Modulation of excitatory synaptic transmissions by TRPV1 in the spinal trigeminal subnucleus caudalis neurons of neuropathic pain rats.

The present study examined contribution of the transient receptor potential vanilloid 1 channel (TRPV1) to the chronic orofacial pain. Bilateral partial nerve ligation (PNL) of the mental nerve, a branch of trigeminal nerve, was performed to induce neuropathic pain. The withdrawal threshold in response to mechanical stimulation of the lower lip skin was substantially reduced after the surgery in the PNL rats while it remained unchanged in the sham rats. This reduction in the PNL rats was alleviated by pregabalin injected intraperitoneally (10 mg/kg) and intracisternally (10, 30, 100 µg). Furthermore, an intracisternal injection of AMG9810, an antagonist of TRPV1, (1.5, 5.0 µg) attenuated the reduction of withdrawal threshold. Spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) were recorded from the spinal trigeminal subnucleus caudalis (Vc) neurons in the brainstem slice, which receive the orofacial nociceptive signals. In the PNL rats, superfusion of capsaicin (0.03, 0.1 µM) enhanced their frequency without effect on the amplitude and the highest concentration (0.3 µM) increased both the frequency and amplitude. In the sham rats, only 0.3 µM capsaicin increased their frequency. Thus, capsaicin-induced facilitation of sEPSCs and mEPSCs in the PNL rats was significantly stronger than that in the sham rats. AMG9810 (0.1 µM) attenuated the capsaicin's effect. Capsaicin was ineffective on the trigeminal tract-evoked EPSCs in the PNL and sham rats. These results suggest that the chronic orofacial pain in the PNL model results from facilitation of the spontaneous excitatory synaptic transmission in the Vc region through TRPV1 at least partly.

Long-Term Depression Induced by Optogenetically Driven Nociceptive Inputs to Trigeminal Nucleus Caudalis or Headache Triggers.

The peripheral trigeminovascular pathway mediates orofacial and craniofacial pain and projects centrally to the brainstem trigeminal nucleus caudalis (TNc). Sensitization of this pathway is involved in many pain conditions, but little is known about synaptic plasticity at its first central synapse. We have taken advantage of optogenetics to investigate plasticity selectively evoked at synapses of nociceptive primary afferents onto TNc neurons. Based on immunolabeling in the trigeminal ganglia, TRPV1-lineage neurons comprise primarily peptidergic and nonpeptidergic nociceptors. Optical stimulation of channelrhodopsin-expressing axons in the TRPV1/ChR2 mouse in TNc slices thus allowed us to activate a nociceptor-enriched subset of primary afferents. We recorded from lamina I/II neurons in acutely prepared transverse TNc slices, and alternately stimulated two independent afferent pathways, one with light-activated nociceptive afferents and the other with electrically-activated inputs. Low-frequency optical stimulation induced robust long-term depression (LTD) of optically-evoked EPSCs, but not of electrically-evoked EPSCs in the same neurons. Blocking NMDA receptors or nitric oxide synthase strongly attenuated LTD, whereas a cannabinoid receptor 1 antagonist had no effect. The neuropeptide PACAP-38 or the nitric oxide donors nitroglycerin or sodium nitroprusside are pharmacologic triggers of human headache. Bath application of any of these three compounds also persistently depressed optically-evoked EPSCs. Together, our data show that LTD of nociceptive afferent synapses on trigeminal nucleus neurons is elicited when the afferents are activated at frequencies consistent with the development of central sensitization of the trigeminovascular pathway.SIGNIFICANCE STATEMENT Animal models suggest that sensitization of trigeminovascular afferents plays a major role in craniofacial pain syndromes including primary headaches and trigeminal neuralgia, yet little is known about synaptic transmission and plasticity in the brainstem trigeminal nucleus caudalis (TNc). Here we used optogenetics to selectively drive a nociceptor-enriched population of trigeminal afferents while recording from superficial laminae neurons in the TNc. Low-frequency optical stimulation evoked robust long-term depression at TRPV1/ChR2 synapses. Moreover, application of three different headache trigger drugs also depressed TRPV1/ChR2 synapses. Synaptic depression at these primary afferent synapses may represent a newly identified mechanism contributing to central sensitization during headache.

