Murine Dorsal Root Ganglion Neurons Research Articles

GPR55‐Dependent Excitation of Dorsal Root Ganglion Neurons by Lysophosphatidylcholine

Irritable Bowel Syndrome is a functional gastrointestinal (GI) disorder that leads to chronic abdominal pain. The perception of this visceral pain involves peripheral mechanisms, such as nociceptors with cell bodies in dorsal root ganglia (DRG) and axons in the gut wall, and central mechanisms, such as neuroplasticity within the spinal cord and brain. Lysophosphatidylcholine (LPC), which has recently been shown to bind to GPR55 in human prostate carcinoma cells, is released from cell membranes and is elevated in patients with IBS. Dorsal root ganglion (DRG) neurons express GPR55, and activation of GPR55 has been implicated in inflammatory pain. Therefore, we hypothesised that elevated LPC during IBS contributes to pain due to its activation of GPR55 on dorsal root ganglion (DRG) neurons, leading to the excitation of visceral pain pathways. Current clamp recordings revealed that application of LPC (10 µM) to murine DRG neurons depolarised the resting membrane potential (p = 0.0001) by approximately 8 mV and decreased the rheobase (p < 0.05) by approximately 20%. Using ratiometric Ca2+ imaging using FURA‐2 AM, LPC (10 µM) doubled intracellular [Ca2+]i. This effect was significantly reduced by the selective GPR55 antagonist CID16020046 (10 µM) (p < 0.05), suggesting the response to LPC is at least partially mediated by GPR55. The source of the [Ca2+] elevation following LPC application was elucidated using cyclopiazonic acid (CPA; 10 µM), which depletes intracellular Ca2+ stores, and a 0‐Ca2+ external solution to remove the contribution of Ca2+ influx from extracellular sources. While both significantly decreased the Ca2+ influx elicited by LPC, the 0‐Ca2+ external solution almost abolished the effect (p < 0.0001) of LPC. Together, these data suggest that the increased [Ca2+] elicited by LPC activation of GPR55 is partially mediated through the release of Ca2+ from intracellular stores but is mostly due to influx of extracellular Ca2+.

Interleukin-31: The "itchy" cytokine in inflammation and therapy.

The cytokine interleukin-31 has been implicated in the pathophysiology of multiple atopic disorders such as atopic dermatitis (AD), allergic rhinitis, and airway hyper-reactivity. In AD, IL-31 has been identified as one of the main "drivers" of its cardinal symptom, pruritus. Here, we summarize the mechanisms by which IL-31 modulates inflammatory and allergic diseases. TH 2 cells play a central role in AD and release high levels of TH 2-associated cytokines including IL-31, thereby mediating inflammatory responses, initiating immunoregulatory circuits, stimulating itch, and neuronal outgrowth through activation of the heterodimeric receptor IL-31 receptor A (IL31RA)/Oncostatin M receptor (OSMRβ). IL31RA expression is found on human and murine dorsal root ganglia neurons, epithelial cells including keratinocytes and various innate immune cells. IL-31 is a critical cytokine involved in neuroimmune communication, which opens new avenues for cytokine modulation in neuroinflammatory diseases including AD/pruritus, as validated by recent clinical trials using an anti-IL-31 antibody. Accordingly, inhibition of IL-31-downstream signaling may be a beneficial approach for various inflammatory diseases including prurigo. However, as to whether downstream JAK inhibitors directly block IL-31-mediated-signaling needs to be clarified. Targeting the IL-31/IL31RA/OSMRβ axis appears to be a promising approach for inflammatory, neuroinflammatory, and pruritic disorders in the future.

Differential impact of keratinocytes and fibroblasts on nociceptor degeneration and sensitization in small fiber neuropathy.

Peripheral denervation and pain are hallmarks of small fiber neuropathy (SFN). We investigated the contribution of skin cells on nociceptor degeneration and sensitization. We recruited 56 patients with SFN and 31 healthy controls and collected skin punch biopsies for immunohistochemical and immunocytochemical analysis of netrin-1 (NTN1) and proinflammatory and anti-inflammatory cytokine expression patterns. We further applied coculture systems with murine dorsal root ganglion (DRG) neurons for skin cell-nerve interaction studies and patch-clamp analysis. Human keratinocytes attract murine DRG neuron neurites, and the gene expression of the axon guidance cue NTN1 is higher in keratinocytes of patients with SFN than in controls. NTN1 slows and reduces murine sensory neurite outgrowth in vitro, but does not alter keratinocyte cytokine expression. In the naive state, keratinocytes of patients with SFN show a higher expression of transforming growth factor-β1 (P < 0.05), while fibroblasts display higher expression of the algesic cytokines interleukin (IL)-6 (P < 0.01) and IL-8 (P < 0.05). IL-6 incubation of murine DRG neurons leads to an increase in action potential firing rates compared with baseline (P < 0.01). Our data provide evidence for a differential effect of keratinocytes and fibroblasts on nociceptor degeneration and sensitization in SFN compared with healthy controls and further supports the concept of cutaneous nociception.

