Rat Trigeminal Ganglion Neurons Research Articles

Functional expression of bradykinin B1 and B2 receptors in neonatal rat trigeminal ganglion neurons.

Bradykinin (BK) and its receptors (B1 and B2 receptors) play important roles in inflammatory nociception. However, the patterns of expression and physiological/pathological functions of B1 and B2 receptors in trigeminal ganglion (TG) neurons remain to be fully elucidated. We investigated the functional expression of BK receptors in rat TG neurons. We observed intense immunoreactivity of B2 receptors in TG neurons, while B1 receptors showed weak immunoreactivity. Expression of the B2 receptor colocalized with immunoreactivities against the pan-neuronal marker, neurofilament H, substance P, isolectin B4, and tropomyosin receptor kinase A antibodies. Both in the presence and absence of extracellular Ca2+ ([Ca2+]o), BK application increased the concentration of intracellular free Ca2+ ([Ca2+]i). The amplitudes of BK-induced [Ca2+]i increase in the absence of [Ca2+]o were significantly smaller than those in the presence of Ca2+. In the absence of [Ca2+]o, BK-induced [Ca2+]i increases were sensitive to B2 receptor antagonists, but not to a B1 receptor antagonist. However, B1 receptor agonist, Lys-[Des-Arg9]BK, transiently increased [Ca2+]i in primary cultured TG neurons, and these increases were sensitive to a B1 receptor antagonist in the presence of [Ca2+]o. These results indicated that B2 receptors were constitutively expressed and their activation induced the mobilization of [Ca2+]i from intracellular stores with partial Ca2+ influx by BK. Although constitutive B1 receptor expression could not be clearly observed immunohistochemically in the TG cryosection, cultured TG neurons functionally expressed B1 receptors, suggesting that both B1 and B2 receptors involve pathological and physiological nociceptive functions.

Expression and function of purinergic P2Y12 receptors in rat trigeminal ganglion neurons

Purinergic receptors play key signaling roles in neuropathic pain in the orofacial region, which is innervated by trigeminal ganglion (TG) neurons. The neuropathology of purinergic P2Y12 receptors is well characterized in glia; however, their physiological role in TG neurons remains to be fully elucidated. The present study investigated the expression and function of P2Y12 receptors in rat TG neurons. P2Y12 receptor immunoreactivity was intense in the soma, dendrites, and axons, and colocalized with a pan-neuronal marker, neurofilament H, isolectin B4, and substance P. In the presence of extracellular Ca2+, 2-methylthio-ADP (an agonist of P2Y1, 12, 13 receptors) transiently increased intracellular free Ca2+ concentrations ([Ca2+]i), an effect that was abolished by P2Y12 receptor antagonists. In the absence of extracellular Ca2+, ryanodine receptor/channel inhibitors diminished the 2-methylthio-ADP-induced increases in [Ca2+]i. A sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor gradually increased [Ca2+]i, and after a plateau, application of 2-MeS-ADP induced a rapid and transient, but additive increase in [Ca2+]i. An adenylate cyclase inhibitor transiently increased [Ca2+]i, while a phosphodiesterase inhibitor prevented the 2-methylthio-ADP-induced increase in [Ca2+]i. Our study shows that P2Y12 receptors are expressed in TG neurons, and act via a cAMP-dependent pathway to release intracellular Ca2+ from ryanodine-sensitive Ca2+ stores.

Mechanotransduction of trigeminal ganglion neurons innervating inner walls of rat anterior eye chambers.

