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.

Role of rostral medulla in serotonin-induced changes of respiratory rhythm in newborn rat brainstem-spinal cord preparations.

Abnormalities of the serotonin (5-hydroxytryptamine, 5-HT) system may induce respiratory disorders. We examined which regions in the rostral medulla are important for the effect of 5-HT on the frequency of respiratory-like nerve (fR-like) activity by transecting the preparations at different levels near the facial nucleus (nVII) in newborn rat brainstem-spinal cord preparations. The fR-like activity at the fourth cervical ventral root (C4) of the Pons-medulla-spinal cord preparations in 2-3-day-old rats (n=25) was monitored at 26°C, and the change in fR-like activity in response to application of 10µM 5-HT before and after transection was compared among three groups, in which nVII was retained (group A, n=10), partially retained (group B, n=7), or eliminated (group C, n=8) by the transection. Before transection, the resting fR-like activity (set to 100%) and stimulant effect of 5-HT (+101-143%) were similar among the groups. After transection, resting fR-like activity increased in all groups, but the facilitatory effects of 5-HT on the fR-like activity were abolished in groups A and C (fR-like activity of -4% and +7%, respectively). In group B, 5-HT became inhibitory (fR-like activity of -28%). In conclusion, a distinct part of the rostral medulla in the absence of pontine influences may mediate the inhibitory effects of 5-HT on the respiratory rhythm.

Somatostatin enhances tooth-pulp-evoked cervical dorsal horn neuronal activity in the rat via inhibition of GABAergic interneurons.

A recent in vitro electrophysiological analysis combined with anatomical approach suggests that a potential disinhibitory mechanism involving somatostatin (SST), which is released by interneurons in the superficial dorsal horn, contributes to nociceptive transmission (Yasaka et al., 2010); however, whether this mechanism occurs in vivo remains to be determined. The aim of the present study was to investigate whether iontophoretic application of SST facilitates the excitability of nociceptive upper cervical spinal dorsal horn (C1) neurons through GABAergic disinhibiton, using extracellular electrophysiological recording with multibarrel electrodes and immunohistochemical techniques. Immunoreactivity of SST2A receptors was found in layer II of the C1 dorsal horn in the rat and most of these neurons co-expressed the GABA synthesizing enzyme glutamate decarboxylase 67. Single-unit recordings were made from C1 neurons responding to tooth-pulp (TP) electrical stimulation in pentobarbital anesthestized rats. Iontophoretic application of SST significantly increased TP-evoked C1 neuronal discharges in layers I and II of the spinal dorsal horn and this effect occurred in a current-dependent manner. The facilitation of this discharge by SST application was abolished with co-application of the SST2 receptor antagonist, Cyanamid 154806. Iontophoretic application of GABAA receptor antagonist, bicuculline, induced facilitation of TP-evoked C1 neuronal discharges. There was no significant difference in the relative number of spikes between SST and bicuculline applications. These results suggest that a local release of SST facilitates the excitability of trigeminal nocicepitve C1 neuronal activity via inhibition of GABAergic neurons. Therefore, SST2A receptors expressed in layer II GABAergic inhibitory interneurons play an important role in trigeminal nociceptive transmission and are a potential therapeutic target in the treatment of trigeminal pain, including hyperalgesia.

Brain-derived neurotrophic factor enhances the excitability of small-diameter trigeminal ganglion neurons projecting to the trigeminal nucleus interpolaris/caudalis transition zone following masseter muscle inflammation.

