Receptors In Dorsal Root Ganglion Neurons Research Articles

Functional expression of glycine receptors in DRG neurons of mice

Glycine receptor is one of the chloride-permeable ion channels composed of combinations of four α subunits and one β subunit. In adult spinal cord, the glycine receptor α1 subunit is crucial for the generation of inhibitory neurotransmission. The reduced glycinergic inhibition is regarded as one of the key spinal mechanisms underlying pathological pain symptoms. However, the expression and function of glycine receptors in the peripheral system are largely unknown as yet. Here we found that glycine receptor α1 subunit was prevalent in the dorsal root ganglia (DRG) neurons as well as in the sciatic nerves of adult mice. Intraganglionar or intraplantar injection of glycine receptor antagonist strychnine caused the hypersensitivity to mechanical, thermal and cold stimuli, suggesting the functional importance of peripheral glycine receptors in the control of nociceptive signal transmission. Our data showed that peripheral inflammation induced by formalin decreased the expression of glycine receptor α1 subunit on the plasma membrane of DRG neurons, which was attributed to the activation of protein kinase C signaling. Intraplantar application of glycine receptor agonist glycine or positive modulator divalent zinc ion alleviated the first-phase painful behaviors induced by formalin. These data suggested that peripheral glycine receptor might serve as an effective target for pain therapy.

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

SummaryThe immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in Pd1−/− mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.

Gi-coupled receptor activation potentiates Piezo2 currents via Gβγ.

Mechanically activated Piezo2 channels are key players in somatosensory touch, but their regulation by cellular signaling pathways is poorly understood. Dorsal root ganglion (DRG) neurons express a variety of G-protein-coupled receptors that modulate the function of sensory ion channels. Gi-coupled receptors are generally considered inhibitory, as they usually decrease excitability. Paradoxically, activation of Gi-coupled receptors in DRG neurons sometimes induces mechanical hypersensitivity, the mechanism of which is not well understood. Here, we find that activation of Gi-coupled receptors potentiates mechanically activated currents in DRG neurons and heterologously expressed Piezo2 channels, but inhibits Piezo1 currents in heterologous systems in a Gβγ-dependent manner. Pharmacological inhibition of kinases downstream of Gβγ, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) also abolishes the potentiation of Piezo2 currents. Local injection of sumatriptan, an agonist of the Gi-coupled serotonin 1B/1D receptors, increases mechanical sensitivity in mice, and the effect is abolished by inhibiting PI3K and MAPK. Hence, our studies illustrate an indirect mechanism of action of Gβγ to sensitize Piezo2 currents and alter mechanosensitivity after activation of Gi-coupled receptors.

2 Hz EA Reduces Heroin Withdrawal-Induced Hyperalgesia and Heroin Relapse by Downregulating P2X3 Receptors in DRG Neurons.

Electroacupuncture (EA) has effective analgesic effects. Our previous study demonstrated that the upregulation of P2X3 receptors in the dorsal root ganglia (DRG) might participate in heroin withdrawal-induced hyperalgesia. The aim of this study is to further explore whether 2 Hz EA reduces heroin relapse associated with its analgesic effect and whether P2X3 receptors in the DRG are involved in this process. 2 Hz EA was adopted to treat the heroin SA rats in the present study. Heroin-seeking and pain sensitivity were evaluated. The expression of P2X3 receptors in the DRG was detected. Our results showed that compared with the control group, the reinstatement, thermal hyperalgesia, and mechanical allodynia of the heroin-addicted group were increased significantly. The expression of P2X3 receptors in the DRG was increased markedly. After being treated using 2 Hz EA, reinstatement was reduced, hyperalgesia was decreased, and the upregulated expression of P2X3 receptors in the DRG had decreased significantly compared to that in the heroin-addicted group. Consequently, our results indicated that 2 Hz EA was an effective method for treating heroin-induced hyperalgesia and helping prevent relapse, and the potential mechanism might be related to the downregulation of P2X3 receptor expression in the DRG.

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth.SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits.

Presynaptic inhibition of transient receptor potential vanilloid type 1 (TRPV1) receptors by noradrenaline in nociceptive neurons.