To address mechanoreceptive roles of trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior eye chambers, we investigated the mechanotransduction process and mechanosensitive (MS) channel on somata of TG neurons innervating this area in vitro. Rat TG neurons innervating inner walls of anterior chambers were labeled by anterior chamber injection of 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI). The neuronal cell bodies were voltage clamped using a whole cell patch-clamp technique, while it was deformed by ejection of bath solution to verify mechanotransduction. Immunofluorescence staining was performed on sections of TG ganglia to determine the specific MS channel proteins. Mechanical stimuli induced MS currents in 55 out of 96 FAST DiI-labeled TG neurons. The MS currents exhibited mechanical intensity-dependent and clamp voltage-dependent characteristics. Mechanical stimulation further enhanced the membrane potential and increased the frequency of action potentials. Transient receptor potential ankyrin 1 (TRPA1), TRP vanilloid 4 (TRPV4), acid-sensing ion channel (ASIC) 2 and ASIC3 channel proteins were expressed in FAST DiI-labeled TG neurons. The inhibitory effect of HC-030031, a specific inhibitor of TRPA1, on MS currents demonstrated that TRPA1 was an essential MS channel protein. Taken together, our results show that mechanical stimuli induce MS currents via MS channels such as TRPA1 to trigger mechanotransduction in TG neurons innervating inner walls of anterior chambers. Our results indicate the existence of mechanoreceptive TG nerve endings in inner walls of anterior chambers. Whether the mechanoreceptive TG nerve endings play a role in intraocular pressure sensation warrants further investigation.

Phenotypes of ATP-activated current associated with their genotypes of P2X1-6 subunits in neurons innervating tooth-pulp

To explore the association of the phenotype of ATP-activated current with the genotype of P2X1-6 subunits in nociceptors, we developed a method that allows us to label nociceptive neurons innervating tooth-pulp in rat trigeminal ganglion (TG) neurons using a retrograde fluorescence-tracing method, to record ATP-activated current in freshly isolated fluorescence-labeled neurons, and then to conduct single cell immunohistochemical staining for P2X1-6 subunits in the same neuron. We found that fast application of 100 μM ATP to fluorescence-traced TG neurons produced robust inward current in 87% (96/110) of cells tested. The diameter of cells varied from 16 to 56 μm. Three types of ATP-activated current (F, I and S) were recorded with distinct rise times of the current (R10–90, P < 0.05). There was a positive correlation between the cell diameter and the value of R10–90 (P < 0.05): the value of R10-90 increased with increases in the cell diameter. Cells responsive to ATP with the type F current mainly showed positive staining for P2X3 and P2X5, but negative staining for P2X2; cells responsive to ATP with the type I current showed positive staining for P2X1-3 and P2X5, but negative staining for P2X4; and cells responsive to ATP with the type S current showed positive staining for P2X1-5, but negative staining for P2X6. The present findings suggest that in addition to P2X3 subunits, P2X5 subunits are also involved in the generation of the F type of ATP-activated current in small-sized nociceptive neurons. In addition to the P2X2/3 subunit-containing channels, more complex uncharacterized combinations of P2X1-5 subunits exist in native medium-sized nociceptive neurons exhibiting the I and S types of ATP-activated current. In addition, the P2X6 subunit is not a main subunit involved in the nociceptive signal in rat TG neurons innervating tooth-pulp.

The expression of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) and HCN2 in the rat trigeminal ganglion, sensory root, and dental pulp

Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) and 2 (HCN2) are abundantly expressed in primary sensory neurons and contribute to neuronal excitability and pathological pain. We studied the expression of HCN1 and HCN2 in the rat trigeminal ganglion (TG) neurons and axons in the dental pulp, and the changes in their expression following inflammation, using light- and electron-microscopic immunocytochemistry and quantitative analysis.HCN1 and HCN2 were expressed predominantly in large-sized, neurofilament 200-immunopositive (+) or parvalbumin+ soma in the TG whereas they were expressed mostly in unmyelinated and small myelinated axons in the sensory root. The expression was particularly strong along the plasma membrane in the soma. In the dental pulp, majority of HCN1+ and HCN2+ axons coexpressed calcitonin gene-related peptide. They were expressed mainly in the peripheral pulp and pulp horn where the axons branch extensively in the dental pulp. The expression of HCN1 and HCN2 in TG neurons increased significantly in rats with experimentally induced inflammation of the dental pulp.Our findings support the notion that HCN1 and HCN2 are expressed mainly by both the soma of mechanosensitive neurons in the TG and peripheral axons of nociceptive neurons in the sensory root, and may play a role in the mechanisms of inflammatory pain from the dental pulp.

