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Method Article

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

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DOI:

10.3791/62945

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September 20th, 2021

 , 
1Center for Hypothalamic Research and Department of Internal Medicine, UTSouthwestern Medical Center at Dallas

In This Article

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 ganglia1,2,3. Their axons travel together via several branches of the vagus nerve to craniocervical, thoracic, and abdominal territories4,5,6,7. From their visceral endings, vagal afferents can respond to a wide range of physiological and noxious stimuli8,9,10. However, the distribution of signaling molecules and receptors involved in vagal sensing remains poorly characterized. This is partly because the vagal ganglia, in spite of their small size, express a broad spectrum of receptors, including a large number of GPCRs8,11,12,13. Moreover, vagal afferent neurons are inherently heterogeneous and display distinct molecular profiles14. To complicate the matter, the jugular, petrosal, and nodose ganglia are attached in the mouse, thereby forming a single ganglionic mass. Lastly, in a subset of animals, the nodose ganglion is attached to the sympathetic superior cervical ganglion15.

In the past, investigators have turned to immunohistochemistry to study the neurochemical make-up of vagal afferent neurons16,17,18. While immunohistochemistry using validated antibodies is useful, the results of immunohistochemical studies must be interpreted with caution. For example, numerous efforts to identify specific antibodies against GPCRs have failed19,20,21,22,23,24,25, leading investigators to conclude that the majority of antibodies against GPCRs are unreliable. To circumvent these issues, quantitative PCR (qPCR) has been widely used for assessing gene expression in the rodent vagal ganglionic mass26,27,28,29. However, examining gene expression using qPCR occurs at the cost of a loss of spatial information. In particular, it cannot be predicted how many cells or what cell type(s) express a particular gene of interest (e.g., nodose vs. jugular cells). Recurring issues also include the contamination with adjacent tissues and the inclusion of variable lengths of the vagus nerve, superior cervical, and jugular ganglia during dissection15. As a result of the above difficulties, controversy surrounds the expression and distribution of several GPCRs in vagal afferent neurons. One particularly puzzling example relates to the ghrelin receptor (Ghsr). Whereas some studies have found widespread expression of this receptor in vagal afferent neurons30,31,32, others have found Ghsr mRNA to be nearly undetectable in the nodose ganglion11,14. Detailed mapping of Ghsr mRNA in the vagal ganglionic mass is therefore warranted.

In situ hybridization (ISH) has also been used to assess gene expression patterns in the vagal ganglionic mass7,11,12,33,34,35. Because RNA-based techniques remain more reliable and specific than antibody-based techniques under most circumstances36,37, ISH studies have proven valuable for better understanding of the neurochemical coding of vagal afferent neurons. Nonetheless, traditional ISH techniques themselves are not without caveats. Radioactive ISH is sensitive but generates background and remains cumbersome38. Non-radioactive ISH is less complicated but also less sensitive38. In contrast, the recently developed RNAscope ISH method is highly sensitive and generates minimal background39. The current study applied multiplex fluorescent RNAscope to the detection of GPCRs in vagal afferent neurons of the mouse. We focused on mapping the distribution of Ghsr and compared its distribution to that of the cholecystokinin receptor (Cck1r), another GPCR well known to be expressed in the nodose ganglion34. Lastly, the two transcription factors, paired-like homeobox 2b (Phox2b) and PR domain zinc finger protein 12 (Prdm12), were used as selective markers for nodose and jugular afferent neurons, respectively14. Without visualizing Phox2b or Prdm12, it would be challenging to identify jugular vs. nodose afferents with certainty. Potential technical pitfalls are also discussed throughout the article.

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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.

