Takeshi Tanimoto

Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation

Abstract Peripheral nerve injury activates satellite cells to produce interleukin 1β (IL‐1β) which mediates inflammation and hyperalgesia. This study investigated the hypothesis that activation of satellite glial cells modulates the excitability of trigeminal ganglion (TRG) neurons via IL‐1β following inflammation. Inflammation was induced by injection of complete Freund’s adjuvant (CFA) into the whisker pad area. The threshold for escape from mechanical stimulation applied to the whisker pad in inflamed rats was significantly lower than that in control. Two days post‐CFA injection, the mean percentage of TRG neurons encircled by glial fibrillary acidic protein (GFAP)‐/IL‐1β‐immunoreactive cells was significantly increased compared to controls. GFAP and IL‐1β immunoreactivities were coexpressed in the same cells. Fluorogold (FG) labeling identified the site of inflammation. The number of FG‐labeled IL‐receptor type I (IL‐1RI) TRG neurons in inflamed rats was significantly greater than in controls. In FG‐labeled small TRG neurons, the size of IL‐1β (1 nM) induced‐depolarization in inflamed rats was larger than in controls. IL‐1β application significantly increased firing rates evoked by depolarizing pulses in the neurons of inflamed rats, compared to controls. The response to IL‐1β was abolished by treatment with the IL‐1RI antagonist. These results suggest that activation of satellite glial cells modulates the excitability of small‐diameter TRG neurons via IL‐1β following inflammation, and that the upregulation of IL‐1RI in the soma may contribute to the mechanism underlying inflammatory hyperalgesia. Therefore IL‐1β blockers are potential therapeutic agents for prevention of trigeminal hyperalgesia.

Alteration of the Second Branch of the Trigeminal Nerve Activity Following Inferior Alveolar Nerve Transection in Rats

Abstract After transection of the inferior alveolar nerve (IAN), the whisker pad area, which is innervated by the infraorbital nerve (ION) that was not injured, showed hypersensitivity to mechanical stimulation. Two days after IAN transection, threshold intensity for escape behavior to mechanical stimulation of the ipsilateral whisker pad area was less than 4.0 g, indicating mechanical allodynia. A total of 68 single fiber discharges were recorded from ION fibers at 3 days after IAN transection. The responses of C‐ and A‐fibers were classified according to their conduction velocity. The C‐fiber activities were not affected by IAN transection, whereas A‐fiber activities were significantly enhanced by IAN transection as indicated by an increase in background activity and mechanically evoked response. Since the A‐fiber responses were significantly affected by IAN transection, patch clamp recording was performed from middle to large diameter retrogradely labeled and acutely dissociated trigeminal ganglion (TRG) neurons. The IK (sustained) and IA (transient) currents were significantly smaller and hyperpolarization‐activated current (Ih) was significantly larger in TRG neurons of rats with IAN transection as compared to those of naive rats. Furthermore, current injection into TRG neurons induced high frequency spike discharges in rats with IAN transection. These data suggest that changes in K+ current and Ih observed in the uninjured TRG neurons reflect an increase in excitability of TRG neurons innervated by the ION after IAN transection, resulting in the development of mechano‐allodynia in the area adjacent to the injured IAN innervated region.

Activation of NK1 receptor of trigeminal root ganglion via substance P paracrine mechanism contributes to the mechanical allodynia in the temporomandibular joint inflammation in rats

&NA; The aim of this study was to investigate whether under in vivo conditions, temporomandibular joint (TMJ) inflammation alters the excitability of Aβ‐trigeminal root ganglion (TRG) neuronal activity innervating the facial skin by using extracellular electrophysiological recording with multibarrel‐electrodes. Complete Freunds adjuvant (CFA) was injected into the rat TMJ. Threshold for escape from mechanical stimulation applied to the whisker pad area in inflamed rats (2 days) was significantly lower than that in control rats. A total of 36 Aβ‐TRG neurons responding to electrical stimulation of the whisker pad was recorded in pentobarbital‐anesthetized rats. The number of Aβ‐TRG neurons with spontaneous firings and their firing rate in TMJ inflamed rats were significantly larger than those in control rats. The firing rates of their spontaneous activity in the Aβ‐TRG neurons were current‐dependently decreased by local iontophoretic application of an NK1 receptor antagonist (L‐703,606) in inflamed, but not non‐inflamed rats. Their spontaneous activities were current‐dependently increased by local iontophoretic application of substance P (SP) in control and inflamed rats. The mechanical response threshold of Aβ‐TRG neurons in inflamed rats was significantly lower than that in control rats. The mechanical response threshold in inflamed rats after iontophoretic application of L‐703,606 was not different from that in control rats. These results suggest that TMJ inflammation modulate the excitability of Aβ‐TRG neurons innervating the facial skin via paracrine mechanism due to SP released from TRG neuronal cell body. Such a SP release may play an important role in determining the trigeminal inflammatory allodynia concerning the temporomandibular disorder.

