Manouela V. Valtcheva

Protein kinase Cδ mediates histamine-evoked itch and responses in pruriceptors

BackgroundItch-producing compounds stimulate receptors expressed on small diameter fibers that innervate the skin. Many of the currently known pruritogen receptors are Gq Protein-Coupled Receptors (GqPCR), which activate Protein Kinase C (PKC). Specific isoforms of PKC have been previously shown to perform selective functions; however, the roles of PKC isoforms in regulating itch remain unclear. In this study, we investigated the novel PKC isoform PKCδ as an intracellular modulator of itch signaling in response to histamine and the non-histaminergic pruritogens chloroquine and β-alanine.ResultsBehavioral experiments indicate that PKCδ knock-out (KO) mice have a 40% reduction in histamine-induced scratching when compared to their wild type littermates. On the other hand, there were no differences between the two groups in scratching induced by the MRGPR agonists chloroquine or β-alanine. PKCδ was present in small diameter dorsal root ganglion (DRG) neurons. Of PKCδ-expressing neurons, 55% also stained for the non-peptidergic marker IB4, while a smaller percentage (15%) expressed the peptidergic marker CGRP. Twenty-nine percent of PKCδ-expressing neurons also expressed TRPV1. Calcium imaging studies of acutely dissociated DRG neurons from PKCδ-KO mice show a 40% reduction in the total number of neurons responsive to histamine. In contrast, there was no difference in the number of capsaicin-responsive neurons between KO and WT animals. Acute pharmacological inhibition of PKCδ with an isoform-specific peptide inhibitor (δV1-1) also significantly reduced the number of histamine-responsive sensory neurons.ConclusionsOur findings indicate that PKCδ plays a role in mediating histamine-induced itch, but may be dispensable for chloroquine- and β-alanine-induced itch.

Enhanced nonpeptidergic intraepidermal fiber density and an expanded subset of chloroquine-responsive trigeminal neurons in a mouse model of dry skin itch.

UNLABELLED Chronic pruritic conditions are often associated with dry skin and loss of epidermal barrier integrity. In this study, repeated application of acetone and ether followed by water (AEW) to the cheek skin of mice produced persistent scratching behavior with no increase in pain-related forelimb wiping, indicating the generation of itch without pain. Cheek skin immunohistochemistry showed a 64.5% increase in total epidermal innervation in AEW-treated mice compared to water-treated controls. This increase was independent of scratching, because mice prevented from scratching by Elizabethan collars showed similar hyperinnervation. To determine the effects of dry skin treatment on specific subsets of peripheral fibers, we examined Ret-positive, calcitonin gene-related peptide (CGRP)-positive, and glial cell line-derived neurotrophic factor family receptor α3 (GFRα3)-positive intraepidermal fiber density. AEW treatment increased Ret-positive fibers but not CGRP-positive or GFRα3-positive fibers, suggesting that a specific subset of nonpeptidergic fibers could contribute to dry skin itch. To test whether trigeminal ganglion neurons innervating the cheek exhibited altered excitability after AEW treatment, primary cultures of retrogradely labeled neurons were examined using whole-cell patch clamp electrophysiology. AEW treatment produced no differences in measures of excitability compared to water-treated controls. In contrast, a significantly higher proportion of trigeminal ganglion neurons was responsive to the nonhistaminergic pruritogen chloroquine after AEW treatment. We conclude that nonpeptidergic, Ret-positive fibers and chloroquine-sensitive neurons may contribute to dry skin pruritus. PERSPECTIVE This study examines the underlying neurobiological mechanisms of persistent dry skin itch. Our results indicate that nonpeptidergic epidermal hyperinnervation and nonhistaminergic pruritic receptors are potential targets for chronic pruritus.

Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures

Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

Group II mGluRs suppress hyperexcitability in mouse and human nociceptors

Abstract We introduce a strategy for preclinical research wherein promising targets for analgesia are tested in rodent and subsequently validated in human sensory neurons. We evaluate group II metabotropic glutamate receptors, the activation of which is efficacious in rodent models of pain. Immunohistochemical analysis showed positive immunoreactivity for mGlu2 in rodent dorsal root ganglia (DRG), peripheral fibers in skin, and central labeling in the spinal dorsal horn. We also found mGlu2-positive immunoreactivity in human neonatal and adult DRG. RNA-seq analysis of mouse and human DRG revealed a comparative expression profile between species for group II mGluRs and for opioid receptors. In rodent sensory neurons under basal conditions, activation of group II mGluRs with a selective group II agonist produced no changes to membrane excitability. However, membrane hyperexcitability in sensory neurons exposed to the inflammatory mediator prostaglandin E2 (PGE2) was prevented by (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC). In human sensory neurons from donors without a history of chronic pain, we show that PGE2 produced hyperexcitability that was similarly blocked by group II mGluR activation. These results reveal a mechanism for peripheral analgesia likely shared by mice and humans and demonstrate a translational research strategy to improve preclinical validation of novel analgesics using cultured human sensory neurons.

Metabotropic Glutamate Receptor 2/3 (mGluR2/3) Activation Suppresses TRPV1 Sensitization in Mouse, But Not Human, Sensory Neurons

Abstract The use of human tissue to validate putative analgesic targets identified in rodents is a promising strategy for improving the historically poor translational record of preclinical pain research. We recently demonstrated that in mouse and human sensory neurons, agonists for metabotropic glutamate receptors 2 and 3 (mGluR2/3) reduce membrane hyperexcitability produced by the inflammatory mediator prostaglandin E2 (PGE2). Previous rodent studies indicate that mGluR2/3 can also reduce peripheral sensitization by suppressing inflammation-induced sensitization of TRPV1. Whether this observation similarly translates to human sensory neurons has not yet been tested. We found that activation of mGluR2/3 with the agonist APDC suppressed PGE2-induced sensitization of TRPV1 in mouse, but not human, sensory neurons. We also evaluated sensory neuron expression of the gene transcripts for mGluR2 (Grm2), mGluR3 (Grm3), and TRPV1 (Trpv1). The majority of Trpv1 + mouse and human sensory neurons expressed Grm2 and/or Grm3, and in both mice and humans, Grm2 was expressed in a greater percentage of sensory neurons than Grm3. Although we demonstrated a functional difference in the modulation of TRPV1 sensitization by mGluR2/3 activation between mouse and human, there were no species differences in the gene transcript colocalization of mGluR2 or mGluR3 with TRPV1 that might explain this functional difference. Taken together with our previous work, these results suggest that mGluR2/3 activation suppresses only some aspects of human sensory neuron sensitization caused by PGE2. These differences have implications for potential healthy human voluntary studies or clinical trials evaluating the analgesic efficacy of mGluR2/3 agonists or positive allosteric modulators.

Evidence from cluster analysis for differentiation of antisaccade performance groups based on speed/accuracy trade-offs

Anti and pro-saccade performance in single or mixed contexts was explored in a large sample of young adults (n=281). ANOVAs were first conducted to evaluate trial type, context and gender effects. A cluster analysis was then used to determine whether subgroups could be identified based on saccadic performance variables. Increased antisaccade errors were observed among females and during mixed-saccade runs. Cluster analysis identified two groups: 1) increased errors clustered with faster latencies and 2) decreased errors clustered with slower latencies. These data offer justification for examining subgroups based on saccadic performance and may help elucidate mechanisms underlying response variability within and between different populations.

Macrophage-to-sensory neuron crosstalk mediated by Angiotensin II type-2 receptor elicits neuropathic pain

Peripheral nerve damage initiates a complex series of cellular and structural processes that culminate in chronic neuropathic pain. Our study defines local angiotensin signaling via activation of the Angiotensin II (Ang II) type-2 receptor (AT2R) on macrophages as the critical trigger of neuropathic pain. An AT2R-selective antagonist attenuates neuropathic, but not inflammatory pain hypersensitivity in mice, and requires the cell damage-sensing ion channel transient receptor potential family-A member-1 (TRPA1). Mechanical and cold pain hypersensitivity that are characteristic of neuropathic conditions can be attenuated by chemogenetic depletion of peripheral macrophages and AT2R-null hematopoietic cell transplantation. Our findings show no AT2R expression in mouse or human sensory neurons, rather AT2R expression and activation in macrophages triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on sensory neurons. Our study defines the precise neuro-immune crosstalk underlying nociceptor sensitization at the site of nerve injury. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic neuropathic pain, and therefore identifies multiple analgesic targets.