Eugenol inhibits the GABAA current in trigeminal ganglion neurons.

Eugenol has sedative, antioxidant, anti-inflammatory, and analgesic effects, but also serves as an irritant through the regulation of a different set of ion channels. Activation of gamma aminobutyric acid (GABA) receptors on sensory neurons leads to the stabilization of neuronal excitability but contributes to formalin-induced inflammatory pain. In this study, we examined the effect of eugenol on the GABA-induced current in rat trigeminal ganglia (TG) neurons and in human embryonic kidney (HEK) 293 cells expressing the GABAA receptor α1β2γ2 subtype using the whole-cell patch clamp technique. RT-PCR and Western blot analysis were used to confirm the expression of GABAA receptor γ2 subunit mRNA and protein in the TG and hippocampus. Eugenol decreased the amplitude ratio of the GABA-induced current to 27.5 ± 3.2% (p < 0.05) in TG neurons, which recovered after a 3-min washout. In HEK 293 cells expressing the α1β2γ2 subtype, eugenol inhibited GABA-induced currents in a dose-dependent manner. Application of eugenol also decreased the GABA response in the presence of a G-protein blocker. Eugenol pretreatment with different concentrations of GABA resulted in similar inhibition of the GABA-induced current in a non-competitive manner. In conclusion, eugenol inhibits the GABA-induced current in TG neurons and HEK 293 cells expressing the GABAA receptor in a reversible, dose-dependent, and non-competitive manner, but not via the G-protein pathway. We suggest that the GABAA receptor could be a molecular target for eugenol in the modulation of nociceptive information.

Expression of transient receptor potential vanilloid 1 and anoctamin 1 in rat trigeminal ganglion neurons innervating the tongue.

Transient receptor potential vanilloid 1 (TRPV1) is a polymodal sensor that is activated by heat (>43 °C), acid, or capsaicin, the pungent ingredient of hot peppers. Reports that mice lacking TRPV1 display heat avoidance behaviors and TRPV1-negative neurons respond to heat suggest that an additional heat sensor is present. Anoctamin 1 (ANO1; also known as transmembrane protein 16A [TMEM16A]), is a component of Ca(2+)-activated chloride channels (CaCCs), and has been recently identified as a heat sensor, activated by temperatures over 44 °C. ANO1 is highly co-localized with TRPV1 in small-diameter dorsal root ganglion (DRG) neurons. The aim of the present study was to investigate co-expression of ANO1 and TRPV1 in rat trigeminal ganglion (TG) neurons innervating the tongue by using retrograde labeling and immunohistochemical techniques. Fluoro-gold (FG) retrograde labeling was used to identify the TG neurons innervating the anterior two thirds of the tongue; as expected, most labeling was detected in the mandibular division of the TGs. The FG-labeled TG neurons showed TRPV1 immunoreactivity (17.9%) and ANO1 immunoreactivity (13.7%), indicating that TRPV1- and ANO1-expressing neurons were present in the mandibular division of the TGs. Seventy-six percent of the ANO1-immunoreactive TG neurons were also immunoreactive for TRPV1; this co-expression was mainly detected in small- to medium-diameter TG neurons. The high degree of co-expression of TRPV1 and ANO1 suggests that cooperation between ANO1 and TRPV1 plays a role in the signaling pathways of nociceptive TG neurons.

