Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation

Neuroscience 138 (2006) 621– 630

ENHANCED EXCITABILITY OF RAT TRIGEMINAL ROOT GANGLION NEURONS VIA DECREASE IN A-TYPE POTASSIUM CURRENTS FOLLOWING TEMPOROMANDIBULAR JOINT INFLAMMATION Key words: voltage-dependent Kⴙ currents, inflammation, trigeminal root ganglion, temporomandibular joint disorder, allodynia, perforated patch-clamp.

M. TAKEDA,a* T. TANIMOTO,a M. IKEDA,a M. NASU,b J. KADOI,a S. YOSHIDAa AND S. MATSUMOTOa a Department of Physiology, School of Dentistry at Tokyo, Nippon Dental University, 1-9-20, Fujimi-cho, Chiyoda-ku, Tokyo 102-8159, Japan b Research Center for Odontology, School of Dentistry at Tokyo, Nippon Dental University, 1-9-20, Fujimi-cho, Chiyoda-ku, Tokyo 1028159, Japan

Chronic pathological conditions such as tissue inflammation can change the properties of somatic sensory pathways, leading to hyperalgesia and allodynia (Scholz and Woolf, 2002). Changes in the excitability of primary afferent neurons (peripheral sensitization) and/or altered information processing in the spinal cord or higher centers contribute to increased pain sensation (Millan, 1999). Complete Freund’s adjuvant (CFA) models of inflammation in the orofacial region have been developed in rats to study the trigeminal nervous system (Imbe et al., 2001; Iwata et al., 1999). Temporomandibular joint (TMJ) inflammation is associated with spreading pain and hyperalgesia (Sessle and Hu, 1999), and TMJ disorder patients complain of pain from innoxious vibrotactile stimulation (Fillingim et al., 1998). We recently reported that TMJ inflammation modulates the excitability of A␤-trigeminal root ganglion (TRG) neurons innervating the facial skin via a paracrine mechanism due to the release of substance P (SP) from the TRG neuronal cell body. Such a release may be important in determining the trigeminal inflammatory allodynia associated with TMJ disorders (Takeda et al., 2005b,c). Although inflammation increases the fraction of small-diameter and SP-immunoreactive TMJ neurons (A␦-, C-fibers), the ionic mechanism underlying TMJ inflammation-induced change in the SP-related hyperexcitability of TMJ neurons remains to be determined. Voltage-dependent K⫹ (Kv) channels are important physiological regulators of membrane potential in excitable tissues, including sensory ganglia (Ficker and Heinemann, 1992). Dorsal root ganglion (DRG) and TRG neurons express three distinct classes of K⫹ current in varying quantities: dominant-sustained (K-current; IK), fast-inactivating transient (A-current; IA), and slow-inactivating transient (D-current; ID) currents (Everill et al., 1998; Takeda et al., 2004a,b; Yoshida and Matsumoto 2005). Kv channel subunits, Kv 1.4, 4.2 and 4.3 mediate the IA (Pearce and Duchen, 1994; Winkelman et al., 2005). These currents are often defined by their sensitivity to 4-aminopyridine (4-AP). Since Kv 1.4 and 4.3 channel subunits are expressed in the small-diameter (A␦-, C-fiber types) neurons of the DRG (Rasband et al., 2001; Winkelman et al., 2005), IA may be significant in regulating the activity of nociceptive neurons. For example, 4-AP depolarizes the resting membrane potential and markedly increases the action poten-

Abstract—The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund’s adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (Vhⴝⴚ60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 ␮m), voltagedependent transient Kⴙ current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltagedependent transient Kⴙ current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at twofold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5 mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund’s adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient Kⴙ current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder. © 2005 Published by Elsevier Ltd on behalf of IBRO. *Corresponding author. Tel: ⫹81-3-3261-8740; fax: ⫹81-3-3261-8740. E-mail address: [email protected] (M. Takeda). Abbreviations: BDNF, brain-derived neurotrophic factor; CFA, complete Freund’s adjuvant; DRG, dorsal root ganglion; DTx, dendrotoxin; EGTA, ethylene glycol-bis-␤-aminoethyl ether N,N,N=, N=-tetraacetic acid; FG, Fluorogold; HEPES, N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid; IA, voltage-dependent fast inactivating transient currents; ID, voltage-dependent dominant sustained currents; IK, voltagedependent dominant sustained transient; I–V, current–voltage; k, slope factor; Kv, voltage-dependent K⫹; NT3, neurotrophin-3; SP, substance P; TEA, tetraethylammonium; TMJ, temporomandibular joint; TRG, trigeminal root ganglion; TTX, tetrodotoxin; TTX-R, tetrodotoxin resistant; TTX-R-INa, TTX-resistance Na⫹ currents; Vm, test pulse voltage; VR1, vanilloid receptor 1; V1/2, membrane potential half activation; 4-AP, 4-aminopyridine. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.11.024

