Nociceptive Dorsal Root Ganglion Research Articles

(209) Characterization of the Na+/Ca2+ Exchanger (NCS) in Human DRG Neurons

Because of the importance of intracellular Ca2+ to processes ranging from protein-interactions to cell death, the concentration of cytosolic Ca2+ ([Ca2+]c) is tightly regulated by a complex array of pumps, transporters, exchangers, and binding proteins. We have recently demonstrated that the Na+/Ca2+ exchanger (NCX), in particular NCX3, plays an important role in attenuating large-increases in [Ca2+]c in a subpopulation of putative nociceptive rat dorsal root ganglion (DRG) neurons. Interestingly, we subsequently demonstrated that inflammation-induced dysregulation of [Ca2+]c in this subpopulation of putative nociceptors was due, at least in part, to a decrease in NCX activity in the sensory neuron cell body. Given the potential importance of this exchanger to nociceptive processing as well as injury-induced changes in nociceptive processing, in the face of a growing list of differences between rodent and human sensory neurons, the purpose of the present study was to characterize NCX activity in human sensory neurons. We employed electrical field stimulation to evoke Ca2+ transients in the presence and absence of LiCl or Choline-Cl bath to block NCX activity. Interestingly, electrical field stimulation of human DRG neurons, at the same parameters used in rat, evoked a much larger Ca2+ transient in both magnitude and duration. We obtained evidence of NCX activity in the majority of neurons tested. Similar to the role of NCX in rat DRG neurons, block of NCX in human DRG neurons increased the duration, but not the magnitude of the electrically evoked Ca2+ transient. However, in contrast to the rat, there appeared to be constitutive NCX activity. Consistent with a number of recent studies, these results highlight potentially important similarities and differences between rodent and human sensory neurons, underscoring the importance of human sensory neurons as a pre-clinical screen of novel therapeutics targeting the primary afferent. Research funded by NIH T32NS073548, 1R01NS083347, and R01DK107966.

Partial sciatic nerve ligation leads to an upregulation of Ni2+-resistant T-type Ca2+ currents in capsaicin-responsive nociceptive dorsal root ganglion neurons.

BackgroundNeuropathic pain resulting from peripheral nerve lesions is a common medical condition, but current analgesics are often insufficient. The identification of key molecules involved in pathological pain processing is a prerequisite for the development of new analgesic drugs. Hyperexcitability of nociceptive DRG-neurons due to regulation of voltage-gated ion-channels is generally assumed to contribute strongly to neuropathic pain. There is increasing evidence, that T-type Ca2+-currents and in particular the Cav3.2 T-type-channel isoform play an important role in neuropathic pain, but experimental results are contradicting.PurposeTo clarify the role of T-type Ca2+-channels and in particular the Cav3.2 T-type-channel isoform in neuropathic pain.MethodsThe effect of partial sciatic nerve ligation (PNL) on pain behavior and the properties of T-type-currents in nociceptive DRG-neurons was tested in wild-type and Cav3.2-deficient mice.ResultsIn wild-type mice, PNL of the sciatic nerve caused neuropathic pain and an increase of T-type Ca2+-currents in capsaicin-responsive neurons, while capsaicin-unresponsive neurons were unaffected. Pharmacological experiments revealed that this upregulation was due to an increase of a Ni2+-resistant Ca2+-current component, inconsistent with Cav3.2 up-regulation. Moreover, following PNL Cav3.2-deficient mice showed neuropathic pain behavior and an increase of T-Type Ca2+-currents indistinguishable to that of PNL treated wild-type mice.ConclusionThese data suggest that PNL induces an upregulation of T-Type Ca2+-currents in capsaicin-responsive DRG-neurons mediated by an increase of a Ni2+-insensitive current component (possibly Cav3.1 or Cav3.3). These findings provide relevance for the development of target specific analgesic drugs.

