Generation of resonance-dependent oscillation by mGluR-I activation switches single spiking to bursting in mesencephalic trigeminal sensory neurons.

The primary sensory neurons supplying muscle spindles of jaw-closing muscles are unique in that they have their somata in the mesencephalic trigeminal nucleus (MTN) in the brainstem, thereby receiving various synaptic inputs. MTN neurons display bursting upon activation of glutamatergic synaptic inputs while they faithfully relay respective impulses arising from peripheral sensory organs. The persistent sodium current (IN aP ) is reported to be responsible for both the generation of bursts and the relay of impulses. We addressed how IN aP is controlled either to trigger bursts or to relay respective impulses as single spikes in MTN neurons. Protein kinase C (PKC) activation enhanced IN aP only at low voltages. Spike generation was facilitated by PKC activation at membrane potentials more depolarized than the resting potential. By injection of a ramp current pulse, a burst of spikes was triggered from a depolarized membrane potential whereas its instantaneous spike frequency remained almost constant despite the ramp increases in the current intensity beyond the threshold. A puff application of glutamate preceding the ramp pulse lowered the threshold for evoking bursts by ramp pulses while chelerythrine abolished such effects of glutamate. Dihydroxyphenylglycine, an agonist of mGluR1/5, also caused similar effects, and increased both the frequency and impedance of membrane resonance. Immunohistochemistry revealed that glutamatergic synapses are made onto the stem axons, and that mGluR1/5 and Nav1.6 are co-localized in the stem axon. Taken together, glutamatergic synaptic inputs onto the stem axon may be able to switch the relaying to the bursting mode.

Predictability of various serial subtractions on global deterioration scale according to education level.

BACKGROUND: The serial 100-7s subtraction, an item on the Mini-Mental State Examination (MMSE), is well known for being difficult for uneducated people. Therefore, we investigated into alternative serial subtractions for serial 100-7s subtraction in uneducated people. METHODS: One hundred sixty-nine subjects were enrolled by neurologic or neuropsychiatric out-patient clinics in 4 university medical centers. The subjects were divided into two groups: an uneducated group and an educated group (at least primary schooling) by questionnaire. We investigated the correlation between incorrect number of serial subtractions and Global Deterioration Scale (GDS) score in both groups and undertook receiver operating characteristic (ROC) curve analysis. MMSE including serial 40-4s subtraction, serial 20-2s subtraction, and serial 10-1s subtraction instead of serial 100-7s subtraction were arbitrally named MMSE4, MMSE2, and MMSE1. RESULTS: In the educated group, serial 100-7s subtraction showed the highest correlation with GDS score (correlation coefficient, 0.465; P < 0.001). In the uneducated group, serial 40-4s subtraction showed the highest correlation with GDS score (correlation coefficient, 0.608; P < 0.001), and serial 100-7s indicated the lowest correlation (correlation coefficient, 0.378; P = 0.023). In ROC curve analysis for MMSE, MMSE4, MMSE2, and MMSE1 to assess the presence of dementia (GDS score ≥ 3) in uneducated subjects, the area under the curve (AUC) was 0.648, 0.770, 0.758, and 0.711, respectively, and in educated subjects, AUC for MMSE, MMSE4, MMSE2, and MMSE1 was 0.729, 0.719, 0.716, and 0.714, respectively. CONCLUSION: Out of MMSE items, serial 100-7s is adequate in the educated elderly, but may be less adequate in the uneducated elderly. Serial 40-4s seems to be more appropriate for MMSE in the uneducated elderly.

A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons.

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (I(h)) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 µm in diameter) exhibiting tonic firing and prominent I(h), SKL-NP inhibited I(h) and spike firings in a concentration dependent manner (IC(50)=7.85 µM). SKL-NP-induced inhibition of I(h) was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates I(h) in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I(h)) in DRG neurons of rats.

Neurochemical properties of dental primary afferent neurons.

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Aß fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Aδ fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Aß fibers. We suggest that these Aß fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

TRPV1 in GABAergic interneurons mediates neuropathic mechanical allodynia and disinhibition of the nociceptive circuitry in the spinal cord.