NGF-induced mechanical sensitization of the masseter muscle is mediated through peripheral NMDA receptors

Intramuscular injection of nerve growth factor (NGF) in healthy humans mimics some of the symptoms of myofascial temporomandibular disorders (M-TMD). We hypothesized that NGF induces a prolonged myofascial mechanical sensitization by increasing peripheral N-methyl-d-aspartate (NMDA) receptor expression, leading to an enhanced response of muscle nociceptors to endogenous glutamate. Behavioral experiments with an injection of NGF (25μg/ml, 10μl) into the masseter muscle reduced the mechanical withdrawal threshold for 1day in male rats and 5days in female rats. These results mirror the sex-related differences found in NGF-induced mechanical sensitization in humans. Intramuscular injection with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV, 0.020g/ml, 10μl) reversed the mechanical sensitization in male but not in female rats. NGF increased the number of NMDA receptor subtype 2B (NR2B)-expressing rat trigeminal masseter ganglion neurons in both sexes, which peaked at 3days post injection. There was an association between the levels of NR2B expression and NGF-induced mechanical sensitization. The average soma size of NR2B-expressing neurons increased significantly. Increased expression of neuropeptides (CGRP and SP) was observed in NR2B-expressing masseter ganglion neurons in female but not in male rats. In healthy men and women, comparable basal expression levels of NR2B and SP were found in peripheral fibers from masseter muscle microbiopsies. This study suggests that NGF-induced sensitization of masseter nociceptors is mediated, in part, by enhanced peripheral NMDA receptor expression. Measurement of peripheral NMDA receptor expression may be useful as a biomarker for M-TMD pain.

Investigating the expression of metabotropic glutamate receptors in trigeminal ganglion neurons and satellite glial cells: implications for craniofacial pain

Context/objective: Previous studies have demonstrated that various subtypes of the metabotropic glutamate receptors (mGluRs) are expressed in the dorsal root ganglion (DRG) of the peripheral nervous system (PNS), implicating that glutamate potentially contributes to sensory transmission through these receptors. While mGluR expression has been investigated largely in the DRG, the present study focused on mGluR expression on neurons and satellite glial cells (SGCs) of the trigeminal ganglion (TG). Materials and methods: To address the presence of mGluRs in rat TG neurons and their corresponding SGCs, the trigeminal ganglia from six adult male Wistar rats were isolated and immunohistochemistry and immunocytochemistry were performed. The expression of mGluR1α-, mGluR2/3- and mGluR8 on TG neurons and SGCs was investigated in tissue slices and isolated cells. Results: 35.1 ± 6.0% of the TG neurons were positive for mGluR1α, whereas 39.9 ± 7.7% and 55.5 ± 6.3% were positive for mGluR2/3 and mGluR8, respectively. Immunoreactive neurons expressing mGluRs were mainly medium- to large sized, with a smaller population of small-sized neurons showing immunoreactivity. The SGCs showed immunoreactivity toward mGluR1α and mGluR8, but not mGluR2/3, both in the tissue and in isolated cells. Conclusions: Findings from the present study showed that trigeminal neurons express mGluR1α, mGluR2/3 and mGluR8, while SGCs only express mGluR1α and mGluR8. This novel evidence may advance investigations on a possible role of mGluRs in relation to trigeminal pain transmission within the craniofacial region.

Activation of transient receptor potential ankyrin 1 by eugenol

Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5μM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol.

Transient Receptor Potential Channels Encode Volatile Chemicals Sensed by Rat Trigeminal Ganglion Neurons

Primary sensory afferents of the dorsal root and trigeminal ganglia constantly transmit sensory information depicting the individual’s physical and chemical environment to higher brain regions. Beyond the typical trigeminal stimuli (e.g. irritants), environmental stimuli comprise a plethora of volatile chemicals with olfactory components (odorants). In spite of a complete loss of their sense of smell, anosmic patients may retain the ability to roughly discriminate between different volatile compounds. While the detailed mechanisms remain elusive, sensory structures belonging to the trigeminal system seem to be responsible for this phenomenon. In order to gain a better understanding of the mechanisms underlying the activation of the trigeminal system by volatile chemicals, we investigated odorant-induced membrane potential changes in cultured rat trigeminal neurons induced by the odorants vanillin, heliotropyl acetone, helional, and geraniol. We observed the dose-dependent depolarization of trigeminal neurons upon application of these substances occurring in a stimulus-specific manner and could show that distinct neuronal populations respond to different odorants. Using specific antagonists, we found evidence that TRPA1, TRPM8, and/or TRPV1 contribute to the activation. In order to further test this hypothesis, we used recombinantly expressed rat and human variants of these channels to investigate whether they are indeed activated by the odorants tested. We additionally found that the odorants dose-dependently inhibit two-pore potassium channels TASK1 and TASK3 heterologously expressed In Xenopus laevis oocytes. We suggest that the capability of various odorants to activate different TRP channels and to inhibit potassium channels causes neuronal depolarization and activation of distinct subpopulations of trigeminal sensory neurons, forming the basis for a specific representation of volatile chemicals in the trigeminal ganglia.