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tial firing in TRG neurons (Puil et al., 1989). In addition, Yoshimura et al. (1999) demonstrated that during cyclophosphamide-induced chronic cystitis, bladder afferent neurons with tetrodotoxin (TTX)-resistant spikes exhibited a lower threshold for spike activation with enhanced firing properties because of steady-state inactivation and reduction in the current density of IA channels. More recently, Dang et al. (2004) also reported that a reduction in IA, but not in IK, contributes to increased excitability in gastric sensory neurons following gastric ulcers. Taken together, these results led us to suggest that TMJ inflammation may modify the A-currents, thereby contributing to the hyperexcitability of TRG neurons innervating the TMJ. The present study aimed to test this possibility for the first time.

EXPERIMENTAL PROCEDURES The experiments were approved by the animal use and care committee of Nippon Dental University and were consistent with the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983). Double-blind experiments were carried out and all possible efforts were made to minimize the number of animals used and their suffering.

Induction of TMJ inflammation The experiments were performed on 39 adult male Wistar rats (100 –170 g, body weight). Each animal was anesthetized with sodium pentobarbital (45 mg/kg, i.p.), and CFA (0.05 ml 1:1 oil/ saline suspension) was injected into the left side of the TMJ capsule, as described in previous studies (Ren, 1999; Takeda et al., 2005b,c). For control rats, vehicle (0.9% NaCl) was injected into the left side of the TMJ capsule. In some experiments (n⫽3), the CFA-induced inflammation was verified with Evan’s Blue dye (50 mg/ml, 1 ml/kg, i.v.) extravasation. Postmortem examination of the injected TMJ region found accumulation of blue dye in the TMJ and periarticular tissue, indicating that the plasma protein extravasation was due to localized inflammation (Imbe et al., 2001). In preliminary experiments, we found no sign of inflammation of the overlying skin after CFA injection into a TMJ using the conventional histological method. Thus, CFA induced only a local inflammatory reaction and did not affect the overlying skin. One day after CFA injection, the animals were checked for abnormal pain sensations by probing the injected sites and surrounding orofacial skin with von Frey filaments. The animals often responded to a threshold stimulation with vocalization and scratching behavior, suggesting nociceptive stimulation, as described previously (Ren, 1999). Two days after CFA or saline injection, the von Frey hair (calibrated with a force transducer) was applied to the whisker pad (Takeda et al., 2000) and the mechanical threshold for escape behavior was measured in inflamed and control rats.

Retrograde-labeling of TRG neurons innervating TMJ On the day of CFA/vehicle injection, to identify TRG neurons innervating TMJ (second and third branches), we used the Fluorogold (FG)-labeling method (Takeda et al., 2004b, 2005a). FG solution (2% in distilled water, 20 ␮l) was injected into the TMJ in the ipsilateral region of CFA/vehicle injection using a 31-gauge needle. CFA or saline was injected slowly over a 3 min period.

Acute cell dissociation and whole-cell patch-clamp recording The patch-clamp recording was conducted two days after CFA or vehicle injection. For electrophysiological study, 15 control and 15