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PC-FACS

Differential use-dependent inactivation of Nav1.8 in the subpopulation of cultured dorsal root ganglion

Sodium channel Nav1.8 is expressed preferentially in members of the peripheral nervous system such as nociceptive dorsal root ganglion (DRG) neurons. Using immunocytochemistry and eletrophysiological recording, we found that a subpopulation of small DRG neurons, grouped by isolectin B4 (IB4) immunoreactivity, showed a different use-dependent inactivation of Nav1.8 current, and this situation changed over time during in vitro culture. The IB 4 - immunoreactivity of DRG neurons was not changed during in vitro culture with the exception of this small population of IB4-negative small-diameter DRG neurons. The Nav1.8 channel in IB 4 + neurons underwent a level of use-dependent inactivation that was significantly stronger than that seen in IB 4 - neurons at 1 and 2 days-in-vitro (DIV). The use-dependent inactivation of the Nav1.8 channel in IB 4 + neurons at 1 DIV was significantly attenuated at 2 DIV. The values for voltage dependency of activation and steady-state inactivation of Nav1.8 were similar in all subpopulations of DRG neurons and did not change over time. The time constant for entry into slow inactivation of Nav1.8 in IB 4 + neurons was significantly faster than in IB 4 - neurons at 1 and 2 DIV, while the rate of recovery from slow inactivation of Nav1.8 in IB 4 + neurons was slower than that seen in IB 4 - neurons. Moreover, the time constant for entry into the slow inactivation of Nav1.8 in IB 4 + neurons after 1 DIV was significantly faster than at 2 DIV, and the rate of recovery from the slow inactivation of Nav1.8 in IB 4 + neurons at 1 DIV was slower than that at 2 DIV, which indicated that the strong use-dependent inactivation in IB 4 + neurons at 1 DIV was the result of a greater preference for the slow inactivation state than at 2 DIV. Our data suggest that the time-dependent change of the use-dependent inactivation of the Nav1.8 channel in DRG neurons cultured in vitro would contribute to the excitability of a subpopulation of DRG neurons and could play an important role in the development of inflammatory and neuropathic pain.

A-Type KV Channels in Dorsal Root Ganglion Neurons: Diversity, Function, and Dysfunction.

A-type voltage-gated potassium (Kv) channels are major regulators of neuronal excitability that have been mainly characterized in the central nervous system. By contrast, there is a paucity of knowledge about the molecular physiology of these Kv channels in the peripheral nervous system, including highly specialized and heterogenous dorsal root ganglion (DRG) neurons. Although all A-type Kv channels display pore-forming subunits with similar structural properties and fast inactivation, their voltage-, and time-dependent properties and modulation are significantly different. These differences ultimately determine distinct physiological roles of diverse A-type Kv channels, and how their dysfunction might contribute to neurological disorders. The importance of A-type Kv channels in DRG neurons is highlighted by recent studies that have linked their dysfunction to persistent pain sensitization. Here, we review the molecular neurophysiology of A-type Kv channels with an emphasis on those that have been identified and investigated in DRG nociceptors (Kv1.4, Kv3.4, and Kv4s). Also, we discuss evidence implicating these Kv channels in neuropathic pain resulting from injury, and present a perspective of outstanding challenges that must be tackled in order to discover novel treatments for intractable pain disorders.

Reducing CXCR4-mediated nociceptor hyperexcitability reverses painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.

Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Horn.

