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Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, axonal degeneration, and loss of cutaneous innervation. However, the complete molecular profile underlying the hyper-excitable cellular phenotype of DRG nociceptors in PDN has not been elucidated. This gap in our knowledge is a critical barrier to developing effective, mechanism-based, and disease-modifying therapeutic approaches which are urgently needed to relieve the symptoms of PDN. Using single-cell RNA sequencing we demonstrated an increased expression of the Mas-related G Protein-Coupled Receptor d (Mrgprd) in a subpopulation of DRG neurons in the well-established High-Fat Diet (HFD) mouse model of PDN.In vivocalcium imaging allowed us to demonstrate that activation of Mrgprd receptors expressed by cutaneous afferents produced DRG neuron hyper-excitability and oscillatory calcium waves. Furthermore, Mrgprd-positive cutaneous afferents persist in diabetic mice skin. Importantly, limiting Mrgprd signaling or Mrgprd-positive DRG neuron excitability, reversed mechanical allodynia in the HFD mouse model of PDN. Taken together, our data highlights a key role of Mrgprd-mediated DRG neuron excitability in the generation and maintenance of neuropathic pain in a mouse model of PDN. Hence, we propose Mrgprd as a promising accessible target for developing effective therapeutics currently unavailable for treating neuropathic pain in PDN. Furthermore, understanding which DRG neurons cell type is mediating mechanical allodynia in PDN is of fundamental importance to our basic understanding of somatosensation and may provide an important way forward for identifying cell-type-specific therapeutics to optimize neuropathic pain treatment and nerve regeneration in PDN.

The extracellular matrix (ECM) is a dynamic structure composed of a large number of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological and pathological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are often associated with painful human conditions and murine experimental pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. To expand the knowledge on ECM composition and synthesis in the peripheral nervous system, we used a transcriptomal approach to investigate the expression and cellular origin of matrisome genes in murine and human dorsal root ganglia (DRG), containing the cell bodies of sensory neurons. Bulk RNA sequencing data showed that over 60% 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). Examination of the cellular origin of matrisome expression by single cell RNA sequencing on murine DRG revealed that core matrisome genes, especially collagens, were expressed by vascular leptomeningeal-like (fibroblast) cell types whereas matrisome-associated genes were mainly expressed by neuronal cell types. We analyzed cell-cell communication networks with the CellChat R package and predicted an important role for the Collagen signaling pathway in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscopein situhybridization and immunohistochemistry confirmed expression of collagens in fibroblasts surrounding nociceptors in human DRG. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human. The identification of the cellular distribution of murine and human matrisome genes provides a framework to study the role of the ECM in peripheral nervous tissue and its effects on pain signaling.HighlightsTranscriptomal analyses of mouse and human dorsal root ganglia (DRG) revealed that over 60% of matrisome genes are expressed by murine and human dorsal root ganglia (DRG), with over 85% of the genes with orthologues overlapping between both species.Matrisome-associated genes had the highest expression in both species and included conserved expression of annexins, S100 calcium binding proteins and cathepsins.Collagens and collagen receptors are expressed by distinct cell types in murine and human DRG, suggesting that the collagen signaling pathway could be involved in cell-cell signaling.

Microelectrode arrays (MEAs) have become important tools in high throughput assessment of neuronal activity. However, geometric and electrical constraints largely limit their ability to detect action potentials to the neuronal soma. Enhancing the resolution of these systems to detect axonal action potentials has proved both challenging and complex. In this study, we have bundled sensory axons from dorsal root ganglia through a capillary alginate gel (Capgel™) interfaced with an MEA and observed an enhanced ability to detect spontaneous axonal activity compared with two-dimensional cultures. Moreover, this arrangement facilitated the long-term monitoring of spontaneous activity from the same bundle of axons at a single electrode. Finally, using waveform analysis for cultures treated with the nociceptor agonist capsaicin, we were able to dissect action potentials from multiple axons on an individual electrode, suggesting that this model can reproduce the functional complexity associated with sensory fascicles in vivo. This novel three-dimensional functional model of the peripheral nerve can be used to study the functional complexities of peripheral neuropathies and nerve regeneration as well as being utilized in the development of novel therapeutics.

Satellite glial cells (SGCs) are glial cells in the peripheral nervous system that form sheaths around the neuronal cell body. This unique arrangement of SGCs allows it to exert a highly regulated control over the neuronal microenvironment. Not much is known about the origin of SGCs. In this study, we examine the development of SGCs. We show that rat SGCs develop postnatally and these cells express a number of markers associated with Schwann cell precursors, in particular cadherin-19 (CDH19) even in adult DRGs. We developed a method for the purification of SGCs and showed that they are transcriptionally and morphologically very similar to adult rat Schwann cells (SCs). Finally, we demonstrate that purified SGCs are capable of myelinating embryonic axons when cocultured with those axons. Based on these observations we hypothesize that SGCs represent a population of cells in the SC lineage, whose further differentiation appears to be arrested through contact with DRG neuronal soma.