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Eugenol reverses mechanical allodynia after peripheral nerve injury by inhibiting hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Mechanical allodynia is a common symptom found in neuropathic patients. Hyperpolarization-activated cyclic nucleotide-gated channels and their current, I(h), have been suggested to play an important role in neuropathic pain, especially in mechanical allodynia and spontaneous pain, by involvement in spontaneous ectopic discharges after peripheral nerve injury. Thus, I(h) blockers may hold therapeutic potential for the intervention of mechanical allodynia under diverse neuropathic conditions. Here we show that eugenol blocks I(h) and abolishes mechanical allodynia in the trigeminal system. Eugenol produced robust inhibition of I(h) with IC(50) of 157 µM in trigeminal ganglion (TG) neurons, which is lower than the dose of eugenol that inhibits voltage-gated Na channels. Eugenol-induced I(h) inhibition was not mediated by G(i/o)-protein activation, but was gradually diminished by an increase in intracellular cAMP concentration. Eugenol also inhibited I(h) from injured TG neurons which were identified by retrograde labeling with DiI and reversed mechanical allodynia in the orofacial area after chronic constriction injury of infraorbital nerve. We propose that eugenol could be potentially useful for reversing mechanical allodynia in neuropathic pain patients.

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 µM) and capsaicin (1 µM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 µM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 µM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.

Effects of saccharin intake on hippocampal and cortical plasticity in juvenile and adolescent rats.

The sensory system is developed and optimized by experiences given in the early phase of life in association with other regions of the nervous system. To date, many studies have revealed that deprivation of specific sensory experiences can modify the structure and function of the central nervous system; however, the effects of sensory overload remains unclear. Here we studied the effect of overloading the taste sense in the early period of life on the synaptic plasticity of rat hippocampus and somatosensory cortex. We prepared male and female Sprague Dawley rats with ad libitum access to a 0.1% saccharin solution for 2 hrs per day for three weeks after weaning on postnatal day 22. Saccharin consumption was slightly increased in males compared with females; however, saccharin intake did not affect chow intake or weight gain either in male or in female rats. We examined the effect of saccharin-intake on long term potentiation (LTP) formation in hippocampal Schaffer collateral pathway and somatosensory cortex layer IV - II/III pathways in the 6-week old saccharin-fed rats. There was no significant difference in LTP formation in the hippocampus between the control group and saccharin-treated group in both male and female rats. Also in the somatosensory cortex, we did not see a significant difference in LTP among the groups. Therefore, we conclude that saccharin-intake during 3~6 weeks may not affect the development of physiological function of the cortical and hippocampal synapses in rats.

Selectively targeting pain in the trigeminal system.

We tested whether it is possible to selectively block pain signals in the orofacial area by delivering the permanently charged lidocaine derivative QX-314 into nociceptors via TPRV1 channels. We examined the effects of co-applied QX-314 and capsaicin on nociceptive, proprioceptive, and motor function in the rat trigeminal system. QX-314 alone failed to block voltage-gated sodium channel currents (I(Na)) and action potentials (APs) in trigeminal ganglion (TG) neurons. However, co-application of QX-314 and capsaicin blocked I(Na) and APs in TRPV1-positive TG and dental nociceptive neurons, but not in TRPV1-negative TG neurons or in small neurons from TRPV1 knock-out mice. Immunohistochemistry revealed that TRPV1 is not expressed by trigeminal motor and trigeminal mesencephalic neurons. Capsaicin had no effect on rat trigeminal motor and proprioceptive mesencephalic neurons and therefore should not allow QX-314 to enter these cells. Co-application of QX-314 and capsaicin inhibited the jaw-opening reflex evoked by noxious electrical stimulation of the tooth pulp when applied to a sensory but not a motor nerve, and produced long-lasting analgesia in the orofacial area. These data show that selective block of pain signals can be achieved by co-application of QX-314 with TRPV1 agonists. This approach has potential utility in the trigeminal system for treating dental and facial pain.

R-type Calcium Channel Isoform in Rat Dorsal Root Ganglion Neurons.

R-type Ca(v)2.3 high voltage-activated Ca(2+) channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six Ca(v)2.3 isoforms (Ca(v)2.3a~Ca(v)2.3e), the Ca(v)2.3e isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of Ca(v)2.3 isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, Ca(v)2.3a and Ca(v)2.3e, in DRG neurons. Single-cell RT-PCR detected the expression of Ca(v)2.3e mRNA in 20% (n=14/70) of DRG neurons, relative to Ca(v)2.3a expression in 2.8% (n=2/70) of DRG neurons. Ca(v)2.3e mRNA was mainly detected in small-sized neurons (n=12/14), but in only a few medium-sized neurons (n=2/14) and not in large-sized neurons, indicating the prominence of Ca(v)2.3e in nociceptive DRG neurons. Moreover, Ca(v)2.3e was preferentially expressed in tyrosine-kinase A (trkA)-positive, isolectin B4 (IB4)-negative and transient receptor potential vanilloid 1 (TRPV1)-positive neurons. These results suggest that Ca(v)2.3e may be the main R-type Ca(2+) channel isoform in nociceptive DRG neurons and thereby a potential target for pain treatment, not only in the trigeminal system but also in the spinal system.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells.

