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).

The functional difference between transient and sustained K+ currents on the action potentials in tetrodotoxin-resistant adult rat trigeminal ganglion neurons.

To determine whether there is a difference between a transient K(+) current (I(A)) and a sustained K(+) current (I(K)) regarding the neuronal function in small-diameter adult rat trigeminal ganglion (TG) neurons, which were insensitive to tetrodotoxin (TTX, 1 microM), we performed two different types of experiments. Primary cultures of dissociated TG neurons were prepared, and electrophysiological recordings were performed with the whole-cell configuration using the patch-clamp technique. In the voltage-clamp mode, two distinct K(+) current components, (I(A)) and (I(K)), were identified, and two different components (59.5% and 96.3%) of I(K) to the total K(+) current were observed at a +50 mV step-pulse. The IC(50) value for 4-aminoprydine (4-AP, 0.05-50 mM), which inhibited the I(A) by 50%, was 0.7 mM. That for tetraethylammonium (TEA, 0.02-20 mM) to inhibit 50% of I(K) was 1.5 mM. In the current-clamp mode, we used 0.5 mM 4-AP and 2 mM TEA at each concentration nearly equal to the IC(50) value. Irrespective of the absence or presence of TEA (2 mM), 0.5 mM 4-AP application increased the number of action potentials due to the decreased duration of the depolarization phase (DDP). TEA in the presence and absence of 4-AP prolonged the duration of action potentials as well as the duration of repolarization phase (DRP). These results suggested that I(A) and I(K) had independent effects regulating the intrinsic firing properties of the action potential number and timing, respectively, in adult rat TTX-R TG neurons.

Prostaglandin E2 potentiates the excitability of small diameter trigeminal root ganglion neurons projecting onto the superficial layer of the cervical dorsal horn in rats.

The aim of the present study was to investigate how prostaglandin E2 (PGE2) affects the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, using fluorescence retrograde tracing and perforated patch-clamp techniques. TRG neurons were retrogradely labeled with fluorogold (FG). The cell diameter of FG-labeled neurons was small (< 30 microm). Under the voltage-clamp mode, application of PGE2 (0.01-10 microM) concentration-dependently increased the magnitude of the peak tetrodotoxin-resistant sodium current (TTX-R I(Na)) and this current was maximal at a concentration of 1 microM. One micromolar PGE2 application caused a hyperpolarizing shift of 8.3 mV in the activation curve for TTX-R I(Na). In the current-clamp mode, the PGE2 (1 microM) application significantly increased the number of action potentials during the depolarizing step pulses as well as the level of overshoot but had no significant effect on the resting membrane potential. These results suggest that the excitability of small diameter TRG neurons seen after 1 microM PGE2 application is involved in an increase in the

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

Peripheral nerve injury activates satellite cells to produce interleukin 1beta (IL-1beta) 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-1beta 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-1beta-immunoreactive cells was significantly increased compared to controls. GFAP and IL-1beta 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-1beta (1 nM) induced-depolarization in inflamed rats was larger than in controls. IL-1beta application significantly increased firing rates evoked by depolarizing pulses in the neurons of inflamed rats, compared to controls. The response to IL-1beta 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-1beta following inflammation, and that the upregulation of IL-1RI in the soma may contribute to the mechanism underlying inflammatory hyperalgesia. Therefore IL-1beta blockers are potential therapeutic agents for prevention of trigeminal hyperalgesia.

Mechanisms involved in modulation of trigeminal primary afferent activity in rats with peripheral mononeuropathy.