Different processing of meningeal and cutaneous pain information in the spinal trigeminal nucleus caudalis.

Introduction Within superficial trigeminal nucleus caudalis (Sp5C) (laminae I/II), meningeal primary afferents project exclusively to lamina I, whereas nociceptive cutaneous ones distribute in both lamina I and outer lamina II. Whether such a relative absence of meningeal inputs to lamina II represents a fundamental difference from cutaneous pathways in the central processing of sensory information is still unknown. Methods We recorded extracellular field potentials in the superficial Sp5C of anesthetised rats evoked by electrically stimulating the dura mater, to selectively assess the synaptic transmission between meningeal primary afferents and second-order Sp5C neurons, the first synapse in trigeminovascular pathways. We tested the effect of systemic morphine and local glycinergic and GABAAergic disinhibition. Results Meningeal stimulation evokes two negative field potentials in superficial Sp5C. The conduction velocities of the activated primary afferents are within the Aδ- and C-fibre ranges. Systemic morphine specifically suppresses meningeal C-fibre-evoked field potentials, and this effect is reversed by systemic naloxone. Segmental glycinergic or GABAAergic disinhibition strongly potentiates meningeal C-fibre-evoked field potentials but not Aδ-fibre ones. Interestingly, the same segmental disinhibition conversely potentiates cutaneous Aδ-fibre-evoked field potentials and suppresses C-fibre ones. Conclusion These findings reveal that the different anatomical organization of meningeal and cutaneous inputs into superficial Sp5C is associated with a different central processing of meningeal and cutaneous pain information within Sp5C. Moreover, they suggest that the potentiation upon local disinhibition of the first synapse in trigeminovascular pathways may contribute to the generation of headache pain.

Baicalin Activates Glycine and γ-Aminobutyric Acid Receptors on Substantia Gelatinosa Neurons of the Trigeminal Subsnucleus Caudalis in Juvenile Mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives nociceptive afferent inputs from thin-myelinated A[Formula: see text] fibers and unmyelinated C fibers and has been shown to be involved in the processing of orofacial nociceptive information. Scutellaria baicalensis Georgi (Huang-Qin, SbG), one of the 50 fundamental herbs of Chinese herbology, has been used historically as anti-inflammatory and antineoplastic medicine. Baicalin, one of the major compounds of SbG, has been reported to have neuroprotective, anti-inflammatory and analgesic effects. However, the receptor type activated by baicalin and its precise action mechanism on the SG neurons of Vc have not yet been studied. The whole-cell patch clamp technique was performed to examine the ion channels activated by baicalin on the SG neurons of Vc. In high Cl[Formula: see text] pipette solution, the baicalin (300[Formula: see text][Formula: see text]M) induced repeatable inward currents ([Formula: see text][Formula: see text]pA, [Formula: see text]) without desensitization on all the SG neurons tested. Further, the inward currents showed a concentration (0.1-3[Formula: see text]mM) dependent pattern. The inward current was sustained in the presence of tetrodotoxin (0.5[Formula: see text][Formula: see text]M), a voltage sensitive Na[Formula: see text] channel blocker. In addition, baicalin-induced inward currents were reduced in the presence of picrotoxin (50[Formula: see text][Formula: see text]M), a GABAA receptor antagonist, flumazenil (100[Formula: see text][Formula: see text]M), a benzodiazepine-sensitive GABAA receptor antagonist, and strychnine (2[Formula: see text][Formula: see text]M), a glycine receptor antagonist, respectively. These results indicate that baicalin has inhibitory effects on the SG neurons of the Vc, which are due to the activation of GABAA and/or the glycine receptor. Our results suggest that baicalin may be a potential target for orofacial pain modulation.

Synaptic ultrastructure changes in trigeminocervical complex posttrigeminal nerve injury.