inflamed rats were used. Acute dissociation of TRG neurons was performed using the same technique described previously (Takeda et al., 2005b). For acute dissociation of TRG neurons, adult rats were decapitated. The left TRG was rapidly removed and incubated for 15–25 min at 37 °C in modified Hanks’ balanced salt solution (130 mM NaCl, 5 mM KCl, 0.3 mM KH2PO4, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM glucose, 10 mM HEPES, pH 7.3) containing collagenase type XI (2 mg/ml) (Sigma, St. Louis, MO, USA) and type II (2 mg/ml) (Sigma). The cells were dissociated by trituration with a fire-polished Pasteur pipette and were subsequently plated onto poly-L-lysine-coated coverslips in 35-mm dishes. The plating medium contained Leibovitz’s L-15 solution (Invitrogen, Carlsbad, CA, USA) supplemented with 10% newborn calf serum (Invitrogen), 26 mM NaHCO3 and 30 mM glucose. The cells were maintained in 5% CO2 at 37 °C and were used for recording between 2 h and 8 h after plating. After incubation, the coverslips were transferred to the recording chamber in a standard external solution containing 155 mM NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 20 mM glucose, pH 7.3. The recording chamber (volume, 0.5 ml) was mounted onto an inverted microscope (Nikon) equipped with phase contrast, video camera and two micromanipulators. The chamber was perfused under gravity with a standard external solution at approximately 0.5 ml/min. Whole cell recordings were conducted with the rapid perforatedpatch technique (Takeda et al., 2004a,b, 2005b). Fire-polished patch-pipettes (2–5 M⍀) were filled with 120 mM potassium methanesulphonate, 20 mM KCl, 7.5 mM HEPES and 2 mM EGTA, pH 7.3, containing amphotericin B (100 ␮g/ml). To study the total outward K⫹ current, the solution was changed to a K⫹ current isolation solution containing 150 mM choline chloride, 3 mM KCl, 1 mM MgCl2, 10 mM HEPES and 20 mM glucose (pH 7.35). To distinguish IA and IK from total K⫹ currents, we used the following steps as modified in previous studies (Everill and Kocsis, 1999; Takeda et al., 2004a,b; Tsuboi et al., 2004). Outward potassium currents were elicited by stepping to a conditioning voltage of either ⫺40 mV or ⫺120 mV from the holding potential of ⫺60 mV for 300 ms; then the membrane was depolarized from ⫺40 mV to ⫹60 mV in increments of 10 mV; ⫹60 mV produced the largest peak current in each recording. In some experiments, we compared IA densities between control and inflamed rats in the presence of 0.5 ␮M ␣-dendrotoxin (DTx). In this study, the following criteria were used for identification of IA. The A-currents were determined by subtraction of the ⫺40 mV protocol from the ⫺120 mV protocol. If no changes (less than 5%) were found in the peak A-currents after 4-AP (6 mM) application, the neurons were defined as IA-negative. Activation of the currents in standard solution was rapid and decayed only partially during 300-ms depolarization pulses. Amplitudes and rates of rise in the absolute current become more prominent with the magnitude of depolarization. For obtaining IA activation and inactivation data, the external solution was changed to the following solution: 70 mM choline chloride, 80 mM tetraethylammonium (TEA), 2 mM MgCl2, 3 mM KCl, 10 mM HEPES and 20 mM glucose, pH⫽7.4. In a preliminary experiment, no significant differences were observed in activation and inactivation kinetics for FG-labeled and non-labeled cells. In current-clamp mode, action potentials (overshoot of action potential ⬎0 mV) were elicited by depolarizing current pulses (10 –500 pA, 10 pA steps, 300 ms). We firstly determined the threshold for action potentials. The threshold was defined as the currents required for eliciting a single spike. The TTX (1 ␮M) sensitivity for FG-labeled TRG neurons was then tested. Neurons were defined as tetrodotoxin-resistant (TTX-R) if there was no change of spike amplitude elicited by threshold depolarizing current pulses in the presence of TTX. The action potential firing rates were assessed by counting the number of action potentials elicited by depolarizing pulses (two-times threshold currents). Spike duration was determined as the duration of the first spike at the level of half amplitude. Current- and voltage-clamp recordings were conducted with an Axopatch 200B amplifier (Molecular Devices,

M. Takeda et al. / Neuroscience 138 (2006) 621– 630 Foster City, CA, USA). Signals were low-pass filtered at 1 or 5 kHz and digitized at 10 kHz. To evaluate changes in cell membrane resistance during recordings in the current-clamp mode, negative current pulses (50 – 600 pA, 250 ms, 0.2 Hz) were injected through the patch pipette. Access resistance was checked throughout the experiments, and no significant changes were found during experiments. All recordings were performed at room temperature.