In order to clarify the mechanisms underlying the changes in primary afferent neurons in trigeminal neuropathic pain, a chronic constriction nerve injury model of the infraorbital nerve (ION-CCI) was developed in rats. Mechanical allodynia was observed at 3 days after ION-CCI and lasted more than 14 days. Single-unit activities were recorded from the ION of anesthetized rats. C-, Abeta- and Adelta-units were identified on the basis of their conduction velocity. Adelta-units were frequently encountered at a later period after ION-CCI. The highest Adelta-spontaneous activity was recorded at 3 days after ION-CCI and progressively decreased after that, but spontaneous activity was still higher at 14 days after ION-CCI than that of naïve rats. Mechanical-evoked responses of Adelta-units were also highest at 3 days after ION-CCI and then gradually decreased. In consideration of these data, patch-clamp recordings were performed on medium to large size neurons of the dissociated trigeminal ganglion (TRG). Patch-clamp recordings revealed that the IK (sustained) and IA (transient) in rats with ION-CCI were significantly smaller than those of naïve rats, and correlated with an increase in duration of repolarization phase and a decrease in duration of depolarization phase, respectively. The hyperpolarization-activated current (Ih) was significantly larger in TRG neurons of rats with ION-CCI as compared with those of naïve rats. The present results suggest that Ih, IK and IA in Adelta-afferent neurons in TRG are significantly involved in the changes in afferent spontaneous activity and mechanically evoked activity that accompany mechanical allodynia produced by trigeminal nerve injury.

The effect of PKC activity on the TTX-R sodium currents from rat nodose ganglion neurons.

To determine how protein kinase C (PKC) activity influences properties of the tetrodotoxin-resistant sodium current (TTX-R I(Na)) in neonatal rat nodose ganglion (NG) neurons, we assessed the effects of phorbol,-12-myristate,13-acetate (PMA), one of the PKC activators, and staurosporine, one of the PKC inhibitors, on the current. PMA (30 and 100 nM) induced an increase in the peak current amplitude of normalized current-voltage curves, a leftward shift in the potential for half activation (V(1/2)) of normalized conductance-voltage curves and a leftward shift of V(1/2) potential for steady-state inactivation curves. The effects of staurosporine (0.1 and 1 muM) on the peak current amplitude and the V(1/2) potential for activation were opposite compared with those seen after PMA application. Staurosporine (1 muM) antagonized PMA (100 nM)-induced modification of TTX-R I(Na). These results suggest that the basal TTX-R I(Na) obtained from neonatal NG neurons is controlled by the level of PKC activity.

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.

The aim of this study was to investigate whether under in vivo conditions, temporomandibular joint (TMJ) inflammation alters the excitability of Abeta-trigeminal root ganglion (TRG) neuronal activity innervating the facial skin by using extracellular electrophysiological recording with multibarrel-electrodes. Complete Freund's 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 Abeta-TRG neurons responding to electrical stimulation of the whisker pad was recorded in pentobarbital-anesthetized rats. The number of Abeta-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 Abeta-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 Abeta-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 Abeta-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.

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.

Temporomandibular joint inflammation potentiates the excitability of trigeminal root ganglion neurons innervating the facial skin in rats.

The aim of this study was to test the hypothesis that temporomandibular joint (TMJ) inflammation alters the excitability of trigeminal root ganglion (TRG) neurons innervating the facial skin, by using behavioral, electrophysiological, molecular, and immunohistochemical approaches. Complete Freund's adjuvant (CFA) was injected into the rat TMJ to produce inflammation. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ-inflamed rats was significantly lower than that in naïve rats. The TRG neurons innervating the inflamed TMJ were labeled by 2% Fluorogold (FG) injection into the TMJ. The number of FG-labeled substance P (SP)-immunoreactive neurons in the inflamed rats was significantly increased compared with that in the naïve rats. On the other hand, medium- and large-diameter TRG neurons (>30 microm) innervating the facial skin were labeled by FG injection into the facial skin. In the FG-labeled cutaneous TRG neurons, the occurrence of SP (100 nM) induced membrane depolarization in inflamed rats (medium: 73.3%, large : 85.7%) was larger than that in the naïve rats (medium: 29.4%, large : 0%). In addition, SP application significantly increased the firing rate evoked by depolarizing pulses in the neurons of inflamed rats compared with those of naïve rats. Quantitative single-cell RT-PCR analysis showed the increased expression of mRNA for the NK1 receptor in FG-labeled TRG neurons in inflamed rats compared with that in naive rats. The numbers of SP and NK1 receptors/neurofilament 200 positive immunoreactive TRG neurons innervating the facial skin (FG-labeled) in the inflamed rats were significantly increased compared with those seen in naïve rats. These results suggest that TMJ inflammation can alter the excitability of medium- and large-diameter TRG neurons innervating the facial skin and that an increase in SP/NK1 receptors in their soma may contribute to the mechanism underlying the trigeminal inflammatory allodynia in the TMJ disorder.