Presynaptic voltage-gated K+ (Kv) channels in dorsal root ganglion (DRG) neurons are thought to regulate nociceptive synaptic transmission in the spinal dorsal horn. However, the Kv channel subtypes responsible for this critical role have not been identified. The Kv3.4 channel is particularly important because it is robustly expressed in DRG nociceptors, where it regulates action potential (AP) duration. Furthermore, Kv3.4 dysfunction is implicated in the pathophysiology of neuropathic pain in multiple pain models. We hypothesized that, through their ability to modulate AP repolarization, Kv3.4 channels in DRG nociceptors help to regulate nociceptive synaptic transmission. To test this hypothesis, we investigated Kv3.4 immunoreactivity (IR) in the rat cervical superficial dorsal horn (sDH) in both sexes and implemented an intact spinal cord preparation to investigate glutamatergic synaptic currents from second order neurons in the sDH under conditions that selectively inhibit the Kv3.4 current. We found presynaptic Kv3.4 IR in peptidergic and nonpeptidergic nociceptive fibers of the sDH. The Kv3.4 channel is hypersensitive to 4-aminopyridine and tetraethylammonium (TEA). Accordingly, 50 μm 4-aminopyridine and 500 μm TEA significantly prolong the AP, slow the maximum rate of repolarization in small-diameter DRG neurons, and potentiate monosynaptic excitatory postsynaptic currents (EPSCs) in dorsal horn laminae I and II through a presynaptic mechanism. In contrast, highly specific inhibitors of BK, Kv7, and Kv1 channels are less effective modulators of the AP and have little to no effect on EPSCs. The results strongly suggest that presynaptic Kv3.4 channels are major regulators of nociceptive synaptic transmission in the spinal cord.SIGNIFICANCE STATEMENT Intractable neuropathic pain can result from disease or traumatic injury and many studies have been conducted to determine the underlying pathophysiological changes. Voltage-gated ion channels, including the K+ channel Kv3.4, are dysregulated in multiple pain models. Kv3.4 channels are ubiquitously expressed in the dorsal root ganglion (DRG), where they are major regulators of DRG excitability. However, little is known about the ionic mechanisms that regulate nociceptive synaptic transmission at the level of the first synapse in the spinal cord, which is critical to pain transmission in both intact and pathological states. Here, we show that Kv3.4 channels have a significant impact on glutamatergic synaptic transmission in the dorsal horn, further illuminating its potential as a molecular pain therapeutic target.

EIF4E Phosphorylation Influences Bdnf mRNA Translation in Mouse Dorsal Root Ganglion Neurons.

Plasticity in dorsal root ganglion (DRG) neurons that promotes pain requires activity-dependent mRNA translation. Protein synthesis inhibitors block the ability of many pain-promoting molecules to enhance excitability in DRG neurons and attenuate behavioral signs of pain plasticity. In line with this, we have recently shown that phosphorylation of the 5′ cap-binding protein, eIF4E, plays a pivotal role in plasticity of DRG nociceptors in models of hyperalgesic priming. However, mRNA targets of eIF4E phosphorylation have not been elucidated in the DRG. Brain-derived neurotrophic factor (BDNF) signaling from nociceptors in the DRG to spinal dorsal horn neurons is an important mediator of hyperalgesic priming. Regulatory mechanisms that promote pain plasticity via controlling BDNF expression that is involved in promoting pain plasticity have not been identified. We show that phosphorylation of eIF4E is paramount for Bdnf mRNA translation in the DRG. Bdnf mRNA translation is reduced in mice lacking eIF4E phosphorylation (eIF4ES209A) and pro-nociceptive factors fail to increase BDNF protein levels in the DRGs of these mice despite robust upregulation of Bdnf-201 mRNA levels. Importantly, bypassing the DRG by giving intrathecal injection of BDNF in eIF4ES209A mice creates a strong hyperalgesic priming response that is normally absent or reduced in these mice. We conclude that eIF4E phosphorylation-mediated translational control of BDNF expression is a key mechanism for nociceptor plasticity leading to hyperalgesic priming.

Signalling transduction events involved in agonist-induced PGE2/EP4 receptor externalization in cultured rat dorsal root ganglion neurons.