Trigeminal nerves collecting sensory information from the orofacial area synapse on second-order neurons in the dorsal horn of subnucleus caudalis and cervical C1/C2 spinal cord (Vc/C2, or trigeminocervical complex), which is critical for sensory information processing. Injury to the trigeminal nerves may cause maladaptive changes in synaptic connectivity that plays an important role in chronic pain development. Here we examined whether injury to the infraorbital nerve, a branch of the trigeminal nerves, led to synaptic ultrastructural changes when the injured animals have developed neuropathic pain states. Transmission electron microscopy was used to examine synaptic profiles in Vc/C2 at 3 weeks postinjury, corresponding to the time of peak behavioral hypersensitivity following chronic constriction injury to the infraorbital nerve (CCI-ION). Using established criteria, synaptic profiles were classified as associated with excitatory (R-), inhibitory (F-), and primary afferent (C-) terminals. Each type was counted within the superficial dorsal horn of the Vc/C2 and the means from each rat were compared between sham and injured animals; synaptic contact length was also measured. The overall analysis indicates that rats with orofacial pain states had increased numbers and decreased mean synaptic length of R-profiles within the Vc/C2 superficial dorsal horn (lamina I) 3 weeks post-CCI-ION. Increases in the number of excitatory synapses in the superficial dorsal horn of Vc/C2 could lead to enhanced activation of nociceptive pathways, contributing to the development of orofacial pain states.

Selective elimination of isolectin B4-binding trigeminal neurons enhanced formalin-induced nocifensive behavior in the upper lip of rats and c-Fos expression in the trigeminal subnucleus caudalis.

The functional significance of non-peptidergic C-fibers in orofacial pain processing is largely unknown. The present study examined the effects of the selective elimination of isolectin B4 (IB4)-binding (IB4(+)) neurons on formalin-induced face rubbing behavior (FRB) in the upper lip of rats and c-Fos-immunoreactive (c-Fos-IR) cells in the trigeminal subnucleus caudalis (Vc). IB4 conjugated to neurotoxin, saporin (IB4-Sap), blank-saporin (Bl-Sap), or saline (Sal) was injected into the cisterna magna. IB4-Sap treatments significantly decreased IB4(+) terminals in lamina II of Vc and IB4(+) trigeminal ganglia neurons, whereas Sal- and BI-Sap treatments did not. The number of formalin-induced FRB 15-30 min after the formalin injection was significantly higher in IB4-Sap-treated rats than in Sal- or Bl-Sap-treated rats, and was associated with an increase in c-Fos-IR cells. The systemic preadministration of the GABAA antagonist, bicuculline, and agonist, muscimol, had stronger decreasing effects on FRB and c-Fos-IR cells in IB4-Sap-treated rats than the preadministration of Sal, whereas the opposite effects were observed in Sal- and Bl-Sap-treated rats. These results indicate that IB4(+) neurons in the trigeminal nerve play antinociceptive regulatory roles in formalin-induced orofacial pain processing and that GABAA receptor functions at segmental and supratrigeminal sites have complex modulatory influences on antinociceptive roles.

[A COMPARATIVE STUDY OF CATECHOLAMINERGIC AND NITROXIDERGIC VASOMOTOR NEURONS IN THE NUCLEI OF THE CAUDAL PART OF THE BRAINSTEM OF THE RAT].

Immunohistochemical methods for the demonstration of tyrosine hydrolase (TH) and neuronal form of nitric oxide synthase (nNOS) were used to study the distribution of catecholaminergic and nitroxidergic vasomotor neurons respectively, in the nuclei of the medulla oblongata and the pons of 12 Wistar rats. Most often the expression of TG was found in neurons located in the nucleus and several reticular nuclei (gigantocellular, paragigantocellular, caudal pons nucleus), but the proportion of immunoreactive neurons did not exceed 8-14%. In the other nuclei (reticular parvocellular nucleus and oral pons nucleus, spinal nucleus of the trigeminal nerve) the value of this parameter ranged from 1 to 3%. In a large group of nuclei with proven vasomotor function such neurons were constantly not detected. In the structures with high content of catecholaminergic neurons, nNOS-positive cells were found, as a rule, in fewer numbers than in the nuclei with a limited number of TH-positive neurons.