BACKGROUND: The trigeminal subnuclei interpolaris/caudalis transition zones (Vi/Vc) play an important role in orofacial deep pain, however, the role of primary afferent projections to the Vi/Vc remains to be determined. This study investigated the functional significance of hyperalgesia to the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (trkB) signaling system in trigeminal ganglion (TRG) neurons projecting to the Vi/Vc transition zone following masseter muscle (MM) inflammation. RESULTS: The escape threshold from mechanical stimulation applied to skin above the inflamed MM was significantly lower than in naïve rats. Fluorogold (FG) labeling was used to identify the TRG neurons innervating the MM, while microbeads (MB) were used to label neurons projecting to the Vi/Vc region. FG/MB-labeled TRG neurons were immunoreactive (IR) for BDNF and trkB. The mean number of BDNF/trkB-IR small/medium-diameter TRG neurons was significantly higher in inflamed rats than in naïve rats. In whole-cell current-clamp experiments, the majority of dissociated small-diameter TRG neurons showed a depolarization response to BDNF that was associated with spike discharge, and the concentration of BDNF that evoked a depolarizing response was significantly lower in the inflamed rats. In addition, the relative number of BDNF-induced spikes during current injection was significantly higher in inflamed rats. The BDNF-induced changes in TRG neuron excitability was abolished by tyrosine kinase inhibitor, K252a. CONCLUSION: The present study provided evidence that BDNF enhances the excitability of the small-diameter TRG neurons projecting onto the Vi/Vc following MM inflammation. These findings suggest that ganglionic BDNF-trkB signaling is a therapeutic target for the treatment of trigeminal inflammatory hyperalgesia.

Suppression of ATP-induced excitability in rat small-diameter trigeminal ganglion neurons by activation of GABAB receptor.

The aim of the present study was to investigate whether a GABAB receptor agonist could modulate ATP-activated neuronal excitability of nociceptive TRG neurons using perforated whole-cell patch-clamp and immunohistochemical techniques. Immunohistochemical analysis revealed that 86% of P2X3 receptor-immunoreactive, small-diameter TRG neurons co-expressed GABAB receptor. Under voltage-clamp conditions (Vh=-60mV), application of ATP activated the inward current in acutely isolated rat TRG neurons in a dose-dependent manner (10-50 µM) and this current could be blocked by pyridoxal-phosphate-6-azophenyl-27,47-disulfonic acid (PPADS) (10 µM), a selective P2 purinoreceptor antagonist. The peak amplitude of ATP-activated currents was significantly inhibited after application of GABAB receptor agonist, baclofen (10-50 µM), in a concentration-dependent and reversible manner. The baclofen-induced inhibition of ATP-activated current was abolished by co-application of 3-amino-2 (4-chlorophenyl)-2hydroxypropysufonic acid) saclofen, a GABAB receptor antagonist (50 µM). Under current-clamp conditions, application of 20 µM ATP significantly depolarized the membrane potential resulting in increased mean action potential frequencies, and these ATP-induced effects were significantly inhibited by baclofen and these effects were antagonized by co-application of saclofen. Together, the results suggested that GABAB receptor activation could inhibit the ATP-induced excitability of small-diameter TRG neurons activated through the P2X3 receptor. Thus, the interaction between P2X3 and GABAB receptors of small-diameter TRG neuronal cell bodies is a potential therapeutic target for the treatment of trigeminal nociception.

Glial cell line-derived neurotrophic factor modulates the excitability of nociceptive trigeminal ganglion neurons via a paracrine mechanism following inflammation.

Previous our report indicated that acute application of glial cell line-derived neurotrophic factor (GDNF) enhances the neuronal excitability of adult rat small-diameter trigeminal ganglion (TRG) neurons, which innervate the facial skin in the absence of neuropathic and inflammatory conditions. This study investigated whether under in vivo conditions, GDNF modulates the excitability of nociceptive Aδ-TRG neurons innervating the facial skin via a paracrine mechanism following inflammation. We used extracellular electrophysiological recording with multibarrel-electrodes in this study. Spontaneous Aδ-TRG neuronal activity was induced in control rats after iontophoretic application of GDNF into the trigeminal ganglia (TRGs). Noxious and non-noxious mechanical stimuli evoked Aδ-TRG neuronal firing rate were significantly increased by iontophoretic application of GDNF. The mean mechanical threshold of nociceptive TRG neurons was significantly decreased by GDNF application. The increased discharge frequency and decreased mechanical threshold induced by GDNF were antagonized by application of the protein tyrosine kinase inhibitor, K252b. The number of Aδ-TRG neurons with spontaneous firings and their firing rates in rats with inflammation induced by Complete Freund's Adjuvant were significantly higher than control rats. The firing rates of Aδ-TRG spontaneous neuronal activity were significantly decreased by iontophoretic application of K252b in inflamed rats. K252b also inhibited Aδ-TRG neuron activity evoked by mechanical stimulation in inflamed rats. These results suggest that in vivo GDNF enhances the excitability of nociceptive Aδ-TRG neurons via a paracrine mechanism within TRGs following inflammation. GDNF paracrine mechanism could be important as a therapeutic target for trigeminal inflammatory hyperalgesia.