Prostaglandin E2 (PGE2) enriched in inflamed tissues contributes to chronic pain by sensitizing nociceptive dorsal root ganglion (DRG) neurons (nociceptors). Of four PGE2 receptors (EP1-4), EP4 plays a major role in PGE2-induced nociceptor sensitization. We have previously reported that PGE2 or EP4 agonists stimulated EP4 externalization in cultured DRG neurons and this event contributes to nociceptor sensitization. However, the signalling transduction events governing this event remain unknown. In this study, using antibody-based externalization assay, we examined EP subtypes and multiple signalling transduction events involved in PGE2-induced EP4 externalization in cultured DRG neurons. In addition to EP4 agonist, EP2 agonist, to a lesser extent, also induced EP4 externalization while EP1 and EP3 agonists had no effect. The extracellular and intracellular calcium chelators, the inhibitors of CaMKII, cAMP, PKA, PKC, PKCε, PLC, MAPKs, PI3K and Akt suppressed agonist-induced EP4 externalization. The activator of AC, two PKA-specific cAMP analogues and one Epac-specific cAMP analogue also induced EP4 externalization. ELISA showed that double sequential exposures to EP4 agonists induced a greater release of pain peptide CGRP from cultured DRG neurons than a single exposure, an event blocked by the inhibitor of anterograde transport from ER/Golgi complex to cell surface. Taken together, these data suggest that mobilization of extracellular and intracellular calcium as well as the activation of CaMKII, cAMP/PKA, cAMP/Epac, PKC/PKCε, MAPKs, PI3K-Akt and PLC signalling transduction pathways are involved in agonist-induced EP4 externalization. Agonist-enhanced EP4 externalization increases EP4 cell surface abundance and activity, thus enhancing nociceptor sensitization. This study adds mechanistic information regarding signalling transduction events involved in agonist-induced EP4 cell surface trafficking. EP4 and EP2 (to lesser extent) receptors, extra- and intracellular Ca++ , CaKMII, cAMP, PKA, PKC, PKCε, PLC, MAPK, PI3K and Akt are involved in this event. Agonist-induced EP4 externalization contributes to enhanced nociceptor sensitization.

CCR2 upregulation in DRG neurons plays a crucial role in gastric hyperalgesia associated with diabetic gastropathy.

BackgroundDiabetic gastropathy is a complex neuromuscular dysfunction of the stomach that commonly occurs in diabetes mellitus. Diabetic patients often present with upper gastrointestinal symptoms, such as epigastric discomfort or pain. The aim of this study was to assess gastric sensation in streptozocin-induced diabetes mellitus (DM) rats and to determine the contribution of C-C motif chemokine receptor 2 (CCR2) signaling to gastric hyperalgesia.ResultsDM rats showed signs of neuropathy (cutaneous mechanical hyperalgesia) from two weeks after streptozocin administration until the end of the experiment. Accelerated solid gastric emptying was observed at two weeks after streptozocin administration compared to the controls. Intense gastric hyperalgesia also developed in DM rats at two weeks after streptozocin administration, which was significantly reduced after intrathecal administration of the CCR2 antagonist INCB3344. Immunochemical analysis indicated that CCR2 expression was substantially upregulated in small and medium-sized dorsal root ganglia neurons of DM rats, although the protein level of monocyte chemoattractant protein-1, the preferred ligand for CCR2, was not significantly different between the control and DM groups.ConclusionsThese data suggest that CCR2 activation in nociceptive dorsal root ganglia neurons plays a role in the pathogenesis of gastric hyperalgesia associated with diabetic gastropathy and that CCR2 antagonist may be a promising treatment for therapeutic intervention.

TLR4 receptor expression and function in F11 dorsal root ganglion × neuroblastoma hybrid cells.

TLR4 respond to bacterial LPS to produce inflammatory cytokines. TLR4 are expressed in dorsal root ganglia and play a role in pain. F11 dorsal root ganglia × mouse neuroblastoma cells possess many of the properties seen in nociceptive dorsal root ganglia neuronal cells. Here, we investigated the effect of 2 h and 6 h treatment with LPS upon the expression of inflammatory proteins in undifferentiated and differentiated F11 cells. The cells expressed mRNA for TRL4 (mouse, not rat) and proteins involved in TLR4 signaling. TLR4 expression was confirmed using immunohistochemistry. LPS produced modest increases in mouse and rat IL-6 and in mouse cyclooxygenase-2 levels in undifferentiated cells, but did not significantly affect mouse TNF-α expression. This contrasts with the robust effects of LPS upon cyclooxygenase-2 expression in cultured dorsal root ganglia neurons. F11 cells expressed the endocannabinoid metabolizing enzymes fatty acid amide hydrolase and N-acylethanolamine acid amidase (both murine), which were functionally active. These data suggest that F11 cells are not a useful model for the study of LPS-mediated effects but may be useful for the study of endocannabinoid catabolism.