Connections between EM2-containing terminals and GABA/µ-opioid receptor co-expressing neurons in the rat spinal trigeminal caudal nucleus.

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect via the µ-opioid receptor (MOR). To provide morphological evidence for the pain control effect of EM2, the synaptic connections between EM2-immunoreactive (IR) axonal terminals and γ-amino butyric acid (GABA)/MOR co-expressing neurons in lamina II of the spinal trigeminal caudal nucleus (Vc) were investigated in the rat. Dense EM2-, MOR- and GABA-IR fibers and terminals were mainly observed in lamina II of the Vc. Within lamina II, GABA- and MOR-neuronal cell bodies were also encountered. The results of immunofluorescent histochemical triple-staining showed that approximately 14.2 or 18.9% of GABA-IR or MOR-IR neurons also showed MOR- or GABA-immunopositive staining in lamina II; approximately 45.2 and 36.1% of the GABA-IR and MOR-IR neurons, respectively, expressed FOS protein in their nuclei induced by injecting formalin into the left lower lip of the mouth. Most of the GABA/MOR, GABA/FOS, and MOR/FOS double-labeled neurons made close contacts with EM2-IR fibers and terminals. Immuno-electron microscopy confirmed that the EM2-IR terminals formed synapses with GABA-IR or MOR-IR dendritic processes and neuronal cell bodies in lamina II of the Vc. These results suggest that EM2 might participate in pain transmission and modulation by binding to MOR-IR and GABAergic inhibitory interneuron in lamina II of the Vc to exert inhibitory effect on the excitatory interneuron in lamina II and projection neurons in laminae I and III.

Capsaicin-responsive corneal afferents do not contain TRPV1 at their central terminals in trigeminal nucleus caudalis in rats.

We examined the substrates for ocular nociception in adult male Sprague-Dawley rats. Capsaicin application to the ocular surface in awake rats evoked nocifensive responses and suppressed spontaneous grooming responses. Thus, peripheral capsaicin was able to activate the central pathways encoding ocular nociception. Our capsaicin stimulus evoked c-Fos expression in a select population of neurons within rostral trigeminal nucleus caudalis in anesthetized rats. These activated neurons also received direct contacts from corneal afferent fibers traced with cholera toxin B from the corneal surface. However, the central terminals of the corneal afferents that contacted capsaicin-activated trigeminal neurons did not contain TRPV1. To determine if TRPV1 expression had been altered by capsaicin stimulation, we examined TRPV1 content of corneal afferents in animals that did not receive capsaicin stimulation. These studies confirmed that while TRPV1 was present in 30% of CTb-labeled corneal afferent neurons within the trigeminal ganglion, TRPV1 was only detected in 2% of the central terminals of these corneal afferents within the trigeminal nucleus caudalis. Other TRP channels were also present in low proportions of central corneal afferent terminals in unstimulated animals (TRPM8, 2%; TRPA1, 10%). These findings indicate that a pathway from the cornea to rostral trigeminal nucleus caudalis is involved in corneal nociceptive transmission, but that central TRP channel expression is unrelated to the type of stimulus transduced by the peripheral nociceptive endings.

GABAergic influence on temporomandibular joint-responsive spinomedullary neurons depends on estrogen status.

Sensory input from the temporomandibular joint (TMJ) to neurons in superficial laminae at the spinomedullary (Vc/C1-2) region is strongly influenced by estrogen status. This study determined if GABAergic mechanisms play a role in estrogen modulation of TMJ nociceptive processing in ovariectomized female rats treated with high- (HE) or low-dose (LE) estradiol (E2) for 2days. Superficial laminae neurons were activated by ATP (1mM) injections into the joint space. The selective GABAA receptor antagonist, bicuculline methiodide (BMI, 5 or 50µM, 30µl), applied at the site of recording greatly enhanced the magnitude and duration of ATP-evoked responses in LE rats, but not in units from HE rats. The convergent cutaneous receptive field (RF) area of TMJ neurons was enlarged after BMI in LE but not HE rats, while resting discharge rates were increased after BMI independent of estrogen status. By contrast, the selective GABAA receptor agonist, muscimol (50µM, 30µl), significantly reduced the magnitude and duration of ATP-evoked activity, resting discharge rate, and cutaneous RF area of TMJ neurons in LE and HE rats, whereas lower doses (5µM) affected only units from LE rats. Protein levels of GABAA receptor ß3 isoform at the Vc/C1-2 region were similar for HE and LE rats. These results suggest that GABAergic mechanisms contribute significantly to background discharge rates and TMJ-evoked input to superficial laminae neurons at the Vc/C1-2 region. Estrogen status may gate the magnitude of GABAergic influence on TMJ neurons at the earliest stages of nociceptive processing at the spinomedullary region.