Iontophoretic application of an A-type potassium channel blocker to the trigeminal ganglion neurons enhances the excitability of Aδ- and C-neurons innervating the temporomandibular joint in rats.

The present study tested the hypothesis that under in vivo conditions the iontophoretic application of a I(A) channel blocker, 4-aminopyridine (4-AP), to the TRG neurons changes the properties of Aδ-/C-TRG neurons that innervate the temporomandibular joint (TMJ) region, using extracellular electrophysiological recording with multi-barrel electrodes in pentobarbital-anesthetized rats. A total of twenty-one neurons (Aδ-: 76%; C-: 24%) responded to electrical stimulation of the TMJ region in pentobarbital-anesthetized rats. TMJ electrical stimulation-induced discharges of Aδ/C-neurons were significantly potentiated in current dependent manner (30-70 nA) by iontophoretic application of 4-AP into the TRGs. The spontaneous firing rates of Aδ- and C-neurons were also increased by 4-AP in a current-dependent manner (30-70 nA). The mean threshold current that evoked spontaneous discharges of C-neurons was significantly lower than that of Aδ-neurons. Moreover, the mean relative threshold current for electrical stimulation of TMJ-induced response of C-TRG neurons was significantly lower than that of Aδ-neuron. The relative firing rate of C-neurons induced by 4-AP-treatment (70 nA) was significantly higher than for Aδ-neurons. These results suggest that the application of 4-AP enhanced Aδ/C-TRG neuronal activities innervating the TMJ in vivo and C-neurons had significantly higher sensitivity for 4-AP than Aδ-neurons.

Suppression of neurokinin-1 receptor in trigeminal ganglia attenuates central sensitization following inflammation.

This study examined whether local application of a neurokinin-1 (NK1) receptor antagonist into the trigeminal ganglia (TRGs) modulates hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) wide-dynamic range (WDR) neuron activity innervating both the temporomandibular joint (TMJ) region and facial skin following TMJ inflammation. Extracellular single unit recording combined with multibarrel electrodes was used. TMJ inflammation was induced by the injection of complete Freund's adjuvant (CFA). WDR neurons responding to electrical stimuli of the TMJ region and facial skin were recorded from the SpVc in anesthetized rats. The spontaneous and mechanical stimulation-induced discharge frequencies of WDR neurons were significantly larger in inflamed rats than in control rats. The spontaneous WDR activities were current-dependently decreased by local iontophoretic application of an NK1 receptor antagonist into the TRGs after 1 and 2 days of inflammation. The firing frequency of WDR neurons and threshold evoked by mechanical stimulation of facial skin returned to control levels by application of the NK1 receptor antagonist into TRGs after 1 day, but not 2 days, of inflammation. These results suggest that in the early stages of inflammation suppression of the NK1 receptor mechanism in TRGs may prevent central sensitization of SpVc nociceptive neurons.

TRPV1 receptor expression in cardiac vagal afferent neurons of infant rats.

We examined in infant rats whether cardiac afferent neurons in the nodose ganglia (NG) express transient receptor potential vanilloid 1 (TRPV1) receptors. Anesthetized rats (9-11 days old) were injected with 2 µl of a fluorescent retrograde tracer (Fluoro-Gold, FG) into the ventricular wall through the anterior epicardial surface. After 2 days, the NG were excised under anesthesia for identification of FG-labeled neurons and immunohistochemical analysis. Of 3858 NG neurons, 5% (202 neurons) were labeled with FG. Among the FG-labeled neurons, 180 (89%) were TRPV1 positive, of which 123 co-expressed 200-kDa neurofilaments (NF200), which is specific for myelinated nerve fibers. Among the FG-labeled neurons that expressed both TRPV1 and NF200, 37 had relatively large cell diameters (>26 µm) (range: 12-38 µm). In conclusion, in infant rats, most cardiac afferent neurons (both myelinated and unmyelinated) in the NG may express TRPV1 receptors. Although functional properties such as those related to the arterial baroreflex may vary among the neurons, our results suggest that, in immature animals, TRPV1 receptors help convey cardiac sensations and control autonomic reflexes.