Activation of KCNQ Channels Suppresses Spontaneous Activity in Dorsal Root Ganglion Neurons and Reduces Chronic Pain after Spinal Cord Injury.

A majority of people who have sustained spinal cord injury (SCI) experience chronic pain after injury, and this pain is highly resistant to available treatments. Contusive SCI in rats at T10 results in hyperexcitability of primary sensory neurons, which contributes to chronic pain. KCNQ channels are widely expressed in nociceptive dorsal root ganglion (DRG) neurons, are important for controlling their excitability, and their activation has proven effective in reducing pain in peripheral nerve injury and inflammation models. The possibility that activators of KCNQ channels could be useful for treating SCI-induced chronic pain is strongly supported by the following findings. First, SCI, unlike peripheral nerve injury, failed to decrease the functional or biochemical expression of KCNQ channels in DRG as revealed by electrophysiology, real-time quantitative polymerase chain reaction, and Western blot; therefore, these channels remain available for pharmacological targeting of SCI pain. Second, treatment with retigabine, a specific KCNQ channel opener, profoundly decreased spontaneous activity in primary sensory neurons of SCI animals both in vitro and in vivo without changing the peripheral mechanical threshold. Third, retigabine reversed SCI-induced reflex hypersensitivity, adding to our previous demonstration that retigabine supports the conditioning of place preference after SCI (an operant measure of spontaneous pain). In contrast to SCI animals, naïve animals showed no effects of retigabine on reflex sensitivity or conditioned place preference by pairing with retigabine, indicating that a dose that blocks chronic pain-related behavior has no effect on normal pain sensitivity or motivational state. These results encourage the further exploration of U.S. Food and Drug Administration-approved KCNQ activators for treating SCI pain, as well as efforts to develop a new generation of KCNQ activators that lack central side effects.

Network topology of NaV1.7 mutations in sodium channel-related painful disorders

BackgroundGain-of-function mutations in SCN9A gene that encodes the voltage-gated sodium channel NaV1.7 have been associated with a wide spectrum of painful syndromes in humans including inherited erythromelalgia, paroxysmal extreme pain disorder and small fibre neuropathy. These mutations change the biophysical properties of NaV1.7 channels leading to hyperexcitability of dorsal root ganglion nociceptors and pain symptoms. There is a need for better understanding of how gain-of-function mutations alter the atomic structure of Nav1.7.ResultsWe used homology modeling to build an atomic model of NaV1.7 and a network-based theoretical approach, which can predict interatomic interactions and connectivity arrangements, to investigate how pain-related NaV1.7 mutations may alter specific interatomic bonds and cause connectivity rearrangement, compared to benign variants and polymorphisms. For each amino acid substitution, we calculated the topological parameters betweenness centrality (Bct), degree (D), clustering coefficient (CCct), closeness (Cct), and eccentricity (Ect), and calculated their variation (Δvalue = mutant value-WT value). Pathogenic NaV1.7 mutations showed significantly higher variation of |ΔBct| compared to benign variants and polymorphisms. Using the cut-off value ±0.26 calculated by receiver operating curve analysis, we found that ΔBct correctly differentiated pathogenic NaV1.7 mutations from variants not causing biophysical abnormalities (nABN) and homologous SNPs (hSNPs) with 76% sensitivity and 83% specificity.ConclusionsOur in-silico analyses predict that pain-related pathogenic NaV1.7 mutations may affect the network topological properties of the protein and suggest |ΔBct| value as a potential in-silico marker.

Fatty-acid-binding protein inhibition produces analgesic effects through peripheral and central mechanisms.

BackgroundFatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors.ResultsImmunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund’s adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects.ConclusionsThis study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.

Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors.

Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception.