Suppressive effect of vagal afferents on the activity of the trigeminal spinal neurons related to the jaw-opening reflex in rats : Involvement of the endogenous opioid system

The purpose of the present study is to test the hypothesis that via the endogenous pain control system, vagal afferent input modulates the activity of the trigeminal spinal nucleus oralis (TSNO) related to the tooth pulp (TP)-evoked jaw-opening reflex (JOR). Extracellular single-unit recordings were made from 36 TSNO units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram (dEMG) signal in 26 pentobarbital-anesthetized rats. The activity of 36 TSNO neurons and the amplitude of the dEMG increased proportionally during 1.0-3.5 times the threshold for JOR. Some of these neurons (4 out of 5) were also excited by chemical stimulation (bradykinin, 1-2 microl, 1 mM) of TP. In 31 out of 36 TSNO neurons (86%), their activities during tooth pulp stimulation were suppressed by conditioning stimulation of the right vagus nerve. The suppressive effect of vagal afferent stimulation occurred at conditioning-test intervals of 20-150 ms after the onset of the stimulation, and its maximal suppressive effect occurred at approximately 50 ms. The mean time course of this suppressive effect paralleled that of the dEMG. After administration of naloxone (0.5 and 1.0 mg/kg, i.v.), an opiate receptor blocker, the suppressive effect on the activity of TSNO neurons (6 out of 8) was significantly attenuated at the conditioning-test interval of 50 ms compared to the control (p < 0.01). These results suggested that vagal afferent input inhibits nociceptive transmission in the TSNO related to TP-evoked JOR and this inhibitory effect may occur via the endogenous opioid system in rats.

Prostaglandin E2-induced modification of tetrodotoxin-resistant Na+ currents involves activation of both EP2 and EP4 receptors in neonatal rat nodose ganglion neurones.

1 The aim of the present study was to investigate which EP receptor subtypes (EP1–EP4) act predominantly on the modification of the tetrodotoxin‐resistant Na+ current (INaR) in acutely isolated neonatal rat nodose ganglion (NG) neurones. 2 Of the four EP receptor agonists ranging from 0.01 to 10 μM, the EP2 receptor agonist (ONO‐AE1‐259, 0.1–10 μM) and the EP4 receptor agonist (ONO‐AE1‐329, 1 μM) significantly increased peak INaR. The responses were associated with a hyperpolarizing shift in the activation curve. 3 Neither the EP1 receptor agonist ONO‐DI‐004 nor the EP3 receptor agonist ONO‐AE‐248 significantly modified the properties of INaR. 4 In PGE2 applications ranging from 0.01 to 10 μM, 1 μM PGE2 produced a maximal increase in the peak INaR amplitude. The PGE2 (1 μM)‐induced increase in the GV1/2 baseline (% change in G at baseline V1/2) was significantly attenuated by either intracellular application of the PKA inhibitor PKI or extracellular application of the protein kinase C inhibitor staurosporine (1 μM). However, the slope factor k was not significantly altered by PGE2 applications at 0.01–10 μM. In addition, the hyperpolarizing shift of V1/2 by PGE2 was not significantly altered by either PKI or staurosporine. 5 In other series of experiments, reverse transcription–polymerase chain reaction (RT–PCR) of mRNA from nodose ganglia indicated that all four EP receptors were present. 6 The NG contained many neuronal cell bodies (diameter <30 μm) with intense or moderate EP2, EP3, and EP4 receptor‐immunoreactivities. 7 These results suggest that the PGE2‐induced modification of INaR is mainly mediated by activation of both EP2 and EP4 receptors.

Temporomandibular joint inflammation decreases the voltage-gated K+ channel subtype 1.4-immunoreactivity of trigeminal ganglion neurons in rats.

Voltage‐gated K+ (Kv) channels are one of the important physiological regulators of the membrane potentials in excitable cells, including sensory ganglion neurons. The aim of the present study was to investigate whether temporomandibular joint (TMJ) inflammation alters expression of Kv channel subtype 1.4 (Kv1.4) of trigeminal ganglion (TRG) neurons innervating TMJ relating allodynia (pain caused by normally innoxious stimulation), by using both behavioral and immunohistochemical techniques. TMJ inflammation was induced by injection of Complete Freunds Adjuvant (CFA) into the rat TMJ. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ inflamed rats was significantly lower than that in naïve rats. TMJ afferents were identified by fluorogold (FG) labeling. The mean numbers of Kv1.4‐/neurofilament (NF) 200(myelinated fiber marker) positive‐ and negative‐immunoreactivities FG‐labeled small‐/medium‐diameter TRG neurons in inflamed rats were significantly decreased when compared with those in the naïve rats. These findings suggest that TMJ inflammation reduces the expression of Kv1.4 subunits in the small‐/medium sized (Aδ‐/C‐) TRG neurons and this may contribute to trigeminal inflammatory allodynia in TMJ disorder. These results lead us to suggest that Kv channel openers may be a potential therapeutic agents for prevention of mechanical allodynia.