Peripheral inflammation suppresses inward rectifying potassium currents of satellite glial cells in the trigeminal ganglia.

Previous studies indicate that silencing Kir4.1, a specific inward rectifying K(+) (Kir) channel subunit, in sensory ganglionic satellite glial cells (SGCs) induces behavioral hyperalgesia. However, the function of Kir4.1 channels in SGCs in vivo under pathophysiological conditions remains to be determined. The aim of the present study was to examine whether peripheral inflammation in anesthetized rats alters the SGC Kir4.1 current using in vivo patch clamp and immunohistochemical techniques. Inflammation was induced by injection of complete Freund's adjuvant into the whisker pad. The threshold of escape from mechanical stimulation applied to the orofacial area in inflamed rats was significantly lower than in naïve rats. The mean percentage of small/medium diameter trigeminal ganglion (TRG) neurons encircled by Kir4.1-immunoreactive SGCs in inflamed rats was also significantly lower than in naïve rats. In vivo whole-cell recordings were made using SGCs in the trigeminal ganglia (TRGs). Increasing extracellular K(+) concentrations resulted in significantly smaller potentiation of the mean peak amplitude of the Kir current in inflamed compared with naïve rats. In addition, the density of the Ba(2+)-sensitive Kir current associated with small-diameter TRG neurons was significantly lower in inflamed rats compared with naïve rats. Mean membrane potential in inflamed rats was more depolarized than in naïve rats. These results suggest that inflammation could suppress Kir4.1 currents of SGCs in the TRGs and that this impairment of glial potassium homeostasis in the TRGs contributes to trigeminal pain. Therefore, the Kir4.1 channel in SGCs may be a new molecular target for the treatment of trigeminal inflammatory pain.

Direct inhibition of the transient voltage-gated K(+) currents mediates the excitability of tetrodotoxin-resistant neonatal rat nodose ganglion neurons after ouabain application.

The purpose of the present study was to determine the relationship between the responses of transient and sustained K(+) currents, and action potentials to ouabain, and to compare the immunoreactive expression of alpha Na(+)-K(+)-ATPase isoforms (α(1), α(2) and α(3)) in neonatal rat small-diameter nodose ganglion neurons. We used perforated patch-clamp techniques. We first confirmed that the neurons (n=20) were insensitive to 0.5 µM tetrodotoxin (TTX). Application of 1 µM ouabain 1) decreased the transient K(+) currents in 60% of neurons and the sustained K(+) currents in 20%, 2) increased voltage-gated transient and sustained K(+) currents in 20% of neurons, and 3) had no effect on transient K(+) currents in 20% of neurons and on sustained K(+) currents in 60%. Thirteen of the neurons were of a rapidly adapting type, and the remaining 7 were of a slowly adapting type. In 6 rapidly adapting type neurons (46%), their activity was not significantly altered by ouabain application, but in 4 rapidly adapting type neurons, the activity increased. In the remaining 3 rapidly adapting type neurons, ouabain application hyperpolarized the resting membrane potential. The slowly adapting type 7 neurons each showed increased activity after 1 µM ouabain application. The α(1) isoform of Na(+)-K(+)-ATPase was identified as the predominant immunoreactive isoforms in small-diameter nodose ganglion neurons. These results suggest that the increased activity of small-diameter nodose ganglion neurons seen after application of 1 µM ouabain is mediated by direct inhibition of the transient K(+) current.

Potassium channels as a potential therapeutic target for trigeminal neuropathic and inflammatory pain.

Previous studies in several different trigeminal nerve injury/inflammation models indicated that the hyperexcitability of primary afferent neurons contributes to the pain pathway underlying mechanical allodynia. Although multiple types of voltage-gated ion channels are associated with neuronal hyperexcitability, voltage-gated K+ channels (Kv) are one of the important physiological regulators of membrane potentials in excitable tissues, including nociceptive sensory neurons. Since the opening of K+ channels leads to hyperpolarization of cell membrane and a consequent decrease in cell excitability, several Kv channels have been proposed as potential target candidates for pain therapy. In this review, we focus on common changes measured in the Kv channels of several different trigeminal neuropathic/inflammatory pain animal models, particularly the relationship between changes in Kv channels and the excitability of trigeminal ganglion (TRG) neurons. We also discuss the potential of Kv channel openers as therapeutic agents for trigeminal neuropathic/inflammatory pain, such as mechanical allodynia.

Effects of acetazolamide on transient K+ currents and action potentials in nodose ganglion neurons of adult rats.

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K(+) current (I(A) ) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4-AP attenuated or blocked the CO(2) -induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch-clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT-PCR of mRNA and the expression of voltage-gated K(+) (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I(A) by approximately 27% and the additional application of 4-AP (1 mM) further inhibited I(A) by 48%. The application of 0.1 µM α-dendrotoxin (α-DTX), a slow inactivating transient K(+) current (I(D) ) blocker, inhibited the baseline I(A) by approximately 27%, and the additional application of 1 mM AZ further decreased the I(A) by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage-gated K(+) channel proteins were present, and most (80-90%) of the four Kv channels immunoreactive neurons showed the co-expression of carbonic anhydrase-II (CA-II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I(A) via the inhibition of four voltage-gated K(+) channel (Kv) proteins without affecting I(D).

Flecainide inhibits the stimulatory effect of veratridine on the response of airway mechanoreceptors to maintained inflations in rats.

AIMS: the purpose of the present study was to investigate (a) whether maintained inflations result in the inhibition of slowly adapting pulmonary stretch receptor (SAR) discharge to elicit an abrupt change in receptor activity and (b) whether pretreatment with veratridine, a Na(+) channel opener, and/or flecainide, a Na(+) channel blocker, alters the responses of SAR properties to maintained inflations. MAIN METHODS: we measured the properties of SAR activity during maintained inflations at different pressures in 31 anesthetized, artificially ventilated rats with unilateral vagotomy. KEY FINDINGS: During maintained inflations (approximately 5, 10 and 15 cmH(2)O) for about 5s, the procedures did not produce the induction of inhibition of either 16 low-threshold SARs (firing during both inflation and deflation) or 15 high-threshold SARs (firing during inflation only). In these preparations, the excitatory responses of SARs to maintained inflations at three different pressures were markedly enhanced after administration of veratridine (50 µg/kg), but under such conditions, the inhibition of SAR discharges was not observed. In the same SAR preparations, after flecainide treatment (9 mg/kg) sufficient for the blockade of veratridine (50 µg/kg)-induced SAR stimulation, maintained inflations at higher pressures (10 and 15 cmH(2)O) greatly inhibited SAR discharges. Under these conditions, the inhibition of SAR discharges was not observed during maintained inflations at 5 cmH(2)O. SIGNIFICANCE: These results suggest that neither low-threshold SARs nor high-threshold SARs in the rat lung are deactivated during maintained inflations at higher pressures.

In vivo patch-clamp analysis of response properties of rat primary somatosensory cortical neurons responding to noxious stimulation of the facial skin.

BACKGROUND: Although it has been widely accepted that the primary somatosensory (SI) cortex plays an important role in pain perception, it still remains unclear how the nociceptive mechanisms of synaptic transmission occur at the single neuron level. The aim of the present study was to examine whether noxious stimulation applied to the orofacial area evokes the synaptic response of SI neurons in urethane-anesthetized rats using an in vivo patch-clamp technique. RESULTS: In vivo whole-cell current-clamp recordings were performed in rat SI neurons (layers III-IV). Twenty-seven out of 63 neurons were identified in the mechanical receptive field of the orofacial area (36 neurons showed no receptive field) and they were classified as non-nociceptive (low-threshold mechanoreceptive; 6/27, 22%) and nociceptive neurons. Nociceptive neurons were further divided into wide-dynamic range neurons (3/27, 11%) and nociceptive-specific neurons (18/27, 67%). In the majority of these neurons, a proportion of the excitatory postsynaptic potentials (EPSPs) reached the threshold, and then generated random discharges of action potentials. Noxious mechanical stimuli applied to the receptive field elicited a discharge of action potentials on the barrage of EPSPs. In the case of noxious chemical stimulation applied as mustard oil to the orofacial area, the membrane potential shifted depolarization and the rate of spontaneous discharges gradually increased as did the noxious pinch-evoked discharge rates, which were usually associated with potentiated EPSP amplitudes. CONCLUSIONS: The present study provides evidence that SI neurons in deep layers III-V respond to the temporal summation of EPSPs due to noxious mechanical and chemical stimulation applied to the orofacial area and that these neurons may contribute to the processing of nociceptive information, including hyperalgesia.

Alteration of primary afferent activity following inferior alveolar nerve transection in rats.

BACKGROUND: In order to evaluate the neural mechanisms underlying the abnormal facial pain that may develop following regeneration of the injured inferior alveolar nerve (IAN), the properties of the IAN innervated in the mental region were analyzed. RESULTS: Fluorogold (FG) injection into the mental region 14 days after IAN transection showed massive labeling of trigeminal ganglion (TG). The escape threshold to mechanical stimulation of the mental skin was significantly lower (i.e. mechanical allodynia) at 11-14 days after IAN transection than before surgery. The background activity, mechanically evoked responses and afterdischarges of IAN Adelta-fibers were significantly higher in IAN-transected rats than naive. The small/medium diameter TG neurons showed an increase in both tetrodotoxin (TTX)-resistant (TTX-R) and -sensitive (TTX-S) sodium currents (INa) and decrease in total potassium current, transient current (IA) and sustained current (IK) in IAN-transected rats. The amplitude, overshoot amplitude and number of action potentials evoked by the depolarizing pulses after 1 muM TTX administration in TG neurons were significantly higher, whereas the threshold current to elicit spikes was smaller in IAN-transected rats than naive. Resting membrane potential was significantly smaller in IAN-transected rats than that of naive. CONCLUSIONS: These data suggest that the increase in both TTX-S INa and TTX-R INa and the decrease in IA and Ik in small/medium TG neurons in IAN-transected rats are involved in the activation of spike generation, resulting in hyperexcitability of Adelta-IAN fibers innervating the mental region after IAN transection.

Glial cell line-derived neurotrophic factor acutely modulates the excitability of rat small-diameter trigeminal ganglion neurons innervating facial skin.

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in adult sensory neuron function. However, the acute effects of GDNF on primary sensory neuron excitability remain to be elucidated. The aim of the present study was to investigate whether GDNF acutely modulates the excitability of adult rat trigeminal ganglion (TRG) neurons that innervate the facial skin by using perforated-patch clamping, retrograde-labeling and immunohistochemistry techniques. Fluorogold (FG) retrograde labeling was used to identify the TRG neurons innervating the facial skin. The FG-labeled small- and medium-diameter GDNF immunoreactive TRG neurons, and most of these neurons also expressed the GDNF family receptor alpha-1 (GFRalpha-1). In whole-cell voltage-clamp mode, GDNF application significantly inhibited voltage-gated K(+) transient (I(A)) and sustained (I(K)) currents in most dissociated FG-labeled small-diameter TRG neurons. This effect was concentration-dependent and was abolished by co-application of the protein tyrosine kinase inhibitor, K252b. Under current-clamp conditions, the repetitive firing during a depolarizing pulse were significantly increased by GDNF application. GDNF application also increased the duration of the repolarization phase and decreased the duration of the depolarization phase of the action potential, and these characteristic effects were also abolished by co-application of K252b. These results suggest that acute application of GDNF enhances the neuronal excitability of adult rat small-diameter TRG neurons innervating the facial skin, via activation of GDNF-induced intracellular signaling pathway. We therefore conclude that a local release of GDNF from TRG neuronal soma and/or nerve terminals may regulate normal sensory function, including nociception.

The roles of I(D), I(A) and I(K) in the electrophysiological functions of small diameter rat trigeminal ganglion neurons.

