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.

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.

No abstract

In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E 2, epinephrine and carrageenan. Morover, this effect persisted unattenuated 2 weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000, Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.

Mitochondria are present at high concentration at the site of sensory transduction in the peripheral terminals of nociceptors [14; 31]. Since nerve growth factor (NGF), which induces nociceptor sensitization by acting on the high affinity TrkA receptor [28], also produces local recruitment of mitochondria in DRG neurons [5; 6], we evaluated the role of mitochondria in NGF-induced mechanical hyperalgesia. Inhibition of three major mitochondrial functions—oxidation of nutrients, ATP production, and generation of reactive oxygen species—markedly attenuated NGF-induced mechanical hyperalgesia in the rat. Disruption of microtubules, which are required for the trafficking and subcellular localization of mitochondria, also attenuated NGF hyperalgesia. Our results suggest a contribution of mitochondrial localization and function to NGF-dependent pain syndromes.

Many previous investigators have utilized mechanical rubbing as a method for examining effects of epithelial removal. In the present study, we examined whether this procedure also affects mast cell integrity in the underlying lamina propria. We isolated bronchial rings from six ferrets, and we found that removal of epithelium by rubbing decreased the total number of intact mast cells from 10.0 +/- 1.9 to 2.2 +/- 0.6 (SE) mast cells/mm luminal perimeter (P less than 0.01). In addition, we found a very large number of metachromatic particles that appeared to be mast cell granules unassociated with identifiable mast cells. Their identity was confirmed the presence of free mast cell granules and showed that they contained intact membranes. These effects were not observed when the epithelium was removed by enzymatic digestion, but they were observed after mechanical deformation of the bronchi alone. We suggest that mast cell components released by removing or damaging the epithelium may affect the function of various cells in the airway.

Painful small-fiber peripheral neuropathy is a debilitating complication of chronic alcohol abuse. Evidence from previous studies suggests that neuroendocrine mechanisms, in combination with other, as yet unidentified actions of alcohol, are required to produce this neuropathic pain syndrome. In addition to neurotoxic effects of alcohol, in the setting of alcohol abuse neuroendocrine stress axes release glucocorticoids and catecholamines. Since receptors for these stress hormones are located on nociceptors, at which they can act to cause neuronal dysfunction, we tested the hypothesis that alcohol and stress hormones act on the nociceptor, independently, to produce neuropathic pain. We used a rat model, which allows the distinction of the effects of alcohol from those produced by neuroendocrine stress axis mediators. We now demonstrate that topical application of alcohol and exposure to unpredictable sound stress, each alone, has no effect on nociceptive threshold. However, when animals that had previous exposure to alcohol were subsequently exposed to stress, they rapidly developed mechanical hyperalgesia. Conversely, sound stress followed by topical alcohol exposure also produced mechanical hyperalgesia. The contribution of stress hormones was prevented by spinal intrathecal administration of oligodeoxynucleotides antisense to β2-adrenergic or glucocorticoid receptor mRNA, which attenuates receptor level in nociceptors, as well as by adrenal medullectomy. These experiments establish an independent role of alcohol and stress hormones on the primary afferent nociceptor in the induction of painful peripheral neuropathy.