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 microM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 microM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca(2+)](i) response; a transient [Ca(2+)](i) peak and sustained [Ca(2+)](i) increase secondary to ATP-stimulated Ca(2+) influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons.

Transient receptor potential vanilloid subtype 1 (TRPV1) and metabotropic glutamate receptor 5 (mGluR5) located on peripheral sensory terminals have been shown to play critical roles in the transduction and modulation of pain sensation. To date, however, very little is known regarding the significance of functional expression of mGluR5 and TRPV1 on the central terminals of sensory neurons in the dorsal horn of the spinal cord. Here we show that TRPV1 on central presynaptic terminals is coupled to mGluR5 in a membrane-delimited manner, thereby contributing to the modulation of nociceptive synaptic transmission in the substantia gelatinosa neurons of the spinal cord. Further, our results demonstrate that TRPV1 is involved in the pain behaviors induced by spinal mGluR5 activation, and diacylglycerol produced by the activation of mGluR5 mediates functional coupling of mGluR5 and TRPV1 on the presynaptic terminals. Thus, mGluR5-TRPV1 coupling on the central presynaptic terminals of nociceptive neurons may be an important mechanism underlying central sensitization under pathological pain conditions.

Histamine H1 receptor induces cytosolic calcium increase and aquaporin translocation in human salivary gland cells.

One of the common side effects of antihistamine medicines is xerostomia (dry mouth). The current consensus is that antihistamine-induced xerostomia comes from an antimuscarinic effect. Although the effect of antihistamines on salivary secretion is both obvious and significant, the cellular mechanism whereby this happens is still unclear because of the lack of knowledge of histamine signaling in human salivary glands. Here, we have studied histamine receptors and the effect of antihistamines on human submandibular acinar cells. In primary cultured human submandibular gland and a HSG cell line, histamine increased the intracellular Ca(2+) concentration. The histamine-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) increase was inhibited by histamine H1 receptor-specific antagonists, and the expression of the functional histamine H1 receptor was confirmed by reverse transcription-polymerase chain reaction. Interestingly, histamine pretreatment did not inhibit a subsequent carbachol-induced [Ca(2+)](i) rise without "heterologous desensitization." Chlorpheniramine inhibited a carbachol-induced [Ca(2+)](i) increase at a 100-fold greater concentration than histamine receptor antagonism, whereas astemizole and cetrizine showed more than 1000-fold difference, which in part explains the xerostomia-inducing potency among the antihistamines. Notably, histamine resulted in translocation of aquaporin-5 to the plasma membrane in human submandibular gland cells and green fluorescent protein-tagged aquaporin-5 expressing HSG cells. We found that histidine decarboxylase and the histamine H1 receptor are broadly distributed in submandibular gland cells, whereas choline acetyltransferase is localized only at the parasympathetic terminals. Our results suggest that human salivary gland cells express histamine H1 receptors and histamine-synthesizing enzymes, revealing the cellular mechanism of antihistamine-induced xerostomia.

Molecular mechanism for local anesthetic action of eugenol in the rat trigeminal system.

Eugenol is widely used in dentistry as a local analgesic agent, because of its ability to allay tooth pain. Interestingly, eugenol shares several pharmacological actions with local anesthetics which include inhibition of voltage-gated sodium channel (VGSC) and activation of transient receptor potential vanilloid subtype 1 (TRPV1). In the present study, we investigated the effects of eugenol on pain behaviors in orofacial area, and as an attempt to elucidate its mechanism we characterized inhibitory effects of eugenol on VGSCs in trigeminal ganglion (TG) neurons. TG neurons were classified into four types on the basis of their neurochemical and electrophysiological properties such as cell size, shapes of action potential (AP), isolectin-B(4) (IB(4)) binding, and were analyzed for the association of their distinctive electrophysiological properties and mRNA expression of Na(v)1.8 and TRPV1 by using single-cell RT-PCR following whole-cell recordings. Subcutaneous injection of eugenol reduced the thermal nociception and capsaicin-induced thermal hyperalgesia in a dose-dependent manner. Eugenol also diminished digastric electromyogram evoked by noxious electrical stimulation to anterior tooth pulp, which was attributable to the blockade of AP conduction on inferior alveolar nerve. At cellular level, eugenol reversibly inhibited APs and VGSCs in IB(4)+/TRPV1+/Na(v)1.8+ nociceptive TG neurons (Type I-Type III) and IB(4)-/TRPV1-/Na(v)1.8- nociceptive TG neurons (Type IV). Both TTX-resistant I(Na) in Type I-Type III neurons and TTX-sensitive I(Na) in Type IV neurons were sensitive to eugenol. Taken together, these results suggest that eugenol may serve as local anesthetics for other pathological pain conditions in addition to its wide use in dental clinic.