Bilateral descending hypothalamic projections to the spinal trigeminal nucleus caudalis in rats.

Several lines of evidence suggest that the hypothalamus is involved in trigeminal pain processing. However, the organization of descending hypothalamic projections to the spinal trigeminal nucleus caudalis (Sp5C) remains poorly understood. Microinjections of the retrograde tracer, fluorogold (FG), into the Sp5C, in rats, reveal that five hypothalamic nuclei project to the Sp5C: the paraventricular nucleus, the lateral hypothalamic area, the perifornical hypothalamic area, the A11 nucleus and the retrochiasmatic area. Descending hypothalamic projections to the Sp5C are bilateral, except those from the paraventricular nucleus which exhibit a clear ipsilateral predominance. Moreover, the density of retrogradely FG-labeled neurons in the hypothalamus varies according to the dorso-ventral localization of the Sp5C injection site. There are much more labeled neurons after injections into the ventrolateral part of the Sp5C (where ophthalmic afferents project) than after injections into its dorsomedial or intermediate parts (where mandibular and maxillary afferents, respectively, project). These results demonstrate that the organization of descending hypothalamic projections to the spinal dorsal horn and Sp5C are different. Whereas the former are ipsilateral, the latter are bilateral. Moreover, hypothalamic projections to the Sp5C display somatotopy, suggesting that these projections are preferentially involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in rats. Therefore, our results suggest that the control of trigeminal and spinal dorsal horn processing of nociceptive information by hypothalamic neurons is different and raise the question of the role of bilateral, rather than unilateral, hypothalamic control.

Projections from the insular cortex to pain-receptive trigeminal caudal subnucleus (medullary dorsal horn) and other lower brainstem areas in rats.

This study examined the projections from the rat insular cortex (Ins) to lower brainstem areas which are possibly involved in orofacial pain processing. We first examined distributions of Ins neurons projecting directly to the trigeminal caudal subnucleus (Vc, medullary dorsal horn) and oral subnucleus (Vo) which are known to receive orofacial nociceptive inputs. After injections of a retrograde tracer, Fluorogold (FG), into the medial part and lateral part of laminae I/II of Vc, many neurons were labeled bilaterally with a contralateral predominance in the rostral level of granular Ins (GI) and dysgranular Ins (DI) and the caudal level of GI/DI, respectively, but none in the agranular Ins (AI). After FG injections into laminae III-V of Vc, no Ins neurons were labeled. After FG injections into the Vo, many neurons were labeled bilaterally with a contralateral predominance in the rostral and caudal GI/DI, but none in the AI. We then examined descending projections from the GI/DI to the lower brainstem. After injections of an anterograde tracer, biotinylated dextranamine (BDA), into the rostral GI/DI, many BDA-labeled axons and terminals were seen bilaterally with a contralateral predominance in the medial part of laminae I/II of Vc, dorsomedial Vo, juxtatrigeminal region, rostral ventromedial medulla (RVM), and nucleus of the solitary tract, and with an ipsilateral predominance in the parabrachial nucleus (Pb), Kölliker-Fuse nucleus (KF) and trigeminal mesencephalic nucleus. After BDA injections into the caudal GI/DI, they were seen bilaterally with a contralateral predominance in the lateral part of laminae I/II of Vc, ventrolateral Vo, juxtatrigeminal region and RVM, and with an ipsilateral dominance in the lateral zone (PAGl) of periaqueductal gray, Pb and KF. These results suggest that orofacial nociceptive processing of Vc and Vo neurons may be regulated by GI/DI directly or indirectly through brainstem nuclei such as PAGl, Pb, KF and RVM.