Data analysis Data were stored on a computer disk for off-line analysis (pClamp 8.0). Normalized conductance-voltage (G-V) curves were constructed from current–voltage (I–V) curves by dividing the evoked current by the potential obtained: Vm (test potential)⫺Vr (reversal potential). The reversal potential was determined for each individual current from its instantaneous I–V relationship as described previously (Gold et al., 1996). To evaluate changes in the steadystate inactivation of IA, a 500-ms prepulse was varied between ⫺140 mV to ⫹60 mV in 10-mV increments followed by a 50-ms test pulse to ⫹60 mV. Normalized activation and steady-state inactivation curves were fitted to G/Gmax⫽1[1⫹exp{(V1/2⫺Vm)/k}] and I/Imax⫽1/[1⫹exp{(Vm⫺V1/2)/k}], respectively, using the Boltzmann equation. Vm is the test pulse voltage (activation) or the prepulse potential (steady-state inactivation). V1/2 is the membrane potential at half activation and k is the slope factor. The values are expressed as means⫾S.E.M. Statistical analysis was made with Duncan’s new multiple range test. Data for the electrophysiological studies were analyzed by parametric statistical analysis (a two-tailed Student’s t-test). P⬍0.05 was considered significant.

Drugs used All drugs (stock solutions) were stored at ⫺20 °C, and dissolved in standard external solution. K⫹ channel blockers such as, 4-AP (0.5– 6 mM, Sigma) (Takeda et al., 2004a,b), TEA (80 mM, Sigma), ␣-DTx (0.5–1 ␮M) (Alomone Laboratories, Jerusalem, Israel) and TTX (1 ␮M, Sigma) (Takeda et al., 2004a,b) were added to the perfusion for a period ranging from 30 to 60 s.

RESULTS Induction of TMJ inflammation and allodynia After CFA injection, the animals were tested for abnormal pain sensation by probing the injected site and/or the orofacial skin with von Frey filaments. In the TMJ-inflamed rats, the threshold for escape from mechanical stimulation applied to the whisker pad area was significantly reduced

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from 51.3⫾6.3 mN to 19.1⫾2.1 mN at 48 h after CFA injection (n⫽18, P⬍0.05). No significant changes in the contralateral threshold in the whisker pad area were observed in either group (control vs. inflamed; 48.8⫾5.3 mN vs. 48.3⫾3.2 mN, n⫽18, P⬍0.05). Retrograde labeling of TRG neurons innervating TMJ In this study, TRG cell bodies were classified by size as small (⬍30 ␮m), medium (30 –39 ␮m) or large (⬎40 ␮m). Small- and medium-diameter TRG neurons innervating TMJ were labeled at 2 days after FG injection into the TMJ. We previously observed increased SP immunoreactivity in small TRG neurons after TMJ inflammation (Takeda et al., 2005b), and therefore chose to examine these neurons in the present analysis. The acutely isolated TRG neurons were spherical in shape and bright in appearance with a ‘halo’ around the cell body when viewed by phase contrast microscopy (Fig. 1A). Fig. 1B shows the size distribution of dissociated FG-labeled TRG neurons recorded (⬍30 ␮m) in this study, showing no significant differences in mean cell diameter between control and inflamed rats (control vs. inflamed; 21.3⫾1.5 ␮m vs. 22.1⫾0.5 ␮m, N.S). Following perforation of the cell membrane with amphotericin B in 70 acutely dissociated TRG neurons (voltage-clamp only; n⫽30, current-clamp only; n⫽30; voltage-clamp and current-clamp, n⫽5), the series resistance dropped to below 20 M⍀ (16.1⫾1.3 M⍀, n⫽70) within 5–13 min and remained stable for over 15 min. The values for cell capacitance in control and inflamed rats were not significantly different at 14.6⫾0.5 pF (n⫽32) and 16.2⫾0.8 pF (n⫽38), respectively. Effect of TMJ inflammation on the A-currents In the FG-labeled small TRG neurons (control n⫽15; inflamed n⫽20), separation of K⫹ currents was performed as described in previous studies (Takeda et al., 2004a,b). Typical waveforms of depolarization-activated K⫹ currents in the control and TMJ inflamed rats are shown in Fig. 2A ((a), total currents; (b), sustained currents, (c), currents resulting from (a) minus (b), IA). The neurons were held at ⫺60 mV and then stepped to either ⫺120 or ⫺40 mV for

Fig. 1. Retrograde labeling of small-diameter FG-labeled TRG neurons innervating TMJ. (A) Dissociated small-diameter TRG neurons observed under phase contrast optics (upper) and identified TMJ innervated by fluorescent FG (lower) in the same field. Scale bar⫽20 ␮m. Arrowheads show the target cell following FG injection. (B) Size distribution of dissociated FG-labeled TRG neurons (⬍30 ␮m).