Vasoactive intestinal peptide inhibits toll-like receptor 3-induced nitric oxide production in Schwann cells and subsequent sensory neuronal cell death in vitro.

We have previously reported that polyinosinic-polycytidylic acids [poly(I:C)], a synthetic toll-like receptor 3 (TLR3) agonist, induce Schwann cell activation, which exerts neurotoxic effects on sensory neurons. In this study, we investigated the effects of vasoactive intestinal peptide (VIP), a neuropeptide implicated in nerve regeneration, on TLR3-induced Schwann cell activation. VIP receptors VPAC1 and VPAC2 were constitutively expressed in rat Schwann cells. VIP pretreatment inhibited TLR3-induced inducible nitric oxide synthase (iNOS) gene expression and NO production in Schwann cells. Studies on the intracellular signal transduction pathways indicate that the VIP effect is mediated by protein kinase A activation. VIP also inhibited the poly(I:C)-induced p38 activation that is responsible for the iNOS gene expression in Schwann cells. Finally, VIP inhibited dorsal rooyt ganglion neuronal cell death caused by NO produced in activated Schwann cells. Taken together, our data suggest that VIP exerts a neuroprotective effect by inhibiting neurotoxic Schwann cell activation.

Toll-like receptor 2 contributes to glial cell activation and heme oxygenase-1 expression in traumatic brain injury.

Traumatic brain injury is accompanied by glial cell activation around the site of the injury. In this study, we investigated the role of toll-like receptor 2 (TLR2) in glial cell activation using a stab-wound injury (SWI) model with TLR2 knock-out mice. Penetration of a normal mouse brain with a 26-G needle using a stereotaxic instrument resulted in an 18- and 4-fold upregulation of GFAP and CD11b mRNA, respectively, along the needle track in the injury area. However, in the TLR2 knock-out mice, the induced expression of these genes was reduced by 70% and 40%, respectively. Likewise, there was a reduction in the area of activated glial cells detected by immunohistochemistry and the glial cells had a less-activated morphology in the TLR2 knock-out mice. In addition, the expression of the heme oxygenase-1 (HO-1) gene, a glia-expressing wound-responsive gene, was reduced after SWI in TLR2 knock-out mice. Taken together, these data argue that TLR2 contributes to the glial cell activation and HO-1 gene expression associated with traumatic brain injury.

Differential Changes in TRPV1 expression after trigeminal sensory nerve injury.

UNLABELLED: We have recently demonstrated that inferior alveolar nerve and mental nerve (branches of the mandibular nerve) injury from rats serves as a valid trigeminal neuropathic pain model. In these animals, we found that neuronal loss of trigeminal ganglion (TG) was not correlated with pain hypersensitivity. In this study, we examined changes of transient receptor potential vanilloid 1 (TRPV1) expression in the injured and uninjured TG neurons using immunohistochemical analysis at 3 days after surgery, the time point where we observed significant pain hypersensitivity. Injured neurons were identified by positive immunoreactivity for activating transcription factor 3 (ATF3). ATF3 immunoreactivity was exclusively observed in the nuclei of subpopulation of ipsilateral mandibular TG neurons, whereas no ATF3 expression was found in the naive and contralateral TG neurons. Interestingly, the expression of TRPV1 was increased in the uninjured ipsilateral maxillary TG neurons as well as in the uninjured ipsilateral mandibular TG neurons. The upregulation of TRPV1 and ATF3 expression returned to the basal level at 60 days after surgery. Our results demonstrate that trigeminal sensory nerve injury induced differential changes in TRPV1 expression of the injured and uninjured TG neurons. The upregulation of TRPV1 in uninjured TG neurons may play an important role in pain hypersensitivity after trigeminal nerve injury. PERSPECTIVE: The TRPV1 is a well-known pain transducer molecule and plays crucial roles in the perception of inflammatory and thermal pain. This article presents that TRPV1 expression was increased in uninjured neurons rather than injured neurons after peripheral nerve injury. The upregulation of TRPV1 in uninjured neurons may be associated with the development of neuropathic pain. TRPV1 might be a potential target for the treatment of neuropathic pain.