THIK-1 (K2P13.1) is a small-conductance background K+ channel in rat trigeminal ganglion neurons

The goal of this study was to determine the molecular identity of a small-conductance (~5-pS) background K(+) channel expressed in trigeminal ganglion (TG) neurons. We tested the hypothesis that the 5-pS channel is a K2P channel by comparing the pharmacological and single-channel properties of THIK-1 expressed in HEK293 cells. As reported earlier, whole-cell THIK-1 current was inhibited by halothane and activated by arachidonic acid. Among 25 additional modulators tested, bupivacaine (100 μM), quinidine (50 μM) and Ba(2+) (3 mM) and cold (10 °C) were most effective inhibitors of THIK-1 current (>50 % inhibition). In cell-attached patches with high KCl in the pipette and bath solutions, THIK-1 produced a small-conductance (~5 pS) channel with a weak inwardly rectifying current-voltage relationship. Halothane, bupivacaine and cold inhibited the single-channel activities of both THIK-1 and the 5-pS channel in TG neurons, whereas arachidonic acid augmented them. THIK-1 expressed in HEK293 cells and the 5-pS channels in TG neurons were insensitive to hypoxia. Reverse transcriptase-PCR, Western blot and immunocytochemical analyses suggested that THIK-1 mRNA and protein were expressed in TG neurons. These results show that THIK-1 is functionally expressed in TG neurons and contributes to the background K(+) conductance.

Effects of eugenol on T-type Ca2+ channel isoforms.

Eugenol has been used as an analgesic in dentistry. Previous studies have demonstrated that voltage-gated Na(+) channels and high-voltage-activated Ca(2+) channels expressed in trigeminal ganglion (TG) neurons sensing dental pain are molecular targets of eugenol for its analgesic effects. However, it has not been investigated whether eugenol can affect T-type Ca(2+) channels, which are known to be detected in the afferent neurons. In this report, we investigate how eugenol can influence cloned T-type channel isoforms expressed in HEK293 cells, using whole-cell patch clamp. Application of eugenol inhibited Cav3.1, Cav3.2, and Cav3.3 currents in a concentration-dependent manner with IC50 values of 463, 486, and 708 μM, respectively. Eugenol was found to negatively shift the steady-state inactivation curves of the T-type channel isoforms, but it did not shift their activation curves. In addition, eugenol had little effect on the current kinetics of Cav3.1 and Cav3.2, but it accelerated the inactivation kinetics of Cav3.3 currents. Reduction of channel availability enhanced eugenol inhibition sensitivity for Cav3.1 and Cav3.2, but not for Cav3.3. Moreover, eugenol inhibition of T-type channel isoforms was found to be use dependent. Finally, we show that the T-type currents recorded from rat TG neurons were inhibited by eugenol with a similar potency to Cav3.1 and Cav3.2 isoforms. Taken together, our findings suggest that T-type Ca(2+) channels are additional molecular targets for the pain-relieving effects of eugenol.