The transient receptor potential vanilloid type 1 (TRPV1) receptor is a polymodal molecular integrator in the pain pathway expressed in Aδ- and C-fibre nociceptors and is responsible for the thermal hyperalgesia associated with inflammatory pain. Noradrenaline strongly inhibited the activity of TRPV1 channels in dorsal root ganglia neurons. The effect of noradrenaline was reproduced by clonidine and antagonized by yohimbine, consistent with contribution of α2 adrenergic receptors. The inhibitory effect of noradrenaline on TRPV1 channels was dependent on calcium influx and linked to calcium/calmodulin-dependent protein kinase II. In spinal cord slices, clonidine reduced the frequency of capsaicin-induced miniature EPSCs in the presence of tetrodotoxin and ω-conotoxin-MVIIC, consistent with inhibition of presynaptic TRPV1 channels by α2 adrenergic receptors. We suggest that modulation of presynaptic TRPV1 channels in nociceptive neurons by descending noradrenergic inputs may constitute a mechanism for noradrenaline to modulate incoming noxious stimuli in the dorsal horn of the spinal cord. The transient receptor potential vanilloid type 1 (TRPV1) receptor is a well-known contributor to nociceptor excitability. To address whether noradrenaline can down-regulate TRPV1 channel activity in nociceptors and reduce their synaptic transmission, the effects of noradrenaline and clonidine were tested on the capsaicin-activated current recorded from acutely dissociated small diameter (<27μm) dorsal root ganglia (DRG) neurons and on miniature (m)EPSCs recorded from large lamina I neurons in horizontal spinal cord slices. Noradrenaline or clonidine inhibited the capsaicin-activated current by ∼60%, and the effect was reversed by yohimbine, confirming that it was mediated by activation of α2 adrenergic receptors. Similarly, clonidine reduced the frequency of capsaicin-induced mEPSCs by ∼60%. Inhibition of capsaicin-activated current by noradrenaline was mediated by GTP binding proteins, and was highly dependent on calcium influx. The inhibitory effect of noradrenaline on the capsaicin-activated current was not affected either by blocking the activity of protein kinase A with H89, or by blocking the activity of protein kinase C with bisindolylmaleimide II. In contrast, when the calcium/calmodulin-dependent protein kinase II (CaMKII) was blocked with KN-93, the inhibitory effect of noradrenaline on the capsaicin-activated current was greatly reduced, suggesting that activation of adrenergic receptors in DRG neurons is preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by noradrenaline in nociceptive neurons is a mechanism whereby noradrenaline may suppress incoming noxious stimuli at the primary synaptic afferents in the dorsal horn of the spinal cord.

Activation of TRESK channels by the inflammatory mediator lysophosphatidic acid balances nociceptive signalling.

In dorsal root ganglia (DRG) neurons TRESK channels constitute a major current component of the standing outward current IKSO. A prominent physiological role of TRESK has been attributed to pain sensation. During inflammation mediators of pain e.g. lysophosphatidic acid (LPA) are released and modulate nociception. We demonstrate co-expression of TRESK and LPA receptors in DRG neurons. Heterologous expression of TRESK and LPA receptors in Xenopus oocytes revealed augmentation of basal K+ currents upon LPA application. In DRG neurons nociception can result from TRPV1 activation by capsaicin or LPA. Upon co-expression in Xenopus oocytes LPA simultaneously increased both depolarising TRPV1 and hyperpolarising TRESK currents. Patch-clamp recordings in cultured DRG neurons from TRESK[wt] mice displayed increased IKSO after application of LPA whereas under these conditions IKSO in neurons from TRESK[ko] mice remained unaltered. Under current-clamp conditions LPA application differentially modulated excitability in these genotypes upon depolarising pulses. Spike frequency was attenuated in TRESK[wt] neurons and, in contrast, augmented in TRESK[ko] neurons. Accordingly, excitation of nociceptive neurons by LPA is balanced by co-activation of TRESK channels. Hence excitation of sensory neurons is strongly controlled by the activity of TRESK channels, which therefore are good candidates for the treatment of pain disorders.