Dual opposing actions of STW 5 on TRP receptors mediate neuronal desensitisation in vitro

AimsSTW 5 is an herbal drug combination used for the treatment of functional gastrointestinal disorders (FGIDs) with visceral hypersensitivity as the therapy-resistant hallmark. STW 5 has been clinically proven to alleviate visceral hypersensitivity-related symptoms, including abdominal pain, bloating, nausea, and early satiety. However, the molecular and cellular mechanisms underlying the antinociceptive action of STW 5 remain unknown. Here, we investigate the role of STW 5 in the calcium mobilisation of dorsal root ganglion (DRG) sensory neurons. Main methodsCalcium imaging experiments were performed with freshly dissociated cultured murine DRG neurons isolated from mice by microfluorometry. TRPA1-deficient DRGs, TRPV1-deficient DRGs, TRPA1/V1 double-deficient DRGs, and wild-type DRGs have been used to investigate the role of TRPs ion channels in mediating STW 5 action. Key findingsSTW 5 (1.74 and 5.8 mg/ml) induced calcium ion influx into DRG neurons in a concentration-dependent manner. Calcium transients were desensitised during repeated exposure to STW 5, an effect that was facilitated in TRPA1-deficient DRGs and less pronounced in TRPV1-deficient DRGs compared to wild-type (WT) DRGs. SignificanceRepeated exposure to STW 5 induced desensitisation of sensory neurons and may ultimately contribute to its proven clinical efficacy against sensory-related symptoms in patients with FGID, including abdominal pain, bloating, nausea, and early satiety. This effect is modulated by the two prominent irritant sensors in nociceptors, TRPA1 and TRPV1.

Neurochemical and Ultrastructural Characterization of Unmyelinated Non-peptidergic C-Nociceptors and C-Low Threshold Mechanoreceptors Projecting to Lamina II of the Mouse Spinal Cord

C-nociceptors (C-Ncs) and non-nociceptive C-low threshold mechanoreceptors (C-LTMRs) are two subpopulations of small unmyelinated non-peptidergic C-type neurons of the dorsal root ganglia (DRGs) with central projections displaying a specific pattern of termination in the spinal cord dorsal horn. Although these two subpopulations exist in several animals, remarkable neurochemical differences occur between mammals, particularly rat/humans from one side and mouse from the other. Mouse is widely investigated by transcriptomics. Therefore, we here studied the immunocytochemistry of murine C-type DRG neurons and their central terminals in spinal lamina II at light and electron microscopic levels. We used a panel of markers for peptidergic (CGRP), non-peptidergic (IB4), nociceptive (TRPV1), non-nociceptive (VGLUT3) C-type neurons and two strains of transgenic mice: the TAFA4Venus knock-in mouse to localize the TAFA4+ C-LTMRs, and a genetically engineered ginip mouse that allows an inducible and tissue-specific ablation of the DRG neurons expressing GINIP, a key modulator of GABABR-mediated analgesia. We confirmed that IB4 and TAFA4 did not coexist in small non-peptidergic C-type DRG neurons and separately tagged the C-Ncs and the C-LTMRs. We then showed that TRPV1 was expressed in only about 7% of the IB4+ non-peptidergic C-Ncs and their type Ia glomerular terminals within lamina II. Notably, the selective ablation of GINIP did not affect these neurons, whereas it reduced IB4 labeling in the medial part of lamina II and the density of C-LTMRs glomerular terminals to about one half throughout the entire lamina. We discuss the significance of these findings for interspecies differences and functional relevance.

MicroRNA-137 and its downstream target LSD1 inversely regulate anesthetics-induced neurotoxicity in dorsal root ganglion neurons.