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Fig. 2. Decreased IA current densities of FG-labeled TRG neurons after TMJ inflammation. (A) Separation of total outward currents into IA and IK in both control and inflamed rats. (a) Initiated via a prepulse of ⫺120 mV. (b) Initiated via a ⫺40 mV to ⫹60 mV in a 10-mV step of 200 ms. Subtraction of (a)⫺(b) reveals IA. IA sensitive to 4AP (6 mM). (B) Typical example of IA of TRG neurons in both control and inflamed rats. I–V relationships for IA in both control and inflamed rats. (C) Mean peak IA current densities were significantly decreased after TMJ inflammation. * P⬍0.05 control (n⫽11) vs. inflamed (n⫽12) rats.

300 ms (conditioning prepulse potential; Fig. 2A (a), (b)). Currents sensitive to the conditioning voltage were obtained by subtraction of the ⫺40 mV protocol from the ⫺120 mV protocol. Fig. 2B shows changes in the I–V relationship for IA in TRG neurons following TMJ inflammation. In control rats, A-current was observed in 80% (12/15) of FG-labeled neurons, whereas in inflamed rats, only 55% (11/20) of FG-labeled neurons were IA-positive. The total peak K⫹ current density was significantly reduced in rats with TMJ inflammation (control vs. inflamed; 371.6⫾3.1 pA/pF, n⫽12 vs. 216.0⫾5.2 pA/pF, n⫽11, P⬍0.05). However, while the peak A-current density was significantly depressed in rats with TMJ inflammation (control vs. inflamed; 283.8⫾14.2 pA/pF, n⫽12 vs. 148.1⫾13.1 pA/pF, n⫽11, P⬍0.05) (Fig. 2C), the peak IK density did not show any significant difference between control and inflamed rats (control vs. inflamed; 87.8⫾3.3 pA/pF, n⫽12 vs. 67.9⫾4.3 pA/pF, n⫽11, N.S). To determine whether this result reflected a difference in 4-AP sensitivity for A-current between control and inflamed rats, the effect of 4-AP on the IA in the control and inflamed rats was examined. As shown in Fig. 2A, the IA currents identified by the subtraction protocol were 4-AP-sensitive (6 mM) in both control and inflamed animals, consistent with previous studies (Takeda et al., 2004a,b). In the presence of 4-AP (6 mM), the peak A-currents decreased by 79.1⫾9.2% in

control rats (n⫽12) and 74.3⫾11.2% in inflamed rats (n⫽11), while there were no significant differences in the amplitude of remaining currents after 4-AP application between control and inflamed rats (control vs. inflamed; 54.1⫾2.2 pA/pF, n⫽12 vs. 41.7⫾3.2 pA/pF, n⫽11, N.S.). In some TRG neurons (n⫽5), to further examine D-current, we compared D-current density in neurons from control and inflamed rats in the presence of 0.5 ␮M ␣-DTx (Dcurrent blocker). The mean D-current density did not differ between these two groups (control vs. inflamed: 42.1⫾12.8 pA/pF vs. 27.9⫾11.4 pA/pF, n⫽5, NS.) Effect of TMJ inflammation on the voltage-dependence of activation and inactivation of IA The inactivation characteristics of the A-current were examined using a pulse protocol, in which the K⫹ currents were activated by a depolarizing voltage step to 0 mV after 500-ms conditioning prepulses ranging from ⫺140 to ⫹60mV in 10-mV increments. The inactivation curve was plotted as the normalized peak conductance of IA (I/Imax) against the potential of the conditioning prepulse (Vm). The IA in control rats started to inactivate at membrane potentials positive to ⫺120 mV and were nearly totally inactivated by depolarizing pulses to ⫺20 mV. The data were based on the Boltzmann equation fitting, with a half inac-