Novel coupling is painless

The first report that several neurotransmitters, including γ-aminobutyric acid (GABA), decreased neuronal action potential duration in dorsal root ganglia (DRG) neurons appeared more than 35 years ago (Dunlap and Fischbach, 1978). Dunlap and Fischbach (1978) realized that the effects of GABA could not occur via the ionotropic GABA receptor—at that time the only known GABA receptor—and that the target ion channel was most likely a voltage-gated calcium (CaV) channel, rather than a NaV or KV channel. They correctly concluded that another type of GABA receptor must exist, which we now know is the G protein–coupled GABA type B (GABAB) receptor. The modulated channel was later identified as the chick homologue of the N-type Ca2+ channel CaV2.2 (α1B) (Cox and Dunlap, 1992), one of three members of the CaV2 family. GABAB receptors in human and rodent sensory neurons and in various expression systems were shown subsequently to inhibit native N-current and recombinant CaV2.2 current, respectively (Raingo et al., 2007; Callaghan et al., 2008; Adams and Berecki, 2013). Inhibition primarily occurs by a voltage-dependent mechanism common to various neurotransmitters whereby Gβγ binds to CaV2.2 slowing channel opening, whereas positive voltage steps relieve this inhibition (Marchetti et al., 1986). The closely related P/Q-type (α1A) channel, CaV2.1, exhibits similar modulation by GABA (Mintz and Bean, 1993). The third member of the CaV2 family, CaV2.3 (α1E), is less susceptible to direct Gβγ modulation than the other two family members (Shekter et al., 1997). The revelation that mice with a deletion in either CaV2.2 or in CaV2.3 exhibited reduced neuropathic pain–like behavior, indicating that these channels participate in pain sensation signaling (Saegusa et al., 2000, 2001), sparked great interest in the regulation of CaV2 inhibition by GABAB receptors in DRG neurons. Astonishingly, however, the precise mechanism of GABAB receptor modulation of CaV2.3 channels has remained ill defined.

Estrogen modulation of visceral nociception

A large body of literature supports the idea that estrogen modulates nociceptive responses in pelvic pain syndromes; however, whether this hormone is pro- or anti-nociceptive remains unresolved. The dorsal root ganglion (DRG) is an important site of visceral afferent convergence and cross-sensitization. Within the context of our hypothesis visceral nociception and nociceptor sensitization appear to be regulated by purinergic P2X3 and vanilloid TRPV1 receptors and 17β-estradiol modulates DRG neuron response to ATP (P2X agonist) and capsaicin (TRPV1 agonist) suggesting that visceral afferent nociceptors are modulated by estrogen in the DRG. 17-β estradiol (E2), the most common form of estrogen, acts on functional properties of P2X3 and TRPV1 receptors in DRG neurons in vitro. The localization of estrogen receptors (ER) in DRG neurons and the attenuation of ATP/capsaicin-induced intracellular calcium concentration [Ca2+]i strongly suggest that E2 modulates visceral pain processing peripherally. Moreover, E2 appears to have different actions on nociceptive signaling depending on the input. Based on our data we propose that E2 can gate primary afferent response to increase or decrease nociception.

Rapid Impact of Progesterone on the Neuronal Growth Cone

In the last two decades, sensory neurons and Schwann cells in the dorsal root ganglia (DRG) were shown to express the rate-limiting enzyme of the steroid synthesis, cytochrome P450 side-chain cleavage enzyme (P450scc), as well as the key enzyme of progesterone synthesis, 3β-hydroxysteroid dehydrogenase (3β-HSD). Thus, it was well justified to consider that DRG neurons similarly are able to synthesize progesterone de novo from cholesterol. Because direct progesterone effects on axonal outgrowth in peripheral neurons have not been investigated up to now, the present study provides the first insights into the impact of exogenous progesterone on axonal outgrowth in DRG neurons. Our studies including microinjection and laser scanning microscopy demonstrate morphological changes especially in the neuronal growth cones after progesterone treatment. Furthermore, we were able to detect a distinctly enhanced motility only a few minutes after the start of progesterone treatment using time-lapse imaging. Investigation of the cytoskeletal distribution in the neuronal growth cone before, during, and after progesterone incubation revealed a rapid reorganization of actin filaments. To get a closer idea of the underlying receptor mechanisms, we further studied the expression of progesterone receptors in DRG neurons using RT-PCR and immunohistochemistry. Thus, we could demonstrate for the first time that classical progesterone receptor (PR) A and B and the recently described progesterone receptor membrane component 1 (PGRMC1) are expressed in DRG neurons. Antagonism of the classical progesterone receptors by mifepristone revealed that the observed progesterone effects are transmitted through PR-A and PR-B.