Effect of 8-bromo-cAMP on the tetrodotoxin-resistant sodium (Nav 1.8) current in small-diameter nodose ganglion neurons.

We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current in neonatal rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0mM, 8-Br-cAMP at 0.1mM showed a maximal increase in the peak TTX-R Na(+) (Nav1.8) current and produced a hyperpolarizing shift in the conductance-voltage (G-V) curve. The PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5microM) decreased the peak Nav 1.8 current. The Ro-31-8425-induced modulation of the G(V)(1/2) baseline (a percent change in G at baseline V1/2) was not affected by additional 8-Br-cAMP application (0.1mM). The maximal increase in Nav 1.8 currents was seen at 0.1microM after the application of a PKC activator, phorbol 12-myristate 13-acetate (PMA) and forskolin. The PMA-induced increase in Nav 1.8 currents was not significantly affected by additional 0.1mM 8-Br-cAMP application. Intracellular application of a PKA inhibitor, protein kinase inhibitor (PKI, 0.01mM), inhibited the baseline Nav 1.8 current, significantly attenuated the 8-Br-cAMP-and PMA-induced increase in the peak Nav 1.8 current, and caused a significant increase in the slope factor of the inactivation curve. The PKI application at a higher concentration (0.5mM) greatly inhibited the PMA (0.1microM)-induced increase in the peak Nav 1.8 current amplitude and further enhanced the Ro-31-8425-induced decrease in the current. These results suggest that the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated by activation of both PKA and PKC.

Volume expansion suppresses the tooth-pulp evoked jaw-opening reflex related activity of trigeminal neurons in rats

The aim of the present study is to clarify whether physiological stimulation of vagal afferents modulates the activity of the trigeminal spinal nucleus oralis (TSNO) neurons related to the tooth-pulp (TP)-evoked jaw-opening reflex (JOR) in pentobarbital-anesthetized rats. The activity of TSNO neurons and the amplitude of digastric electromyogram (dEMG) increased proportionally during 1.0-3.5 times the threshold for JOR. The amplitude of the dEMG of 14 out of 17 rats was suppressed by physiological stimulation of vagal afferents after intravenous infusion of Ficoll. Out of 23, 18 TSNO unit activities in 14 rats were also suppressed by Ficoll infusion. This suppressive effect of unit and dEMG activities returned to the control level within 25 min. After administration of naloxone (0.5 and 1.0 mg/kg, i.v.) the suppressive effect of Ficoll infusion on the activity of TSNO neurons (5/7) was significantly attenuated compared to the control (p < 0.01). The inhibition TSNO neuronal and dEMG activities by Ficoll infusion was volume-dependent in a range of 5-10% of total blood volume. Furthermore, right vagus nerve ligation greatly inhibited the suppressive effect of Ficoll-induced TSNO activity. These results therefore suggest that low-pressure cardiopulmonary baroreceptors whose afferents travel in the vagus nerve inhibit the pulpal nociceptive transmission.

Immunohistochemical co-expression of carbonic anhydrase II with Kv1.4 and TRPV1 in rat small-diameter trigeminal ganglion neurons

The co-expression of carbonic anhydrase II (CAII) with the voltage-gated potassium channel subtype 1.4 (Kv1.4) or the vanilloid receptor (TRPV1) was examined in adult rat trigeminal ganglion (TG) neurons by using the immunofluorescence method. The small-diameter Kv.1.4-positive TG neurons co-expressed CAII immunoreactivity (47%). Most TRPV1-positive TG neurons (79%) had the CAII immunoreactivity, but showed a lack of immunoreactivity for a neurofilament protein (NF200), a maker of large TG neurons with myelinated axons. The fact that CAII-immunoreactive TG neurons revealed a common expression of both Kv1.4 and TRPV1 leads us to suggest that CAII may be one of the nociceptive neuronal markers.

EXCITATORY MECHANISM OF VERATRIDINE ON SLOWLY ADAPTING PULMONARY STRETCH RECEPTORS IN ANESTHETIZED RABBITS

The excitatory effects of veratridine on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of ouabain (a Na+-K+ ATPase inhibitor) in anesthetized, artificially ventilated rabbits after vagus nerve section. Administration of veratridine (40 microg/kg) stimulated SAR activity but did not significantly alter tracheal pressure. Administration of ouabain (50 microg/kg) initially stimulated SAR activity during both inflation and deflation, but after 20 min, two different types of SAR responses were observed; one became silent at the peak, of inflation only, and the other maintained excitatory activity during both inflation and deflation phases. Veratridine usually inhibited SAR activity in ouabain-treated animals, irrespective of the difference of ouabain effects. These results suggest that veratridine-induced stimulation of SARs is closely related to the change in the Na+ ion gradient, which is regulated by Na+ pump activity.