being digested, prepare density gradients using a particle solution (colloidal silica particles coated with polyvinylpyrrolidone).
   1. Mix 12% particle solution in GaDS, 500 µl per sample.
   2. Mix 28% particle solution in GaDS, 500 µl per sample.
   3. Slowly and carefully add 400 µl 12% density solution on top of 400 µl 28% density solution in a fresh 1.5 ml 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.
7. While ganglia are being digested, label new 1.5 ml 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.
8. After the appropriate digestion time, remove GaDS with digestion enzyme and wash ganglia twice with 1 ml PBS. Add 200 µl of fresh GaDS to each tube.
9. Use a 200 µl pipette, set to 100 µl 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.
10. Pass dissociated cells through a 70 µm cell strainer.
11. Add another 100 µl 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 µl.
12. Carefully layer the 300 µl of cells on top of the previously made density gradient. Avoid bubbles and mixing of cells with the density layers.
13. Centrifuge for 10 min at 2,900 x g at RT.
14. While cells are in the centrifuge, mix lysis buffer (1 ml per experimental sample) as follows: 10 µl 2-mercaptoethanol in 1 ml Buffer RLT (from RNA extraction kit).
    1. Add 300 µl lysis buffer to Fast Blue positive collection tube and 600 µl 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 <2,000 cells, add 300 µl, for >7,000 cells add 600 µl. This will ensure the sorting sheath fluid does not significantly dilute the lysis buffer.
15. Once centrifugation is complete, carefully remove and discard the top 700 µl layer. This layer contains the majority of cell debris that will otherwise interfere with cell sorting.
16. Add 700 µl fresh GaDS to the remaining cell containing solution and pipette up and down several times to mix.
17. Centrifuge for 15 min at 2,900 x g to pellet cells.
18. While cells are in the centrifuge, mix sorting GaDS (500 µl per sample) by adding 5 µl DNase (10 mg/ml) to 1 ml GaDS.
19. Once centrifugation is done, carefully remove and discard supernatant, leaving the cell pellet.
20. Re-suspend cell pellet in 200 - 300 µl sorting GaDS by pipetting up and down with a 1,000 µl pipette set to 200 µl several times. Put samples on ice. Cells are now ready to be sorted.

5. Fluorescence Activated Cell (FAC) Sorting

Note: This section requires operational knowledge of a FACS sorter or the assistance of skilled personnel.

1. Add 1 µl propidium iodide (PI) stock solution (50 µg/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.
2. Transport cells to flow cytometry instrument, e.g., BD FACS AriaII running Diva 8 software, on ice.
3. Since the cell size is variable in this population, use a large nozzle (100 µm) for sorting. To decrease the amount of stress the cells experience and increase cell viability use low pressure (20 psi).
4. Use the FACS sorter software to set up the analysis plots, forward scatter, side scatter (SSC), Fast Blue, and PI, if needed.
5. To re-suspend cells, gently vortex the sample briefly before loading it into the sorter.
6. 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.
7. Sort the positive control sample (incubated with Fast Blue), to set compensation parameters.
8. Once the gates and parameters are set, begin sorting the experimental samples. Samples are sorted into the prepared collection tubes containing RNA lysis buffer.
9. Keep sorted samples on ice until the cell sorting is complete.
10. Begin RNA extraction step immediately after cells are sorted.

6. RNA Extraction

1. Vortex sorted cells in lysis buffer for 1 min to homogenize.
2. Add 1 volume of 70% ethanol and mix by pipetting up and down several times.
3. Transfer up to 700 µl of the sample to a spin column. Centrifuge for 15 sec at 8,000 x g. Discard the flow-through.
   1. Repeat until the total volume of the sample is passed through the spin column.
4. Add 350 µl Buffer RW1 and continue the RNA purification process, including the DNase treatment step, according to the manufacturer's protocol for cells.
5. Once RNA has been extracted, test the RNA quality using a microfluidic electrophoresis system according to the manufacturer's specifications.

Results

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'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and Fluorogold. Lung exposure to DiI labels no...

Discussion

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 ...

Materials

List of materials used in this article

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