Pharmacological target-focused transcriptomic analysis of native vs cultured human and mouse dorsal root ganglia

Abstract

Dorsal root ganglion (DRG) neurons detect sensory inputs and are crucial for pain processing. They are often studied in vitro as dissociated cell cultures with the assumption that this reasonably represents in vivo conditions. However, to the best of our knowledge, no study has directly compared genome-wide transcriptomes of DRG tissue in vivo versus in vitro or between laboratories and culturing protocols. Comparing RNA sequencing-based transcriptomes of native to cultured (4 days in vitro) human or mouse DRG, we found that the overall expression levels of many ion channels and G-protein–coupled receptors specifically expressed in neurons are markedly lower although still expressed in culture. This suggests that most pharmacological targets expressed in vivo are present under the condition of dissociated cell culture, but with changes in expression levels. The reduced relative expression for neuronal genes in human DRG cultures is likely accounted for by increased expression of genes in fibroblast-like and other proliferating cells, consistent with their mitotic status in these cultures. We found that the expression of a subset of genes typically expressed in neurons increased in human and mouse DRG cultures relative to the intact ganglion, including genes associated with nerve injury or inflammation in preclinical models such as BDNF, MMP9, GAL, and ATF3. We also found a striking upregulation of a number of inflammation-associated genes in DRG cultures, although many were different between mouse and human. Our findings suggest an injury-like phenotype in DRG cultures that has important implications for the use of this model system for pain drug discovery.

Global transcriptome analysis of rat dorsal root ganglia to identify molecular pathways involved in incisional pain

“…For peripheral mechanisms of incisional pain, it is critical to identify molecular changes in primary afferent neurons that innervate incised tissues. In this regard, next-generation high-throughput sequencing (NGS) technology have been used to evaluate the gene expression profiles in several preclinical pain models 12 – 16 but not in incisional pain. Because sensory neurons from different tissues respond uniquely to injury, 17 – 19 it is necessary to assess incisional pain-related changes in gene expression.…”

Upregulation of TRPM3 drives hyperexcitability in nociceptors innervating inflamed tissue

“…First, we developed an acute ex vivo preparation of the relevant part of the spinal column, where the cell bodies of sensory neurons were imaged within an intact DRG, but lacking the peripheral input. This preparation avoids the potential loss of specific neuronal populations as well as rapid alterations in their functional properties that are inherent to the isolation and culturing of DRG neurons (18). The use of WGA-AF647-labelling ensured the specificity for afferent neurons that innervated the hind paw tissue, whereas the use of the TRPV1-cre line to drive GCaMP3 expression allowed the specific recording from neurons involved in thermosensation and nociception.…”

“…In a first analysis, we focused on functional expression of TRP channels in the neuronal cell bodies. Since procedures to isolate and culture DRG neurons significanltly alter the expression levels of many ion channels (18), we developed an assay where the DRG was imaged as a whole in situ, using spinning-disk confocal imaging.…”

A pharmacological interactome platform for discovery of pain mechanisms and targets

“…The Bhattacharyya distance (Bhattacharyya, 1943;Wangzhou et al, 2020) was used to calculate the similarity of the probability distributions of scRNA-seq data from 2 different conditions using the following formula. The related Bhattacharyya Coefficient was used to calculate the amount of overlap in the area under the curve of the two sample distributions being compared :…”