The K + channel GIRK2 is both necessary and sufficient for peripheral opioid‐mediated analgesia

The use of opioid agonists acting outside the central nervous system (CNS) is a promising therapeutic strategy for pain control that avoids deleterious central side effects such as apnea and addiction. In human clinical trials and rat models of inflammatory pain, peripherally restricted opioids have repeatedly shown powerful analgesic effects; in some mouse models however, their actions remain unclear. Here, we investigated opioid receptor coupling to K+ channels as a mechanism to explain such discrepancies. We found that GIRK channels, major effectors for opioid signalling in the CNS, are absent from mouse peripheral sensory neurons but present in human and rat. In vivo transgenic expression of GIRK channels in mouse nociceptors established peripheral opioid signalling and local analgesia. We further identified a regulatory element in the rat GIRK2 gene that accounts for differential expression in rodents. Thus, GIRK channels are indispensable for peripheral opioid analgesia, and their absence in mice has profound consequences for GPCR signalling in peripheral sensory neurons.GIRK channels are indispensable for peripheral opioid analgesia. The absence of GIRK channels from mouse dorsal root ganglion neurons questions the predictive validity of mice as a model organism for investigating peripheral GPCRmediated analgesia.

Upregulation of P2X3 receptors by neuronal calcium sensor protein VILIP-1 in dorsal root ganglions contributes to the bone cancer pain in rats

Primary and metastatic cancers that affect bone are frequently associated with severe and intractable pain. The mechanisms underlying the development of bone cancer pain are largely unknown. In this study, we first demonstrated that a functional upregulation of P2X3 receptors in dorsal root ganglion (DRG) neurons is closely associated with the neuronal hyperexcitability and the cancer-induced bone pain in MRMT-1 tumor cell–inoculated rats. Second, we revealed that visinin-like protein 1 (VILIP-1), a member of visinin-like proteins that belong to the family of neuronal calcium sensor proteins is responsible for the observed upregulation of P2X3 receptors in DRG neurons. The interaction between the amino terminus of VLIP-1 and the carboxyl terminus of the P2X3 receptor is critical for the surface expression and functional enhancement of the receptor. Finally, overexpression of VILIP-1 increases the expression of functional P2X3 receptors and enhances the neuronal excitability in naive rat DRG neurons. In contrast, knockdown of VILIP-1 inhibits the development of bone cancer pain via downregulation of P2X3 receptors and repression of DRG excitability in MRMT-1 rats. Taken together, these results suggest that functional upregulation of P2X3 receptors by VILIP-1 in DRG neurons contributes to the development of cancer-induced bone pain in MRMT-1 rats. Hence, P2X3 receptors and VILIP-1 could serve as potential targets for therapeutic interventions in cancer patients for pain management. Pharmacological blockade of P2X3 receptors or knockdown of VILIP-1 in DRGs would be used as innovative strategies for the treatment of bone cancer pain.

Neuronal IL-17 receptor upregulates TRPV4 but not TRPV1 receptors in DRG neurons and mediates mechanical but not thermal hyperalgesia

In addition to the proinflammatory cytokines tumor necrosis factor-α, interleukin-6 and interleukin-1ß, the cytokine interleukin-17 (IL-17) is considered an important mediator of autoimmune diseases such as rheumatoid arthritis. Because tumor necrosis factor-α and interleukin-1ß have the potential to influence the expression of transduction molecules such as transient receptor potential vanilloid 1 (TRPV1) in dorsal root ganglion (DRG) neurons and thus to contribute to pain we explored in the present study whether IL-17A activates DRG neurons and influences the expression of TRPV1. The IL-17A receptor was visualized in most neurons in dorsal root ganglion (DRG) sections as well as in cultured DRG neurons. Upon long-term exposure to IL-17A, isolated and cultured rat DRG neurons showed a significant upregulation of extracellular-regulated kinase (ERK) and nuclear factor κB (NFκB). Long-term exposure of neurons to IL-17A did not upregulate the expression of TRPV1. However, we found a pronounced upregulation of transient receptor potential vanilloid 4 (TRPV4) which is considered a candidate transduction molecule for mechanical hyperalgesia. Upon the injection of zymosan into the paw, IL-17A-deficient mice showed less mechanical hyperalgesia than wild type mice but thermal hyperalgesia was not attenuated in IL-17A-deficient mice. These data show, therefore, a particular role of IL-17 in mechanical hyperalgesia, and they suggest that this effect is linked to an activation and upregulation of TRPV4.