Activation of peripheral P2X receptors is sufficient to induce central sensitization in rat medullary dorsal horn nociceptive neurons.

Central sensitization and purinergic receptor mechanisms have been implicated as important processes in acute and chronic pain conditions following injury or inflammation of peripheral tissues. This study has documented that application of the P2X(1,2/3,3) receptor agonist αß-meATP (100mM) to the rat tooth pulp induces central sensitization in medullary dorsal horn nociceptive neurons that is reflected in significant increases in mechanoreceptive field size and responses to noxious stimuli and decreased mechanical activation threshold. Furthermore, these responses can be blocked by pulp application of the P2X(1,2/3,3) antagonist TNP-ATP and also attenuated by medullary application of TNP-ATP. These results suggest that activation of P2X(1,2/3,3) receptors in orofacial tissues plays a critical role in producing central sensitization in medullary dorsal horn nociceptive neurons.

Functional properties of tooth pulp neurons responding to thermal stimulation.

The response properties of tooth pulp neurons that respond to noxious thermal stimulation of the dental pulp have been not well-studied. The present study was designed to characterize the response properties of tooth pulp neurons to noxious thermal stimulation of the dental pulp. Experiments were conducted on 25 male ferrets, and heat stimulation was applied by a computer-controlled thermode. Only 15% of tooth pulp neurons (n = 39) responded to noxious thermal stimulation of the teeth. Tooth pulp neurons were found in both the superficial and deep nuclear regions of the subnucleus caudalis (Vc) and in the interface between the nucleus caudalis and interpolaris (Vc/Vi). Thirty-seven neurons had cutaneous receptive fields and were classified as either NS (16) or WDR (21) neurons. Repeated heat stimulation of the dental pulp sensitized and increased the number of electrically evoked potentials of tooth pulp neurons. These results provide evidence that both the Vc and Vc/Vi regions contain neurons that respond to noxious thermal stimulation of the dental pulp, and that these cells may contribute to the sensitization process associated with symptomatic pulpitis.

Age-related changes in the effects of 5-hydroxytryptamine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis.

The trigeminal subnucleus caudalis (Vc) is the critical brainstem relay site of orofacial nociceptive processing to higher brain centers. The descending serotonergic pathway from the brainstem exerts inhibitory or facilitatory effects on nociceptive transmission in the spinal dorsal horn and the Vc, and SG neurons of the Vc exhibit hyperpolarization, no response or depolarization in response to 5-hydroxytryptamine (5-HT) application. In this study, we examined age-related changes in the effects of 5-HT on SG neurons of the Vc using immature, peripubertal and adult male mice and gramicidin-perforated patch recordings under the current-clamp mode. In the three age groups, hyperpolarization was the major response in SG neurons exhibiting membrane potential changes in response to 5-HT application. The proportion of the SG neurons responding to 5-HT by hyperpolarization was significantly higher in the immature (20/27) than in the adult mice (10/26; P<0.05). The proportion of SG neurons showing no response to 5-HT was significantly higher in the peripubertal (11/21) and the adult mice (13/26) compared with the immature mice (5/27). The amplitude of 5-HT-induced hyperpolarization significantly decreased with increasing postnatal age (correlation coefficient=-0.43, P<0.05). The mean amplitude of 5-HT-induced hyperpolarization was significantly higher in the immature mice (-9.7±1.1 mV, n=20) than in the peripubertal (-5.3±1.0 mV, n=10) and the adult mice (-5.4±0.9 mV, n=10; both P<0.05). These results suggest that the descending serotonergic modulatory influence over the orofacial nociceptive processing in the Vc may change during postnatal development and postnatal age of three weeks is a critical period for changes in 5-HT-induced hyperpolarizing effects in mice.

Tyramine reduces glycinergic transmission by inhibiting presynaptic Ca(2+) channels in the rat trigeminal subnucleus caudalis.