Rapid effect of ATP on transient outward potassium current (I&lt;SUB&gt;A&lt;/SUB&gt;) in rat trigeminal ganglion neurons

Rapid effect of ATP on transient outward potassium current (I&lt;SUB&gt;A&lt;/SUB&gt;) in rat trigeminal ganglion neurons

P2X3, but not P2X1, receptors mediate ATP-activated current in neurons innervating tooth-pulp

We developed a method that allows us to label nociceptive neurons innervating tooth-pulp in rat trigeminal ganglion neurons using a retrograde fluorescence-tracing method, to record ATP-activated current in freshly isolated fluorescence-labeled neurons and to conduct single cell immunohistochemical staining for P2X1 and P2X3 subunits in the same neuron. Three types of ATP-activated current in these neurons (F, I and S) were recorded. The cells exhibiting the type F current mainly showed positive staining for P2X3, but negative staining for P2X1. The results provide direct and convincing evidence at the level of single native nociceptive neurons for correlation of the characteristics of ATP-activated currents with their composition of P2X1 and P2X3 subunits and cell size. The results also suggest that the P2X3, but not P2X1, is the main subunit that mediates the fast ATP-activated current in nociceptive neurons.

Hemokinin-1 competitively inhibits substance P-induced stimulation of osteoclast formation and function

Hemokinin-1 (HK-1) is a novel member of the tachykinin family that is encoded by preprotachykinin 4 (TAC4) and shares the neurokinin-1 receptor (NK1-R) with substance P (SP). Although HK-1 is thought to be an endogenous peripheral SP-like endocrine or paracrine molecule in locations where SP is not expressed, neither the distribution of HK-1 in the maxillofacial area nor the role HK-1 in bone tissue have been examined. In this study, we investigated the distribution of HK-1 in trigeminal ganglion (TG) and maxillary bone, and assessed the expression of HK-1 during osteoclast differentiation. In vivo, rat molars were loaded for 5days using the Waldo method. In vitro, rat osteoclast-like cells were induced from bone marrow cells. HK-1 distribution and expression were examined by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, HK-1 was localized in rat TG neurons; however, the number of HK-1-positive neurons was less than that of SP-positive neurons. In the maxillary bone, nerve fibers, blood vessels, and osteocytes were immunopositive for HK-1. Furthermore, HK-1-positive immunoreactivity was found in osteoclasts on the pressure side. In vitro, PCR showed that TAC4 and NK1-R mRNA was expressed in osteoclasts as well as in bone marrow cells. Although SP (10−7M) treatment led to an increased number of osteoclasts, HK-1 (10−7M) treatment did not. The numbers of biotin-labeled HK-1 peptides bound osteoclasts significantly decreased upon incubation with unlabeled SP and biotin-labeled HK-1compared with biotin-labeled HK-1 alone. These results suggest that HK-1 may not stimulate the differentiation and function of osteoclasts. SP-stimulated osteoclast formation is competitively regulated by peripheral HK-1 through NK1-Rs.

Tumor necrosis factor regulates the gene for brain-derived neurotrophic factor (BDNF) in rat trigeminal ganglion neurons

Tumor necrosis factor regulates the gene for brain-derived neurotrophic factor (BDNF) in rat trigeminal ganglion neurons

Endothelin Receptor-mediated Responses in Trigeminal Ganglion Neurons

Recent evidence implicates endothelin in nociception, but it is unclear how endothelin activates trigeminal ganglion (TRG) neurons. In the present study, we investigated the expression of the endothelin receptors ETA and ETB and endothelin-induced responses in rat TRG neurons. Double-immunofluorescence studies demonstrated that ETA and ETB were expressed in TRG neurons and that 26% of ETA- or ETB-expressing neurons expressed both receptors. During whole-cell patch-clamp recording, endothelin-1 enhanced an induced current in response to capsaicin, a TRPV1 agonist, in approximately 20% of dissociated neurons. The enhancement was blocked by the PKC inhibitor chelerythrine and by the ETA antagonist BQ-123, but not by the ETB antagonist BQ-788. Ca2+-imaging showed that endothelin-1 increased the intracellular Ca2+ concentration in more than 20% of the dissociated neurons. Importantly, unlike the effect of endothelin-1 on capsaicin-induced current, the Ca2+ response was largely suppressed by BQ-788 but not by BQ-123. These results suggest that ETA-mediated TRPV1 hyperactivation via PKC activation and ETB-mediated Ca2+ mobilization occurs in different subsets of TRG neurons. These endothelin-induced responses may contribute to the induction of orofacial pain. The ETB-mediated function in TRG neurons is a special feature in the trigeminal system because of no ETB expression in dorsal root ganglion neurons.

