Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Summary

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

Abstract

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Introduction

The cell bodies of vagal afferents are contained in the jugular, petrosal, and nodose ganglia^1,2,3. Their axons travel together via several branches of the vagus nerve to craniocervical, thoracic, and abdominal territories^4,5,6,7. From their visceral endings, vagal afferents can respond to a wide range of physiological and noxious stimuli^8,9,10. Ho....

Protocol

NOTE: Mice used in this study were wild-type males on a pure C57BL/6J background. A total of 4 mice were used for multiplex ISH. All mice were approximately 8 weeks old at the time of sacrifice. One male mouse (approximately one-year-old) was also used to demonstrate endogenous fluorescence associated with aging. Animals were housed in ventilated cages within a barrier facility with ad libitum access to food and water. The UT Southwestern Medical Center Institutional Animal Care and Use Committee reviewed and approved the procedures described below. Details about reagents and tools can be found in the Table of Materials.

Results

While RNAScope can be applied to animals of any age, sex, or genetic background, it is advisable to work with young adults (<3 months old). This is because fluorescent artifacts (e.g., lipofuscin) are common findings in neurons of older animals⁴¹. The formalin-fixed ganglia from older mice often contain surprisingly intense endogenous fluorescence that can easily be mistaken for genuine staining (Figure 1A,B). In any case, it is advisable to verify.......

Discussion

The technique of ISH was invented in the late 1960s⁴². However, it is not until the mid-1980s that it was applied for the detection of mRNAs in the central and peripheral nervous systems^43,44. Considering the heterogeneity of the nervous system and recurring issues with antibodies, localizing a particular transcript at the cellular level remains an invaluable tool. Nonetheless, traditional ISH methods have remained laborious and variably s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x PBS	Fisher Scientific	BP399-4
20x SSC	Invitrogen	AM9763
-80°C freezer	PHCBI	MDF-DU901VHA-PA
Adobe Photoshop 2021	Adobe		photo and design software
Baking oven	Thermo Scientific	Model:658
Confocal microscope	Zeiss	LSM880 Airyscan
Cover glass	Brain Research Laboratories	2460-1.5D
Cryostat	Leica	CM 3050 S
Dumont #5 Forceps	F.S.T.	11252-20
Ecomount	Biocare Medical	EM 897L	mounting medium
HybEZ oven			hybridization oven
Hydrophobic pen	Vector Laboratories	H-4000
ImageJ-Fiji	NIH
Large scissors	Henry Schein	100-7561
Micro centrifuge tubes	VWR	20170-333
Minipump variable flow	Fisher Scientific	13-876-1
Opal 520	Akoya biosciences	FP1 1487001KT	Fluorescent biomarker
Opal 570	Akoya biosciences	FP1 1488001KT	Fluorescent biomarker
Opal 690	Akoya biosciences	FP1 1497001KT	Fluorescent biomarker
ProLong Gold Antifade Mountant			mounting medium for fluorescently labeled cells
RNAscope Multiplex Fluorescent Reagent Kit v2	ACD /Bio-Techne	323100	multiplex kit
RNAscope probe Mouse Cck1r-C3	ACD /Bio-Techne	313751-C3
RNAscope probe Mouse DapB	ACD /Bio-Techne	310043
RNAscope probe Mouse Ghsr	ACD /Bio-Techne	426141
RNAscope probe Mouse Phox2b-C2	ACD /Bio-Techne	407861-C2
RNAscope probe Mouse Prdm12-C2	ACD /Bio-Techne	524371-C2
RnaseZap	Sigma	R2020	Rnase decontaminating solution
Small dissecting scissors	Millipore Sigma	Z265977
Superfrost Plus slides	Fisherbrand	1255015
Tissue Tek OCT medium	Sakura	4583
User manual	ACD	323100 USM
Vannas Spring Scissors	Roboz	RS 5620
ZEN Imaging Software	Zeiss