We have recently reported that tyramine acts on putative presynaptic trace amine receptors to inhibit glycinergic transmission in substantia gelatinosa (SG) neurons of the rat trigeminal subnucleus caudalis. However, it is still unknown how tyramine elicits presynaptic inhibition of glycine release. In the present study, therefore, we investigated cellular mechanisms underlying the tyramine-induced inhibition of glycinergic transmission in SG neurons using a conventional whole-cell patch clamp technique. Tyramine (100 µM) reversibly and repetitively decreased the amplitude of action potential-dependent glycinergic inhibitory postsynaptic currents (IPSCs), and increased the paired-pulse ratio. Pharmacological data suggest that the tyramine-induced decrease in glycinergic IPSCs was not mediated by the modulation of adenylyl cyclase, protein kinase A and C, or G-protein coupled inwardly rectifying K(+) channels. On the other hand, glycinergic IPSCs were mainly mediated by the Ca(2+) influx passing through presynaptic N-type and P/Q-type Ca(2+) channels. The tyramine-induced decrease in glycinergic IPSCs was completely blocked by ω-conotoxin GVIA, an N-type Ca(2+) channel blocker, but not ω-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. The results suggest that tyramine acts presynaptically to decrease action potential-dependent glycine release onto SG neurons via the selective inhibition of presynaptic N-type Ca(2+) channels. This tyramine-induced inhibition of glycinergic transmission in SG neurons might affect the process of orofacial nociceptive signals.

Mechanisms involved in extraterritorial facial pain following cervical spinal nerve injury in rats.

BACKGROUND: The aim of this study is to clarify the neural mechanisms underlying orofacial pain abnormalities after cervical spinal nerve injury. Nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) expression and astroglial cell activation in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal dorsal horn (C1-C2) neurons were analyzed in rats with upper cervical spinal nerve transection (CNX). RESULTS: The head withdrawal threshold to mechanical stimulation of the lateral facial skin and head withdrawal latency to heating of the lateral facial skin were significantly lower and shorter respectively in CNX rats compared to Sham rats. These nocifensive effects were apparent within 1 day after CNX and lasted for more than 21 days. The numbers of pERK-like immunoreactive (LI) cells in superficial laminae of Vc and C1-C2 were significantly larger in CNX rats compared to Sham rats following noxious and non-noxious mechanical or thermal stimulation of the lateral facial skin at day 7 after CNX. Two peaks of pERK-LI cells were observed in Vc and C1-C2 following mechanical and heat stimulation of the lateral face. The number of pERK-LI cells in C1-C2 was intensity-dependent and increased when the mechanical and heat stimulations of the face were increased. The decrements of head withdrawal latency to heat and head withdrawal threshold to mechanical stimulation were reversed during intrathecal (i.t.) administration of MAPK/ERK kinase 1/2 inhibitor PD98059. The area of activated astroglial cells was significantly higher in CNX rats (at day 7 after CNX). The heat and mechanical nocifensive behaviors were significantly depressed and the number of pERK-LI cells in Vc and C1-C2 following noxious and non-noxious mechanical stimulation of the face was also significantly decreased following i.t. administration of the astroglial inhibitor fluoroacetate. CONCLUSIONS: The present findings have demonstrated that mechanical allodynia and thermal hyperalgesia occur in the lateral facial skin after CNX and also suggest that ERK phosphorylation of Vc and C1-C2 neurons and astroglial cell activation are involved in orofacial extraterritorial pain following cervical nerve injury.

Neurochemical features of enkephalinergic neurons in the mouse trigeminal subnucleus caudalis.

