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Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.

ABSTRACT: In this protocol, we provide step-by-step instructions for dissection and culture of primary murine dorsal root ganglia (DRG), which provide an opportunity to study the functional properties of peripheral sensory neurons in vitro. Further, we describe the analysis of neuropeptide release by ELISA as a possible downstream application. In addition, isolated DRGs can be used directly for immunofluorescence, flow cytometry, RNA sequencing or proteomic approaches, electrophysiology, and calcium imaging. For complete details on the use and execution of this protocol, please refer to Perner et al. (2020).

SUBMITTER: Perner C

PROVIDER: S-EPMC7876630 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.

Perner Caroline C Sokol Caroline L CL

STAR protocols 20210208 1

In this protocol, we provide step-by-step instructions for dissection and culture of primary murine dorsal root ganglia (DRG), which provide an opportunity to study the functional properties of peripheral sensory neurons <i>in vitro</i>. Further, we describe the analysis of neuropeptide release by ELISA as a possible downstream application. In addition, isolated DRGs can be used directly for immunofluorescence, flow cytometry, RNA sequencing or proteomic approaches, electrophysiology, and calciu ...[more]

PMID: 33615276

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Project description:The extracellular matrix (ECM) is a dynamic structure of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are associated with painful human conditions and murine pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. The goal of this study was to integrate bulk, single-cell, and spatial RNA sequencing (RNAseq) datasets to investigate the expression and cellular origin of matrisome genes in male and female murine and human dorsal root ganglia (DRG). Bulk RNAseq showed that about 65% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators, and secreted factors). Single cell RNAseq on male murine DRG revealed the cellular origin of matrisome expression. Core matrisome genes, especially collagens, were expressed by fibroblasts whereas matrisome-associated genes were primarily expressed by neurons. Cell-cell communication network analysis with CellChat software predicted an important role for collagen signaling pathways in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry demonstrated expression of collagens in fibroblasts surrounding nociceptors in male and female human DRG. Finally, comparing human neuropathic pain samples with non-pain samples also showed differential expression of matrisome genes produced by both fibroblasts and by nociceptors. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human, and that dysregulation of matrisome genes is associated with neuropathic pain.

Dataset Information

Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.

Publications

Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.

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