Eugenol Inhibits ATP-induced P2X Currents in Trigeminal Ganglion Neurons.

Eugenol is widely used in dentistry to relieve pain. We have recently demonstrated voltage-gated Na(+) and Ca(2+) channels as molecular targets for its analgesic effects, and hypothesized that eugenol acts on P2X(3), another pain receptor expressed in trigeminal ganglion (TG), and tested the effects of eugenol by whole-cell patch clamp and Ca(2+) imaging techniques. In the present study, we investigated whether eugenol would modulate 5'-triphosphate (ATP)-induced currents in rat TG neurons and P2X(3)-expressing human embryonic kidney (HEK) 293 cells. ATP-induced currents in TG neurons exhibited electrophysiological properties similar to those in HEK293 cells, and both ATP- and alpha ,beta-meATP-induced currents in TG neurons were effectively blocked by TNP-ATP, suggesting that P2X(3) mediates the majority of ATP-induced currents in TG neurons. Eugenol inhibited ATP-induced currents in both capsaicin-sensitive and capsaicin-insensitive TG neurons with similar extent, and most ATP-responsive neurons were IB4-positive. Eugenol inhibited not only Ca(2+) transients evoked by alpha ,beta-meATP, the selective P2X(3) agonist, in capsaicin-insensitive TG neurons, but also ATP-induced currents in P2X(3)-expressing HEK293 cells without co-expression of transient receptor potential vanilloid 1 (TRPV1). We suggest, therefore, that eugenol inhibits P2X(3) currents in a TRPV1-independent manner, which contributes to its analgesic effect.

Double-stranded RNA induces iNOS gene expression in Schwann cells, sensory neuronal death, and peripheral nerve demyelination.

Inflammation in the peripheral nervous system (PNS) is one of the characteristics of virus-induced peripheral neuropathy. In this inflammatory response, Schwann cells are actively involved. Previously, toll-like receptor 3 (TLR3) was reported as a receptor for double-stranded RNA (dsRNA) that induces antiviral and inflammatory responses in cells of the innate immune system. In this study, we investigated the expression and putative role of TLR3 in Schwann cells. TLR3 was constitutively expressed in Schwann cells. Stimulation with polyinosinic-polycytidylic acid, a synthetic dsRNA analogue, induced the expression of inducible nitric oxide synthase (iNOS) gene in Schwann cells. Studies on the intracellular signal transduction pathways using iSC, an immortalized Schwann cell line, revealed that dsRNA induces the activation of NF-kappaB, p38, and c-Jun N-terminal kinase (JNK). The activation of NF-kappaB, p38, JNK, and dsRNA-dependent protein kinase is required for dsRNA-mediated iNOS gene expression. However, the activation of PI3 kinase and GSK-3beta inhibited iNOS gene induction, a process mediated by their inhibitory effects on NF-kappaB and p38 activation. dsRNA-induced NO production caused neuronal cell death in cultured dorsal root ganglion. Finally, the introduction of dsRNA into the rat sciatic nerve induced iNOS gene expression and peripheral nerve demyelination in vivo. Taken together, these data suggest that viral RNA may induce inflammatory Schwann cell activation via TLR3 and peripheral nerve damage in the PNS.

A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity.

The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular mechanisms underlying glial cell activation, however, have not been clearly elucidated. In this study, we found that damaged sensory neurons induce the expression of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and inducible nitric-oxide synthase genes in spinal cord glial cells, which is implicated in the development of neuropathic pain. Studies using primary glial cells isolated from toll-like receptor 2 knock-out mice indicate that damaged sensory neurons activate glial cells via toll-like receptor 2. In addition, behavioral studies using toll-like receptor 2 knock-out mice demonstrate that the expression of toll-like receptor 2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the toll-like receptor 2 knock-out mice. Similarly, the nerve injury-induced pro-inflammatory gene expression in the spinal cord is also reduced in the toll-like receptor 2 knock-out mice. These data demonstrate that toll-like receptor 2 contributes to the nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity.