Name: detection of g protein-coupled receptor expression in mouse vagal afferent neurons using multiplex in situ hybridization
Uploaded: 2021-09-20T15:00:00
Description: Watch this Scientific Journal Video about Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization at JoVE.com

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.

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 ap...

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...

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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detection of g protein-coupled receptor expression in mouse vagal afferent neurons using multiplex in situ hybridization

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.

While RNAScope can be applied to animals of any age, sex, or genetic background, it is advisable to work with young adults (&#60;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 verif...

Watch this Scientific Journal Video about Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization at JoVE.com

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

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.

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

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...

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