Anesthetic reagents, such as bupivacaine (Bv), induce significant neurotoxicity in dorsal root ganglion neurons (DRGNs). In this study, we investigated the expression, function and cross-association of microRNA-137-3p (miR-137-3p) and lysine (K)-specific demethylase 1A (LSD1) in a murine model of Bv-induced neural injury in DRGNs. Murine DRGNs were culture in vitro and treated with Bv. QPCR was used to evaluate miR-137-3p expression in Bv-injured DRGNs. MiR-137-3p was genetically downregulated to evaluate its rescuing effect on Bv-induced DRGN apoptosis and neurite retraction. The association of miR-137-3p on its downstream target, LSD1 coding gene KDM1A, was evaluated by dual-luciferase activity assay and qPCR. In miR-137-3p-downregulated DRGNs, KDM1A was inhibited to evaluate its involvement in miR-137-3p-mediated modulation on Bv-induced DRGN neurotoxicity. Furthermore, KDM1A expression in Bv-injured DRGN was evaluated by qPCR, and LSD1 was overexpressed in DRGN to evaluate its direct effect on Bv-induced neurotoxicity. MiR-137-3p was upregulated in Bv-injured DRGNs. MiR-137-3p downregulation rescued Bv-induced DRGN apoptosis and neurite retraction. LSD1 was demonstrated to be downstream to, and inversely modulated by miR-137-3p in DRGN. In Bv-injured DRGNs, LSD1 downregulation reversed miR-137-3p-downregualtion-induced neural protection. Furthermore, LSD1 upregulation directly rescued Bv-induced apoptosis and neurite retraction in DRGNs. MiR-137-3p and its downstream target LSD1 are inversely associated to regulate anesthetics-induced neurotoxicity in DRGN. This signaling pathway may be a therapeutic candidate to reduce anesthetics-induced neurological damage in human patients.

Neurotoxic mechanisms of paclitaxel are local to the distal axon and independent of transport defects

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of paclitaxel and other chemotherapeutic agents. Paclitaxel binds and stabilizes microtubules, but the cellular mechanisms that underlie paclitaxel's neurotoxic effects are not well understood. We therefore used primary cultures of adult murine dorsal root ganglion neurons, the cell type affected in patients, to examine leading hypotheses to explain paclitaxel neurotoxicity. We address the role of microtubule hyperstabilization and its downstream effects. Paclitaxel administered at 10–50nM for 1–3days induced retraction bulbs at the tips of axons and arrested axon growth without triggering axon fragmentation or cell death. By correlating the toxic effects and microtubule stabilizing activity of structurally different microtubule stabilizing compounds, we confirmed that microtubule hyperstabilization, rather than an off-target effect, is the likely primary cause of paclitaxel neurotoxicity. We examined potential downstream consequences of microtubule hyperstabilization and found that changes in levels of tubulin posttranslational modifications, although present after paclitaxel exposure, are not implicated in the paclitaxel neurotoxicity we observed in the cultures. Additionally, defects in axonal transport were not implicated as an early, causative mechanism of paclitaxel's toxic effects on dorsal root ganglion neurons. By using microfluidic chambers to selectively treat different parts of the axon with paclitaxel, we found that the distal axon was primarily vulnerable to paclitaxel, indicating that paclitaxel acts directly on the distal axon to induce degenerative effects. Together, our findings point to local effects of microtubule hyperstabilization on the distal-most portion of the axon as an early mediator of paclitaxel neurotoxicity. Because sensory neurons have a unique and ongoing requirement for distal growth in order to reinnervate the epidermis as it turns over, we propose that the ability of paclitaxel to arrest their growth accounts for the selective vulnerability of sensory neurons to paclitaxel neurotoxicity.

Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10μM) or low-intensity blue light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8(-/-)), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and, at higher concentrations, progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure.

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1-induced pruritus.

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

Immunoglobulinfree light chains reduce in an antigen-specific manner the rate of rise of action potentials of mouse non-nociceptive dorsal root ganglion neurons

Plasma B cells secrete immunoglobulinfree light chains (IgLC) which by binding to mast cells can mediate hypersensitivity responses and are involved in several immunological disorders. To investigate the effects of antigen-specific IgLC activation, intracellular recordings were made from cultured murine dorsal root ganglion (DRG) neurons, which can specifically bind IgLC. The neurons were sensitized with IgLC for 90min and subsequently activated by application of the corresponding antigen (DNP-HSA). Antigen application induced a decrease in the rate of rise of the action potentials of non-nociceptive neurons (MANOVA, p=2.10−6), without affecting the resting membrane potential or firing threshold. The action potentials of the nociceptive neurons (p=0.57) and the electrical excitability of both types of neurons (p>0.35) were not affected. We conclude that IgLC can mediate antigen-specific responses by reducing the rate of rise of action potentials in non-nociceptive murine DRG neurons. We suggest that antigen-specific activation of IgLC-sensitized non-nociceptive DRG neurons may contribute to immunological hypersensitivity responses and neuroinflammation.