Intrathecal endothelin-1 has antinociceptive effects in rat model of postoperative pain

Endothelin-1 is known to be a potent vasoconstrictor. Administration of endothelin-1 to the central nervous system (CNS) induces antinociceptive effects. Nociceptive stimuli affect dorsal root ganglion (DRG) neurons and neurons/astrocytes/microglia in the dorsal horn of the spinal cord. Surgical incision in the plantar aspect of the rat hindpaw is a model for postoperative pain, and withdrawal thresholds reportedly decrease around the incision. We hypothesized that intrathecal endothelin-1 would have antinociceptive effects in this model, and affect DRG neurons and microglia/neurons in the dorsal horn. Intrathecal endothelin-1 partially restored the withdrawal threshold (which was decreased by plantar incision). BQ-123, and BQ-788 (specific endothelin ETA- and ETB-receptor antagonists, respectively) attenuated the increase in withdrawal threshold induced by endothelin-1. Phosphorylation of extracellular signal-regulated kinase (ERK) in DRG neurons and microglial activation/ERK phosphorylation in the dorsal horn were observed following the incision. Endothelin-1 decreased the incision-induced increase in the numbers of phosphorylated ERK-positive neurons in DRG and activated microglia in the dorsal horn, without affecting the numbers of phosphorylated ERK-positive neurons in the dorsal horn. BQ-123 or BQ-788 partially suppressed these endothelin-1-induced alterations. Our results show that the pain threshold, which is decreased by surgical stimuli, is partially restored by intrathecal endothelin-1 through both endothelin ETA- and ETB- receptors in DRG neurons and microglia in the spinal cord. Endothelin-1 administration to the CNS may be worth considering as a new candidate for the treatment of postoperative pain and to mitigate prolonged periods of pain.

Alterations in tyrosine kinase receptor (Trk) expression induced by insulin-like growth factor-1 in cultured dorsal root ganglion neurons

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor expressed in small dorsal root ganglion (DRG) neurons. IGF-1 promotes neuronal survival by activating its receptor (IGF-1R). Whether IGF-1 and its signaling pathways influence the expression of tyrosine kinase receptors TrkA, TrkB and TrkC in DRG neurons remains unknown. In the present study, primary cultured DRG neurons were used to determine the effects of IGF-1 on TrkA, TrkB and TrkC expression. The involvement of extracellular signal-regulated protein kinase (ERK1/2) and the effects of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways on IGF-1 were also evaluated. DRG neurons were cultured for 48h and then exposed to IGF-1, PD98059 plus IGF-1, LY294002 plus IGF-1, and PD98059 plus LY294002 plus IGF-1 for an additional 24h. The DRG neurons were continuously exposed to culture medium as a control. All cultures were then processed for detection of mRNA levels of TrkA, TrkB and TrkC using real-time PCR analysis. Protein levels of TrkA, TrkB and TrkC were detected using a Western blot assay. The expression of TrkA, TrkB and TrkC in situ was determined by a fluorescent labeling technique. The levels of phosphorylated ERK1/2 (pERK1/2) and phosphorylated Akt (pAkt) were detected using a Western blot assay. The results indicated that in primary cultured DRG neurons, IGF-1 increased the expression of TrkA and TrkB and their mRNAs but not TrkC or its mRNA. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 alone blocked the effect of IGF-1, but the use of both inhibitors together was effective. IGF-1 may play an important role in regulating the expression of different Trk receptors in DRG neurons through the ERK1/2 and PI3K/Akt signaling pathways. These results suggest that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects.

