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,....

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 t....

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