Enkephalinergic (ENKergic) neurons have been proposed to play crucial roles in pain modulation in the trigeminal subnucleus caudalis (Vc). To assist an advance in the research of ENKergic neurons, here we used preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice, in which all ENKergic neurons were fluorescent. We first performed fluorescent in situ hybridization combined with immunofluorescent histochemistry to confirm the specificity of this transgenic mouse and its advantages in showing ENKergic neurons in the Vc. Then based on this useful transgenic mouse, we examined the phenotypic diversity of PPE-GFP neurons by immunostaining for several markers that characterize ENKergic neuron subtypes. About 25.9±1.9% of GFP-positive neurons were regarded as immunoreactive for glutamic acid decarboxylase (GAD)(67) mRNA and 14.7±1.4% of GFP-positive neurons were positive for γ-aminobutyric acid. The proportions of calbindin-, calretinin-positive cells among the ENKergic neurons were 8.4±1.2% and 7.3±1.7%, respectively. Only 1.1±0.1% of GFP-positive neurons colocalized with parvalbumin and no GFP-positive neurons were found to co-express neuronal nitric oxide synthase. We then injected retrograde tracer into the thalamic regions and observed that a small number of ENKergic neurons in the Vc were retrogradely labeled with the tracer. The present results provide a detailed morphological evidence of the neurochemical features of ENKergic neurons. These results have broad implications for understanding the functional roles of ENKergic neurotransmission in the Vc.

Dietary grape seed polyphenols repress neuron and glia activation in trigeminal ganglion and trigeminal nucleus caudalis.

BACKGROUND: Inflammation and pain associated with temporomandibular joint disorder, a chronic disease that affects 15% of the adult population, involves activation of trigeminal ganglion nerves and development of peripheral and central sensitization. Natural products represent an underutilized resource in the pursuit of safe and effective ways to treat chronic inflammatory diseases. The goal of this study was to investigate effects of grape seed extract on neurons and glia in trigeminal ganglia and trigeminal nucleus caudalis in response to persistent temporomandibular joint inflammation. Sprague Dawley rats were pretreated with 200 mg/kg/d MegaNatural-BP grape seed extract for 14 days prior to bilateral injections of complete Freund's adjuvant into the temporomandibular joint capsule. RESULTS: In response to grape seed extract, basal expression of mitogen-activated protein kinase phosphatase 1 was elevated in neurons and glia in trigeminal ganglia and trigeminal nucleus caudalis, and expression of the glutamate aspartate transporter was increased in spinal glia. Rats on a normal diet injected with adjuvant exhibited greater basal levels of phosphorylated-p38 in trigeminal ganglia neurons and spinal neurons and microglia. Similarly, immunoreactive levels of OX-42 in microglia and glial fibrillary acidic protein in astrocytes were greatly increased in response to adjuvant. However, adjuvant-stimulated levels of phosphorylated-p38, OX-42, and glial fibrillary acidic protein were significantly repressed in extract treated animals. Furthermore, grape seed extract suppressed basal expression of the neuropeptide calcitonin gene-related peptide in spinal neurons. CONCLUSIONS: Results from our study provide evidence that grape seed extract may be beneficial as a natural therapeutic option for temporomandibular joint disorders by suppressing development of peripheral and central sensitization.

Psychophysical stress increases the expression of phospho-CREB, Fos protein and neurokinin-1 receptors in superficial laminae of trigeminal subnucleus caudalis in female rats.

Psychological stress and estrogen status are risk factors to develop painful temporomandibular joint disorders (TMJD); however, the neural basis for this relationship is not known. This study tested the hypothesis that repeated forced swim stress and estradiol treatment alter the phosphorylation of cAMP responsive element-binding protein (pCREB) in trigeminal subnucleus caudalis (Vc), the initial site of sensory input from the TMJ. Ovariectomized female rats were given low or high dose estradiol and subjected to repeated forced swim stress for 3 days and on day 4 an intra-TMJ injection of mustard oil or vehicle was given. Forced swim alone increased the number of pCREB-positive neurons, independent of estradiol treatment or TMJ stimulation, in superficial and deep laminae of Vc. Forced swim also increased the number of Fos-positive neurons in superficial laminae and neurokinin-1 receptor mRNA in whole dorsal Vc, independent of estradiol treatment. These results indicated that persistent psychophysical stress alone was sufficient to increase the expression of pCREB and downstream regulated genes associated with enhanced excitability in the caudal medullary dorsal horn, a brainstem region thought to be critical for TMJD pain.