The Kv currents are divided into three different K+ currents, such as slow inactivating transient K+ current (I(D)), fast inactivating transient K+ current (I(A)) and dominant sustained K+ current (I(K)), in small-diameter rat trigeminal ganglion (TG) neurons. The concentration of alpha-DTX (an I(D) blocker) to evoke the maximal inhibition of I(A) was 0.1 microM, and this concentration caused a 20 % inhibition of I(A) and increased the number of action potentials. Irrespective of the presence of 0.1 microM alpha-DTX, the application of 0.5 mM 4-AP (an IA blocker) caused a 51 % inhibition of I(A) and increased the number of action potentials. The responses were associated with the decreases in the resting membrane potential (RMP) and duration of depolarization phase of action potential (DDP). The application of 2 mM tetraethylammonium (TEA, an I(K) blocker) produced a 55 % inhibition of I(K). Irrespective of the presence of both I(D) and I(A) blockers, the I(K) was the predominant K+ current. The prolongation of duration of action potential was usually observed following TEA treatment, suggesting that I(A) and I(K) had independent effects regulating the intrinsic firing properties of the action potential number and timing, respectively. Furthermore, the response characteristics of action potentials in the presence of both 4-AP and TEA resemble those of TG neurons in rats following chronic constriction nerve injury of the infraorbital nerve as well as after inferior alveolar nerve section. Thus, reducing effects of both I(A) and I(K) may be useful to investigate the mechanism of allodynia.

Ventrolateral lesions at the ponto-medullary junction and the effects of noradrenaline on respiratory rhythm in rat brainstem-spinal cord preparations.

AIMS: We examined whether responses of respiratory frequency (fR) to noradrenaline (NA) were eliminated by mechanical lesions in the ventrolateral area at the ponto-medullary junction in preparations of newborn rat pons-medulla-spinal cord (PMS). MAIN METHODS: Preparations obtained from 2- to 4-day-old rats were superfused with artificial cerebrospinal fluid that was equilibrated with oxygenated (95% O2 plus 5% CO2 gas, and fR was monitored at the C4 ventral root at 24 degrees C. Bilateral lesions were made in the ventrolateral area between the VIth cranial nerve root and the anterior inferior cerebellar artery in PMS (n=11). The resting fR and response to exogenous NA (7 microM) were compared with those of medulla-spinal cord (MS) preparations (n=6). Immunohistochemistry of PMS preparations was performed to detect tyrosine hydroxylase (TH)-positive neurons at the ponto-medullary junction. KEY FINDINGS: PMS preparations with the lesions had (1) a significantly higher resting fR but 2 significantly less fR facilitation after NA application than those of intact PMS preparations, and (3) significantly lower resting fR and (4) significantly less fR reduction after NA application than those of MS preparations. TH-positive neurons were detected in the region from the rostral dorsolateral to the caudal ventrolateral pons (the A5 area), as well as in the ventral area near the facial nucleus. SIGNIFICANCE: Results suggest that ventrolateral area at ponto-medullary junction plays a significant role in exogenous NA-induced fR changes under the influence of pons-induced tonic fR inhibition in newborn rat brainstem-spinal cord preparations.

Systemic administration of lidocaine suppresses the excitability of rat cervical dorsal horn neurons and tooth-pulp-evoked jaw-opening reflex.

Although systemic lidocaine has been demonstrated to have analgesic actions in neuropathic pain conditions, the effect of intravenous lidocaine on trigeminal pain has not been elucidated. The aim of the present study is to investigate the effect of intravenous lidocaine administration on the excitability of the upper cervical dorsal horn (C1) neuron having convergent inputs from both tooth-pulp (TP) and facial skin as well as nociceptive jaw-opening reflex (JOR). After electrical stimulation of TP, extracellular single-unit recordings from 19 C1 neurons and the digastric muscle electromyogram (dEMG) were made in pentobarbital-anesthetized rats. These neurons also responded to non-noxious and noxious mechanical stimulation (touch and pinch) of facial skin, and every neuron was considered to be a wide dynamic range (WDR) neuron. The TP-evoked C1 neuronal and dEMG activities were dose-dependently inhibited by systematic administration of lidocaine (1-2 mg/kg, i.v.). After intravenous injection of lidocaine, the unit discharges induced by both touch and pinch stimuli were inhibited, and the size of the receptive field for pinch was also significantly decreased. The mean spontaneous discharge frequencies were significantly inhibited by the application of lidocaine. These changes were reversed within -20 min. These results suggest that in the absence of neuropathic pain intravenous lidocaine injection suppresses the trigeminal nociceptive reflex as well as the excitability of C1 neurons having convergent inputs from TP and somatic afferents. Systemic lidocaine administration, therefore, may contribute to the alleviation of trigeminal-referred pain associated with tooth pain.