Analgesic effect of electroacupuncture on chronic neuropathic pain mediated by P2X3 receptors in rat dorsal root ganglion neurons

Adenosine 5′-triphosphate disodium (ATP) gated P2X receptors, especially the subtype P2X3, play a key role in transmission of pain signals in neuropathic pain, ATP has been documented to play a significant role in the progression of pain signals, suggesting that control of these pathways through electroacupuncture (EA) is potentially an effective treatment for chronic neuropathic pain. EA has been accepted to effectively manage chronic pain by applying the stimulating current to acupoints through acupuncture needles. To determine the significance of EA on neuropathic pain mediated by P2X3 receptors in the dorsal root ganglion (DRG) neurons, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded, and the expression of P2X3 receptors in the DRG neurons was assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). In addition, the currents which were evoked in DRG neurons isolated from rats following chronic constriction injury (CCI) by the P2X3 receptors agonists i.e. ATP and α,β-methylen-ATP (α,β-meATP) were examined through the experimental use of whole cell patch clamp recording. The present study demonstrates that EA treatment can increase the MWT and TWL values and decrease the expression of P2X3 receptors in DRG neurons in CCI rats. Simultaneously, EA treatment attenuates the ATP and α,β-meATP evoked currents. EA may be expected to induce an apparent induce analgesic effect by decreasing expression and inhibiting P2X3 receptors in DRG neurons of CCI rats. There is a similar effect on analgesic effect between rats with contralateral EA and those with ipsilateral EA.

Botulinum Toxin Decreases Hyperalgesia and Inhibits P2X3Receptor Over-Expression in Sensory Neurons Induced by Ventral Root Transection in Rats

We aim to determine the effects of Botulinum toxin type A (BTX-A) on neuropathic pain behavior and the expression of P2X(3) receptor in dorsal root ganglion (DRG) in rats with neuropathic pain induced by L5 ventral root transection (L5 VRT). Neuropathic pain was induced by L5 VRT in male Sprague-Dawley rats. Either saline or BTX-A was administered to the plantar surface. Behavioral tests were conducted preoperatively and at predefined postoperative days. The expression of P2X(3) receptors in DRG neurons was detected by immunoreactivity at postoperative days 3, 7, 14, and 21. The number of positive P2X(3) neurons in the ipsilateral L5 DRG increased significantly after L5 VRT (P<0.001). This increase persisted for at least 3 weeks after the operation. No significant changes in P2X(3) expression were detected in the contralateral L5, or in the L4 DRGs bilaterally. Subcutaneous administration of BTX-A, performed on the left hindpaw at days 4, 8, or 16 post VRT surgery, significantly reduced mechanical allodynia bilaterally and inhibited P2X(3) over-expression induced by L5 VRT. L5 VRT led to over-expression of P2X(3) receptors in the L5 DRG and bilateral mechanical allodynia in rats. Subcutaneous injection of BTX-A significantly reversed the neuropathic pain behavior and the over-expression of P2X(3) receptor in nociceptive neurons. These data not only show over-expression of purinergic receptors in the VRT model of neuropathic pain but also reveal a novel mechanism of botulinum toxin action on nociceptive neurons.

Effects of prostaglandin D 2 on tetrodotoxin-resistant Na + currents in DRG neurons of adult rat

Tetrodotoxin-resistant (TTX-R) Na + channels play a key role in the generation of action potentials in nociceptive dorsal root ganglion (DRG) neurons and are an important target for the proinflammatory mediator prostaglandin E 2, which augments these currents. Prostaglandin D 2 (PGD 2) is released in the tissue together with prostaglandin E 2, and it was reported to be antiinflammatory, but its effect on primary afferent neurons is unclear. In the present study we localised G s-protein-coupled DP1 and G i-protein-coupled DP2 receptors in DRG neurons, and we assessed the effect of PGD 2 on TTX-R Na + currents in patch-clamp recordings from small- to medium-sized cultured DRG neurons from adult rats. DP1 and DP2 receptor-like immunoreactivity was localised in the vast majority of DRG neurons. In all neurons, PGD 2 shifted conductance to more hyperpolarised potentials, depending on an action at Na v1.9 channels. In about one third of the neurons, PGD 2 additionally influenced Na v1.8 channels by facilitating conductance and by increasing maximal current amplitudes. Selective DP1 receptor activation increased the amplitude of TTX-R Na + currents of most neurons, but this effect was counteracted by DP2 receptor activation, which by itself had no effect. In the current-clamp mode, PGD 2 lowered the threshold for elicitation of an action potential and increased the number of action potentials per stimulus, an effect mainly depending on DP1 receptor activation. Thus, the net effect of PGD 2 on DRG neurons is pronociceptive, although the magnitude of the TTX-R Na + currents depends on the balance of DP1 and DP2 receptor activation. Prostaglandin D 2 regulates conductance and current amplitudes of tetrodotoxin-resistant sodium channels on dorsal root ganglion neurons by a balanced action at DP1 and DP2 receptor subtypes.

The effects of sympathetic outflow on upregulation of vanilloid receptors TRPV1 in primary afferent neurons evoked by intradermal capsaicin

The vanilloid receptor TRPV(1) is a key nociceptive molecule located in primary afferent nociceptive neurons in dorsal root ganglia (DRG) for initiating neurogenic inflammation and pain. Our recent study demonstrates that up-regulation of TRPV(1) receptors by intradermal injection of capsaicin is modulated by activation of the protein kinase C (PKC) cascade. Neurogenic inflammation and pain resulting from capsaicin injection are sympathetically dependent, responding to norepinephrine, adenosine 5'-triphosphate (ATP) and/or neuropeptide Y released from sympathetic efferents. In a rat model of acute neurogenic inflammatory pain produced by capsaicin injection, we used immunofluorescence and Western blots combined with pharmacology and surgical sympathectomies to analyze whether the capsaicin-evoked up-regulation of TRPV(1) in DRG neurons is affected by sympathetic outflow by way of activating the PKC cascade. Sympathetic denervation reduced significantly the capsaicin-evoked expressions of TRPV(1), calcitonin gene-related peptide and/or phosphorylated PKC and their co-expression. These reductions could be restored by exogenous pretreatment with an analog of ATP, alpha,beta-methylene ATP. Inhibition of PKC with chelerythrine chloride prevented the ATP effect. Consistent results were obtained from experiments in which capsaicin-evoked changes in cutaneous inflammation (vasodilation and edema) were examined after sympathetic denervation, and the effects of the above pharmacological manipulations were evaluated. Our findings suggest that the capsaicin-evoked up-regulation of TRPV(1) receptors in DRG neurons is modulated sympathetically by the action of ATP released from sympathetic efferents to activate the PKC cascade. Thus, this study proposes a potential new mechanism of sympathetic modulation of neurogenic inflammation.

Increases in transient receptor potential vanilloid‐1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation

A recent study by our group demonstrates pharmacologically that the transient receptor potential vanilloid-1 (TRPV(1)) is activated by intradermal injection of capsaicin to initiate neurogenic inflammation by the release of neuropeptides in the periphery. In this study, expression of TRPV(1), phosphorylated protein kinase C (p-PKC), and calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons was visualized by using immunofluorescence, real-time PCR, and Western blots to examine whether increases in TRPV(1) mRNA and protein levels evoked by capsaicin injection are subject to modulation by the activation of PKC and to analyze the role of this process in the pathogenesis of neurogenic inflammation. Capsaicin injection into the hindpaw skin of anesthetized rats evoked increases in the expression of TRPV(1), CGRP and p-PKC in mRNA and/or protein levels and in the number of single labeled TRPV(1), p-PKC, and CGRP neurons in ipsilateral L4-5 DRGs. Coexpressions of TRPV(1) with p-PKC and/or CGRP in DRG neurons were also significantly increased after CAP injection. These evoked expressions at both molecular and cellular levels were significantly inhibited after TRPV(1) receptors were blocked by 5'-iodoresiniferatoxin (5 microg) or PKC was inhibited by chelerythrine chloride (5 microg). Taken together, these results provide evidence that up-regulation of TRPV(1) mRNA and protein levels under inflammatory conditions evoked by capsaicin injection is subject to modulation by the PKC cascade in which increased CGRP level in DRG neurons may be related to the initiation of neurogenic inflammation. Thus, up-regulation of TRPV(1) receptors in DRG neurons seems critical for initiating acute neurogenic inflammation.