Stimulating TRPV1 externalization and synthesis in dorsal root ganglion neurons contributes to PGE2 potentiation of TRPV1 activity and nociceptor sensitization.

Persistent peripheral sensitization contributes to chronic pain. Plasticity of nociceptive dorsal root ganglion (DRG) neurons (nociceptors) induced by pro-inflammatory mediators contributes to sensitization. Prostaglandin E2 (PGE2) enriched in injured tissues is known not only directly to sensitize DRG neurons, but also to potentiate sensitizing effects of other pain mediators such as capsaicin and its receptor transient receptor potential vanilloid-1 (TRPV1). It remains unknown whether PGE2 potentiates TRPV1 activity by stimulating its synthesis, cell surface and axonal trafficking in DRG neurons. Combined biochemical, morphological, pharmacological and behavioral approaches have been used to address this issue in both invitro and invivo models. PGE2 increased TRPV1 externalization in cultured rat DRG neurons in a time- and concentration-dependent manner, an event blocked by an inhibitor of protein synthesis or anterograde export. EP1 and EP4, but not EP2 and EP3, mediated this event. EP1 agonist-induced TRPV1 externalization was suppressed by inhibitors of CaMKII, PLC, PKC and PKCε, while EP4 agonist-induced TRPV1 externalization by inhibitors of cAMP/PKA and ERK/MAPK. Pre-exposure to PGE2 potentiated release of calcitonin gene-related peptide from cultured DRG neurons evoked by subsequent capsaicin stimulation. This event was blocked by an inhibitor of protein synthesis or export, suggesting that PGE2-induced TRPV1 synthesis and externalization is coupled to enhanced TRPV1 activity. Pre-exposure to PGE2 not only prolonged tactile allodynia evoked by subsequent capsaicin challenge, but also increased TRPV1 levels in L4-6 DRG, sciatic nerves and plantar skin. Our data indicate that facilitating TRPV1 synthesis, cell surface and axonal trafficking is a novel mechanism underlying PGE2 potentiation of TRPV1 activity.

Stress activates pronociceptive endogenous opioid signalling in DRG neurons during chronic colitis

Aims and backgroundPsychological stress accompanies chronic inflammatory diseases such as IBD, and stress hormones can exacerbate pain signalling. In contrast, the endogenous opioid system has an important analgesic action during...

L5 spinal nerve axotomy induces sensitization of cutaneous L4 Aβ-nociceptive dorsal root ganglion neurons in the rat in vivo

Partial nerve injury often leads to peripheral neuropathic pain (PNP), a major health problem that lacks effective drug treatment. PNP is characterized by ongoing/spontaneous pain, and hypersensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. Preclinical studies using the L5 spinal nerve ligation/axotomy (SNL/SNA) model of PNP suggest that this type of chronic pain results partly from sensitization of ipsilateral L4C-and Aδ-fiber nociceptive dorsal root ganglion (DRG) neurons, but whether L4 β-nociceptors, which constitute a substantial group of DRG neurons, also become sensitized remains unanswered. To address this issue, intracellular recordings from somata of cutaneous Aβ-nociceptors (classified according to their dorsal root conduction velocities (>6.5m/s), and physiologically based on their responses to noxious (but not innocuous) mechanical stimuli) were made from L4-DRGs in normal (control) rats and in rats seven days after L5 SNA in vivo. Compared with control, cutaneous L4 Aβ-nociceptive DRG neurons in SNA rats (that developed mechanical hypersensitivity) exhibited sensitization indicated by: a) decreased mean mechanical threshold (from 57.8±7.1 to 10.3±1.7mN), b) decreased mean dorsal root electrical threshold (from 11.4±0.7 to 4.3±0.4V), c) increased mean response to a suprathreshold mechanical stimulus (from 18.5±1.8 to 34±3.7spikes/sec) and d) an obvious, but non-significant, increase in the incidence of ongoing/spontaneous activity (from 3% to 18%). These findings suggest that cutaneous L4 Aβ-nociceptors also become sensitized after L5 SNA, and that sensitization of this subclass of A-fiber nociceptors may contribute both directly and indirectly to nerve injury-induced PNP.

