Unbiased “omics” techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease.

SummaryStructural variants of an agonist peptide-major histocompatibility complex (MHC) molecule ligand can show partial agonist and/or antagonist properties. A number of such altered ligands appear to act as pure antagonists. They lack any detectable ability to induce T cell effector function and have been described as unable to induce calcium transients and turnover of inositol phosphates. This has been interpreted as an inability of these ligands to initiate any T cell receptor (TCR)-dependent signal transduction, with their antagonist properties ascribed to competition with offered agonist for TCR occupancy. Yet antagonists for mature CD8 + T cells can induce positive selection of thymocytes, implying active induction ofT cell differentiation events, and partial agonists or agonist/antagonist combinations elicit a distinctive pattern of early TCR-associated tyrosine phosphorylation events in CD4 + T cells. We have therefore directly examined proximal TCR signaling in a CD8 + T cell line in response to various related ligands. TCR engagement with natural peptide-MHC class I agonist resulted in the same pattern of early TCR-associated tyrosine phosphorylation events as seen with CD4 + cells, including accumulation of both the p21 and p23 forms of phosphorylated ~, phosphorylation of CD3e, and association ofphosphorylated ZAP-70 with the TCR. Two antagonists that lacked the ability to induce any detectable CTL effector response (cytolysis, esterase release, ~/interferon secretion, interleukin-2 receptor o~ upregulation) were nevertheless found to also induce TCR-dependent phosphorylation events. In these cases, there was preferential accumulation of the p21 form of phospho-~ without net phosphorylation of CD3e, as well as the association of nonphosphorylated ZAP-70 kinase with the receptor. These data show that variant ligands induce similar TCR-dependent phosphorylation events in CD8 + T cells as first observed in CD4 + cells. More importantly, they demonstrate that some putatively pure antagonists are actually a subset of partial agonists able to induce intracellnlar biochemical changes through the TCR. This delivery of a partial signal by antagonists raises the possibility that antagonism in some cases may result from active interference with stimulation ofeffector activity by agonist in mature T cells, while the same variant signal could selectively trigger intracellular events that allow positive without negative selection in thymocytes.

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We have previously shown, in the rat, that neuropathic and inflammatory events produce a neuroplastic change in nociceptor function whereby a subsequent exposure to a proinflammatory mediator (e.g. prostaglandin E2 ; PGE2 ) produces markedly prolonged mechanical hyperalgesia. While the initial approximately 30 min of this prolonged PGE2 hyperalgesia remains PKA-dependent, it subsequently switches to become dependent on protein kinase C epsilon (PKCε). In this study we tested the hypothesis that the delayed onset, PKCε-mediated, component of PGE2 hyperalgesia is generated by the active release of a nucleotide from the peripheral terminal of the primed nociceptor and this nucleotide is then metabolized to produce adenosine, which acts on a Gi-coupled A1 adenosine receptor on the nociceptor to generate PKCε-dependent hyperalgesia. We report that inhibitors of ATP-binding cassette transporters, of ecto-5'-phosphodiesterase and ecto-5'nucleotidase (enzymes involved in the metabolism of cyclic nucleotides to adenosine) and of A1 adenosine receptors each eliminated the late, but not the early, phase of PGE2 -induced hyperalgesia in primed animals. A second model of chronic pain induced by transient attenuation of G-protein-coupled receptor kinase 2, in which the prolongation of PGE2 hyperalgesia is not PKCε-dependent, was not attenuated by inhibitors of any of these mechanisms. Based on these results we propose a contribution of an autocrine mechanism, in the peripheral terminal of the nociceptor, in the hyperalgesic priming model of chronic pain.