Suppressive effects of low-power laser on cytosolic calcium ion changes elicited by bradykinin in cultured murine dorsal root ganglion neurons

Suppressive effects of low-power laser on cytosolic calcium ion changes elicited by bradykinin in cultured murine dorsal root ganglion neurons

Effects of nitric oxide and hypoxia on low-and high-voltage activated calcium currents in murine DRG neurons

The effects of a nitric oxide (NO)-containing aqueous solution (authentic NO) and hypoxia on low-and high-voltage activated calcium currents (I Ca,lva andI Ca,hva , respectively; in the latter transient and sustained portions were differentiated) were studied in enzymatically dispersed medium-sized neuronal somata from the murine dorsal root ganglia (DRG). Authentic NO (10 μM) was found to decrease the mean peak amplitude ofI Ca,lva , from 3.5±0.3 to 1.2±0.2 nA (n=11,p 0.05). The data strongly suggest that NO is capable of reversibly suppressing both types of calcium channels in murine DRG neurons and of modulating in this way their excitability. It seems likely that this ability is based on a direct effect of NO on the corresponding channels and not on NO participation in the induction of hypoxic effects. Yet, a hypothesis that NO is a messenger of hypoxic damage to neural cells still should be suggested.

Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells.

1. Kainate receptors with pharmacological properties similar to those of the GluR5 subunit have been shown to modulate inhibitory synaptic transmission in the CA1 region of the hippocampus. The kinetic properties of currents gated by GluR5 receptors have not been examined in detail. Here we describe several biophysical features of recombinant GluR5 receptors expressed in HEK293 cells. 2. We found that homomeric GluR5 receptors can exhibit striking inter-cell variability in channel kinetics in response to the agonists kainate and glutamate. Desensitization rates in response to kainate varied between individual cells by nearly 1000-fold (range, 1.5 ms to 1.5 s), while glutamate desensitization rates differed by 9-fold (range, 1.0 to 9.0 ms). 3. The time course of recovery from desensitization in response to glutamate also showed inter-cell variation. The majority of glutamate currents in GluR5-expressing cells recovered from desensitization with two widely separated exponential components: 50 +/- 10 ms and 5.1 +/- 1.0 s (contributing 37.6 % and 62.4 % of the sum of the exponential fits, respectively). In contrast, currents with the fastest desensitization kinetics had a recovery time course of 4.8 +/- 0.3 s. 4. Kainate receptors in murine dorsal root ganglion neurons are likely to be composed of homomeric GluR5 subunits. These receptor currents recovered from glutamate desensitization with a biexponential time course of 36 +/- 4 ms and 4.7 +/- 0.7 s. 5. These results suggest that aspects of GluR5 kainate receptor function are modulated by intracellular mechanism(s). At synapses such mechanisms could regulate the frequency- response relationship of synaptic kainate receptors by altering their rate of entry into and recovery from desensitization.

ATP-induced calcium release from the intracellular stores in murine dorsal root ganglion neurons

ATP-induced calcium release from the intracellular stores in murine dorsal root ganglion neurons

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Enhanced neuronal regeneration by retinoic acid of murine dorsal root ganglia and of fetal murine and human spinal cord in vitro

This study demonstrates that retinoic acid (RA), an active metabolite of vitamin A, can act to enhance regeneration of neurites, at physiologic concentrations, in vitro. Explanted fragments of mouse dorsal root ganglia (DRG) and mouse and human spinal cord (SC) were maintained, in vitro, for periods up to 11 d. Murine DRG neurons were exposed to RA concentrations ranging from 100 microM to 1 nM, whereas neurons within murine and human SC explants were exposed to 10 microM to 10 nM RA. Results show that RA significantly (P less than 0.001) increases mean neurite length but not neurite number. Specifically, murine DRG neurons showed increases in mean neurite length of 30.7% with individual explants showing increases of up to 133.5%. Murine and human SC showed mean enhancements of 43.4 and 58.1%, respectively, but did so at lower concentrations of RA. The results indicate that RA may play a potentially critical role in neuronal regeneration.