Paclitaxel-induced increase in NCX activity in subpopulations of nociceptive afferents: A protective mechanism against chemotherapy-induced peripheral neuropathy?

We recently demonstrated, in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), that there is a significant decrease in the duration of the depolarization-evoked Ca2+ transient in isolated somata of putative nociceptive afferents innervating the glabrous skin of the hindpaw, but no change in transient magnitude or the resting concentration of intracellular Ca2+ ([Ca2+]i). Because the Na+-Ca2+ exchanger (NCX) only contributes to the regulation of the duration of the evoked Ca2+ transient, in putative nociceptive dorsal root ganglion (DRG) neurons, we hypothesized that an increase in NCX activity underlies the CIPN-induced change in this subpopulation of neurons. Acutely dissociated retrogradely labeled sensory neurons from naïve, vehicle-, and paclitaxel-treated rats were studied with fura-2 based Ca2+ imaging. There was no difference in the relative level of NCX activity between glabrous neurons from paclitaxel-treated or control rats. However, in contrast to the relatively large and long lasting Ca2+ transients needed to evoke NCX activity in neurons from naïve rats, there was evidence of resting NCX activity in glabrous neurons from both vehicle- and paclitaxel-treated rats. More interestingly, there was a paclitaxel-induced increase in NCX activity in putative nociceptive neurons innervating the thigh, neurons in which there is no evidence of a change in the depolarization-induced Ca2+ transient, or a body site in which there was a change in nociceptive threshold. Furthermore, while the majority of NCX activity in glabrous neurons is sensitive to the NCX3-preferring blocker KB-R7943, the increase in NCX activity in thigh neurons was resistant to KB-R7943 but sensitive to the NCX1-preferring blocker SEA0400. These results suggest that a mechanism(s) other than NCX underlies the paclitaxel-induced decrease in the duration of the evoked Ca2+ transient in putative nociceptive glabrous skin neurons. However, the compensatory response to paclitaxel observed may also explain why only subpopulations of sensory neurons are impacted by paclitaxel, raising the intriguing possibility that CIPN is due to the failure of injured neurons to appropriately compensate for the deleterious consequences of this compound.

Preferred recycling pathway by internalized PGE2 EP4 receptor following agonist stimulation in cultured dorsal root ganglion neurons contributes to enhanced EP4 receptor sensitivity

Prostaglandin E2 (PGE2), a well-known pain mediator abundantly produced in injured tissues, sensitizes nociceptive dorsal root ganglion (DRG) neurons (nociceptors) through its four EP receptors (EP1–4). Our prior study showed that PGE2 or EP4 agonist stimulates EP4 externalization and this event was not only suppressed by the inhibitor of anterograde export, but also by the recycling inhibitor (St-Jacques and Ma, 2013). These data suggest that EP4 recycling also contributes to agonist-enhanced EP4 surface abundance. In the current study, we tested this hypothesis using antibody-feeding-based internalization assay, recycling assay and FITC-PGE2 binding assay. We observed that selective EP4 agonist 1-hydroxy-PGE1 (1-OH-PGE1) or CAY10850 time- and concentration-dependently increased EP4 internalization in cultured DRG neuron. Internalized EP4 was predominantly localized in the early endosomes and recycling endosomes, but rarely in the late endosomes and lysosomes. These observations were confirmed by FITC-PGE2 binding assay. We further revealed that 1-OH-PGE1 or CAY10850 time- and concentration-dependently increased EP4 recycling. Double exposures to 1-OH-PGE1 induced a greater increase in calcitonin gene-related peptide (CGRP) release than a single exposure or vehicle exposure, an event blocked by pre-treatment with the recycling inhibitor monensin. Our data suggest that EP4 recycling contributes to agonist-induced cell surface abundance and consequently enhanced receptor sensitivity. Facilitating EP4 externalization and recycling is a novel mechanism underlying PGE2-induced nociceptor sensitization.