1. Sample collection

  1. On the day of sacrifice, administer an overdose of chloral hydrate (500 mg/kg, i.p.) to the mice. Perfuse the deeply anesthetized mice transcardially using a peristaltic pump with 5 mL of 1x phosphate-buffered saline (PBS) followed by 50 mL of 10% formalin at room temperature.
    NOTE: This is done in a fume hood to prevent inhalation of formalin.
  2. As the nodose, petrosal, and jugular ganglia are fused to form a single ganglionic mass in the mouse15, carefully collect the entire vagal ganglionic mass from each side (left and right) with the help of a dissecting scope and fine spring scissors and forceps (see the Table of Materials).
  3. When decapitating the mouse with a pair of large scissors (see the Table of Materials), avoid crushing the brainstem.
  4. Following a dissection approach described before in the rat40, remove the sternohyoid and omohyoid muscles lateral to the trachea with small forceps (Table of Materials) to expose the occipital bone. Clear the whole region of muscle, adipose tissue, and conjunctive tissue until the carotid artery, vagus nerve, the hypoglossal nerve, and foramen magnum are visible. Cut the entire hypoglossal nerve with small spring scissors (Table of Materials).
  5. Look for the nodose ganglion as a translucent mass close to the foramen magnum15. Using small scissors (Table of Materials), carefully break the occipital bone to further expose the jugular ganglion. Look for black melanocytes at the surface of the jugular as a visual landmark.
  6. Cut nerve attachments between the vagal ganglionic mass and extract the whole ganglionic mass by gently pulling on the peripheral end of the vagus nerve while simultaneously cutting the jugular ganglion towards the brainstem with small spring scissors (Table of Materials).
  7. Remove the conjunctive tissue and fat sticking to the vagus nerve and ganglionic mass as much as possible.
  8. As it is easy to lose one ganglion during the transfer from one tube to the other, keep ~0.5 cm of the vagus nerve to facilitate handling and localization of samples.
  9. Place ganglia with the help of fine forceps in 1.5 mL microcentrifuge tubes and incubate at 4 °C in formalin for 24 h and with 30% sucrose for another 24 h.
  10. Position ganglia inside a small drop (~4 mm Ø) of optimum cutting temperature (OCT) medium on a piece of aluminum foil. Next, freeze the sample on a bed of dry ice.
  11. Cut the samples with a cryostat at -20 °C into sections of 14 µm thickness and collect on glass microscope slides as 3 series 42 µm apart.
    NOTE: Avoid placing sections close to the edges of the slide. To save reagents, keep the tissue sections as tightly packed as possible but without overlapping.
  12. Store the samples in a -80 °C freezer until needed for ISH for at least 6 months.
    ​NOTE: Refer to Figure 1 for troubleshooting for endogenous fluorescence.

2. Pretreatment and ISH

  1. Before the day of the experiments, autoclave laboratory glassware to be used for ISH, including staining dishes and washing buffer jars.
    NOTE: While working under RNase-free conditions is not critical, wear gloves at all times. Keep RNase decontamination solution bottles (Table of Materials) within reach in case contamination is suspected.
  2. On the first day, bring the slides to room temperature on a bench specifically dedicated to ISH. Rinse the slides in 1x PBS and bake them for 30 mins at 60 °C in a baking oven (Table of Materials).
  3. Post-fix the slides in 4% formalin for 15 min at 4 °C.
  4. Bring the reagents in the multiplex kit (Table of Materials) to room temperature.
  5. Dehydrate the slides in 50%, 70%, and 100% ethanol for 5 min each. Apply H2O2 solution to the samples for 10 min and then rinse in double-distilled water (ddH2O).
  6. Treat the samples with the target retrieval reagent for 5 min, rinse in ddH2O followed by 100% ethanol, and air-dry. Using a hydrophobic pen, create a barrier around the samples.
    NOTE: Do not apply the hydrophobic barrier too close to the tissue sections.
  7. Apply protease for 30 min at 40 °C in a hybridization oven (Table of Materials).
  8. In the meantime, prewarm the probes at 40 °C and cool them at room temperature before use. Incubate the slides with the desired combination of target probes (Cck1r-C3, Ghsr-C1, Phox2b-C2; or Cck1r-C3, Ghsr-C1, Prdm12-C2) for 2 h at 40 °C in a hybridization oven.
  9. Incubate negative control tissue with the dihydrodipicolinate reductase (DapB) target C1 probe for 2 h at 40 °C in a hybridization oven.
  10. Rinse the slides in wash buffer and store overnight in 5x saline sodium citrate (SSC) at room temperature. For convenience, split the experiment into two days. On the following day, rinse the slides with wash buffer and incubate them with amplification reagents listed below (see the user manual in Table of Materials).
  11. Apply AMP1 (30 min at 40 °C), AMP2 (30 min at 40 °C), and AMP 3 (15 min at 40 °C). Rinse in wash buffer between each step.
  12. Dissolve each Opal dye (Table of Materials) in dimethyl sulfoxide and store the solutions at 4 °C until needed.
  13. For channel 1, incubate the slides with HRP-C1 (15 min at 40 °C) and Opal 520 (green color; dilution 1/1,500; 30 mins at 40°C). Rinse in wash buffer between each step.
  14. For channel 2, incubate the slides with HRP-C2 (15 min at 40 °C) and Opal 570 (yellow color; dilution 1/1,500; for 30 min at 40 °C). Rinse in wash buffer between each step.
  15. For channel 3, incubate the slides with HRP-C3 (15 min at 40 °C) and Opal 690 (far red color; dilution 1/1,500; for 30 min at 40 °C). Rinse in wash buffer between each step.
  16. Wash the slides and expose them to 4′,6-diamidino-2-phenylindole (DAPI) for 30 s.
  17. Immediately apply mounting medium on each slide and place a coverslip over the tissue section.
  18. Keep the tissues horizontal in a slide holder at 4 °C until imaging.

3. Microscopy and data analysis

NOTE: Multiplex ISH data can be imaged with a wide range of instruments with the appropriate filters. However, a preferred imaging method is confocal microscopy with 20x and 63x (oil) objectives; refer to the discussion for the reasons. Refer to Figure 2 for the determination of the optimal signal-to-noise ratio.

  1. Use a confocal microscope equipped with 488, 561, and 633 nm laser lines. Use the following acquisition parameters (see Table 1): Opal 690 (HeNe 633 nm, detection range 668-696 nm, power 2.0, gain 724); Opal 570 (DPSS laser 561 nm, detection range 579-627 nm, <power 15.0, <gain 450); Opal 520 (argon laser 488 nm, detection range 499-535 nm , <power 6.0, <gain 830); DAPI (diode laser 405, detection range 415-502 nm, <power 12.0, <gain 532).
  2. To improve the quality of the images, acquire with a line averaging of 4 and a pixel size of at least 1024 x 1024.
    NOTE: The above parameters are only provided as an example, and modifications are recommended depending on one instrument, magnification (20x or 63x), expression levels, and endogenous levels of fluorescence for any given tissue.
  3. Once satisfied with the acquisition parameters, collect images using the same settings. Attribute false colors to each channel to facilitate visualization.
    NOTE: For example, red (Phox2b or Prdm12), cyan (Cck1r), yellow (Ghsr), and grey (DAPI).
  4. Perform cell counting using the digital images by identifying the outline of RNAscope-positive profiles with one nucleus lightly stained with DAPI. Calculate the percentage of RNAscope-positive profiles expressing each transcript [Figure 3A-C].
  5. To improve the rigor and accuracy of the estimates, count at least 2,000 neuronal profiles from at least 4 different animals. Perform counting from left and right ganglia separately.
  6. Input the data in a pie chart with the total number of counted profiles.
  7. Use imaging software to acquire images and stitch together 20x images. Use ImageJ-Fiji and another photo and design software to generate the final plates. Apply adjustments to contrast and lightness uniformly to all images.

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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...

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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 systems43,44. 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...

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Disclosures

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the Neuroanatomy/Histology/Brain Injection Core funded by NIH grant #5P01DK119130-02. The authors would like to acknowledge the assistance of the UT Southwestern Live Cell Imaging Facility (headed by Dr. Phelps) and its staff (Abhijit Bugde and Marcel Mettlen), supported in part by the NIH Grant #1S10OD021684-01, a Shared Resource of the Harold C. Simmons Cancer Center, supported in part by an NCI Cancer Center Support Grant, P30 CA142543.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10x PBSFisher ScientificBP399-4
20x SSCInvitrogenAM9763
-80°C freezerPHCBIMDF-DU901VHA-PA
Adobe Photoshop 2021Adobephoto and design software
Baking ovenThermo ScientificModel:658
Confocal microscopeZeissLSM880 Airyscan
Cover glassBrain Research Laboratories2460-1.5D
CryostatLeicaCM 3050 S
Dumont #5 ForcepsF.S.T.11252-20
EcomountBiocare MedicalEM 897Lmounting medium
HybEZ ovenhybridization oven
Hydrophobic penVector LaboratoriesH-4000
ImageJ-FijiNIH
Large scissorsHenry Schein100-7561
Micro centrifuge tubesVWR20170-333
Minipump variable flowFisher Scientific13-876-1
Opal 520Akoya biosciencesFP1 1487001KTFluorescent biomarker
Opal 570Akoya biosciencesFP1 1488001KTFluorescent biomarker
Opal 690Akoya biosciencesFP1 1497001KTFluorescent biomarker
ProLong Gold Antifade Mountantmounting medium for fluorescently labeled cells
RNAscope Multiplex Fluorescent Reagent Kit v2ACD /Bio-Techne323100multiplex kit
RNAscope probe Mouse Cck1r-C3ACD /Bio-Techne313751-C3
RNAscope probe Mouse DapBACD /Bio-Techne310043
RNAscope probe Mouse GhsrACD /Bio-Techne426141
RNAscope probe Mouse Phox2b-C2ACD /Bio-Techne407861-C2
RNAscope probe Mouse Prdm12-C2ACD /Bio-Techne524371-C2
RnaseZapSigmaR2020Rnase decontaminating solution
Small dissecting scissorsMillipore SigmaZ265977
Superfrost Plus slidesFisherbrand1255015
Tissue Tek OCT mediumSakura4583
User manualACD323100 USM
Vannas Spring ScissorsRobozRS 5620
ZEN Imaging SoftwareZeiss

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G Protein Coupled ReceptorsMultiplex ISHJugular Nodose GangliaRNAscope TechnologyConfocal MicroscopyCholecystokinin ReceptorGhrelin ReceptorPhox2b Marker
Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization
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