Sodium-Calcium Exchangers in Rat Trigeminal Ganglion Neurons

BackgroundNoxious stimulation and nerve injury induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via various receptors or ionic channels. While an increase in [Ca2+]i excites neurons, [Ca2+]i overload elicits cytotoxicity, resulting in cell death. Intracellular Ca2+ is essential for many signal transduction mechanisms, and its level is precisely regulated by the Ca2+ extrusion system in the plasma membrane, which includes the Na+-Ca2+ exchanger (NCX). It has been demonstrated that Ca2+-ATPase is the primary mechanism for removing [Ca2+]i following excitatory activity in trigeminal ganglion (TG) neurons; however, the role of NCXs in this process has yet to be clarified. The goal of this study was to examine the expression/localization of NCXs in TG neurons and to evaluate their functional properties.ResultsNCX isoforms (NCX1, NCX2, and NCX3) were expressed in primary cultured rat TG neurons. All the NCX isoforms were also expressed in A-, peptidergic C-, and non-peptidergic C-neurons, and located not only in the somata, dendrites, axons and perinuclear region, but also in axons innervating the dental pulp. Reverse NCX activity was clearly observed in TG neurons. The inactivation kinetics of voltage-dependent Na+ channels were prolonged by NCX inhibitors when [Ca2+]i in TG neurons was elevated beyond physiological levels.ConclusionsOur results suggest that NCXs in TG neurons play an important role in regulating Ca2+-homeostasis and somatosensory information processing by functionally coupling with voltage-dependent Na+ channels.

Hydrogen Sulfide Increases Excitability through Suppression of Sustained Potassium Channel Currents of Rat Trigeminal Ganglion Neurons

BackgroundHydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role and mechanism for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study is to investigate distribution of endogenous H2S synthesizing enzyme cystathionine-β-synthetase (CBS) expression and role of H2S on excitability and voltage-gated potassium channels of TG neurons.MethodsImmunofluorescence studies were carried out to determine whether CBS was co-expressed in Kv1.1 or Kv1.4-positive TG neurons. Whole cell patch clamp recordings were employed on acutely isolated TG neurons from adult male Sprague Dawley rats (6–8 week old). von Frey filaments were used to examine the pain behavioral responses in rats following injection of sodium hydrosulfide.ResultsIn rat TG, 77.3±6.6% neurons were immunoreactive for CBS, 85.1±3.8% for Kv1.1 and 97.8±1.1% for Kv1.4. Double staining showed that all CBS labeled cells were Kv1.1 and Kv1.4 positive, but only 92.2±6.1% of Kv1.1 and 78.2±9.9% of Kv1.4 positive cells contained CBS. Application of H2S donor NaHS (250 μM) led to a significant depolarization of resting membrane potential recorded from TG neurons. NaHS application also resulted in a dramatic reduction in rheobase, hyperpolarization of action potential threshold, and a significant increase in the number of action potentials evoked at 2X and 3X rheobase stimulation. Under voltage-clamp conditions, TG neurons exhibited transient A-type (IA) and sustained outward rectifier K+ currents (IK). Application of NaHS did suppress IK density while did not change IA density of TG neurons (n=6). Furthermore, NaHS, a donor of hydrogen sulfide, produced a significant reduction in escape threshold in a dose dependent manner.ConclusionThese data suggest that endogenous H2S generating enzyme CBS was co-localized well with Kv1.1 and Kv1.4 in TG neurons and that H2S produces the mechanic pain and increases neuronal excitability, which might be largely mediated by suppressing IK density, thus identifying for the first time a specific molecular mechanism underlying pain and sensitization in TG.