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tivation potential (V1/2) and slope factor (k) of ⫺75.0⫾2.7 mV (n⫽6) and 12.3⫾1.4 mV (n⫽6), respectively. This inactivation curve indicates that the 10 –20% of the maximum current was elicited at membrane potentials in the range of ⫺60 to ⫺50 mV, equivalent to the resting membrane potential level (Fig. 3A). In contrast, A-currents in TRG neurons from the inflamed rats were inactivated at more negative membrane potentials. The current was almost negligible when the holding membrane potential was held in the range of ⫺30 to ⫺20 mV. The inactivation curve in TMJ-inflamed rats was therefore displaced to a more hyperpolarized level by ⬃13 mV in comparison with control rats. Mean values for V1/2 and k in the inactivation of TRG neuron A-currents from the inflamed rats were ⫺88⫾3.5 mV (n⫽6) and 15.0⫾2.0 mV, respectively (n⫽6) (Fig. 3B and 3C), showing a significant difference in IA for V1/2 in the IA inactivation curve between control and inflamed rats (P⬍0.01) (Fig. 3B), but no significant difference in the k values (Fig. 3C). The voltage dependence of A-current activation in TRG neurons innervating TMJ was not statistically differ-

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ent between control and inflamed rats (Fig. 3D), as measured by comparing the peak conductance (G) of outward K⫹ currents evoked by voltage steps from ⫺80 mV to ⫹60 mV. G was then normalized with respect to the calculated maximal conductance Gmax. In this manner, IA in the TRG neurons innervating TMJ in the control rats was elicited by depolarizations positive to ⫺60 mV, with V1/2 occurring at the membrane potential of ⫺11.6⫾9.8 mV (n⫽6), with a slope factor of 17.3⫾0.8 mV according to the modified Boltzmann equation. Similarly, V1/2 and k in the IA activation curve of TRG neurons from TMJ-inflamed rats were ⫺11.6⫾5.3 and 19.4⫾0.5 mV, respectively (n⫽6), indicating no significant differences (Fig. 3E, 3F). Changes in the excitability of TRG neurons after TMJ inflammation In 30 of 70 FG-positive TRG neurons, we compared changes in the action potential firing rate evoked by depolarizing pulses in control (n⫽12) and inflamed (n⫽18) rats. In this study, small-diameter neurons had characteristics of

Fig. 3. Steady-state activation and inactivation curves of IA in FG-labeled TRG neurons from control and inflamed rats. (A) Inactivation characteristics of A-current obtained in the neurons from control and inflamed rats. Relative IA conductance normalized to the maximal IA conductance (G/Gmax) were plotted against membrane potential. Vh and k were obtained by fitting curves using the modified Boltzmann equation. Note: TMJ inflammation resulted in a leftward shift in the inactivation curve of IA. (B, C) Effect of TMJ inflammation on the membrane potential half inactivation (V1/2) and slope factor (k) of IA inactivation. * P⬍0.05 control (n⫽6) vs. inflamed (n⫽6) rats. (D) Activation characteristics of A-currents in control and inflamed rats. Relative peak conductance of A-current normalized to the maximal conductance of A-currents (G/Gmax) was plotted against membrane potentials. V1/2 and k were obtained by fitting curves using the modified Boltzmann equation. (E, F) Effect of TMJ inflammation on the membrane potential half activation (V1/2) and k of IA activation. * P⬍0.05 control (n⫽6) vs. inflamed (n⫽6) rats.

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nociceptors: (1) small size (⬍30 ␮m); (2) presence of long-duration action potentials (Waddell and Lawson, 1990); and, (3) resistance of the action potential to TTX (Ritter and Mendell, 1992; Moore et al., 2002). A typical example of this is shown in Fig. 4A. In current-clamp mode, FG-labeled small-diameter TRG neurons exhibited a long duration (5.3–7.0 ms) with a prominent shoulder on the repolarization phase (control, n⫽12; inflamed, n⫽18). All

the neurons tested exhibited action potentials in the presence of TTX (1 ␮M) (Fig. 4B); the mean resting membrane potential in inflamed rats was similar to that in the control rats (control vs. inflamed; ⫺60.2⫾1.2 mV, n⫽12 vs. ⫺58.4⫾1.0 mV, n⫽18, N.S.). The half-height duration of the first action potential was slightly increased by inflammation, but no significant difference was observed between control and inflamed rats (control vs. inflamed;

Fig. 4. Increased excitability of FG-labeled TRG neurons innervating TMJ after TMJ inflammation. (A) Action potential waveform in response to depolarizing current pulses. Note a prominent shoulder on the falling phase (filled triangle). (B) The action potential was induced at the threshold level (300 ms, 60 pA) and this potential was resistant to TTX (1 ␮M). (C) Typical example of current-clamp traces of action potential elicited at threshold and two-times threshold in FG-labeled TRG neurons in control and inflamed rats. TMJ inflammation reduced the threshold current, while increasing the number of spikes evoked by a two-times threshold. (D) Comparison of mean threshold current in control (n⫽12) and inflamed rats (n⫽18). * P⬍0.05. (E) Comparison of mean number of spikes elicited by a two-times threshold current in control (n⫽5) and inflamed rats (n⫽7). * P⬍0.05.

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6.1⫾1.1 ms, n⫽12 vs. 6.9⫾0.5 ms, n⫽18, N.S.). As shown in Fig. 4C, TMJ inflammation caused a significant reduction in the threshold current and increased the number of action potentials evoked by a two-fold threshold. The mean threshold current of the TRG neurons in the inflamed rats was significantly decreased compared with control rats (control vs. inflamed; 81.2⫾7.1 pA, n⫽12 vs. 35.1⫾5.1 pA, n⫽18, P⬍0.05) (Fig. 4D). Interestingly, some of the neurons fired repetitive discharges (control, five of 12; inflamed seven of 18), and in these neurons, the mean number of spikes elicited by the two-fold threshold was significantly increased compared with that seen in control rats (1.7⫾0.2 spikes, n⫽5 vs. 4.4⫾0.3 spikes, n⫽7, P⬍0.05) (Fig. 4E). Effect of 4-AP application on the action potential of TRG neurons Finally, five of 70 FG-positive TRG neurons in control rats were tested to determine whether the IA blocker, 4-AP (0.5 mM) could alter the firing rates evoked by depolarization pulses. Using voltage-clamp mode, we first determined the sensitivity of A-currents to 4-AP (Fig. 5A), and found that the mean A-current density was significantly depressed (251.7⫾13.1 pA/pF vs. 121.3⫾14.5 pA/pF, 48.1⫾4.9%, n⫽5, P⬍0.05 (Fig. 5B). These values are similar to approximately ⬃50% reduction reported in a previous study (Yoshida and Matsumoto 2005). By current-clamp mode, application of 4-AP to the same cells caused a significant reduction in the threshold currents and increased the firing rate of TTX-R action potentials (1 ␮M) evoked by the two-fold threshold (Fig. 5C). The mean threshold of action potentials evoked by current stimulation after 4-AP application was significantly decreased from control values obtained before 4-AP application (before vs. after; 85.1⫾6.4 pA vs. 41.1⫾4.8 pA, n⫽5, P⬍0.05), but was not different from the values obtained for inflamed rats (Fig. 5D). In contrast, the mean firing rates of action potentials evoked by a two-fold current stimulation after 4-AP application was significantly greater than the before values (before vs. after; 2.5⫾0.2 spikes vs. 5.8⫾0.4 spikes, P⬍0.05) but not different from the values obtained from inflamed rats (Fig. 5E). The half-height duration of first action potential was slightly increased by 4-AP application, but no significant difference was observed before and after 4-AP application (before vs. after; 6.5⫾2.1 ms, n⫽5 vs. 7.3⫾1.3 ms, n⫽5, N.S.).

DISCUSSION The results presented herein provide evidence that TMJ inflammation potentiates the excitability of small-diameter TRG neurons innervating TMJ, by suppressing A-currents via a leftward, hyperpolarizing, shift in the inactivation curve without any changes in the activation curve. The IA suppression of the TRG neuron innervating TMJ may contribute to trigeminal inflammatory allodynia in the TMJ disorder.

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Retrograde labeling of small-TRG neurons innervating TMJ The approach of taking electrophysiological recordings of the afferent neurons innervating the inflamed target (i.e. TMJ) enabled us to discriminate the innervation sites of peripheral terminals in small-diameter neurons of the TRG. The TRG neurons recorded in this study were ⬍30 ␮m and all TRG neurons recorded under current-clamp conditions expressed TTX-R. We recently reported that retrogradelabeled TRG neurons derived from the masseter muscle also expressed the vanilloid receptor 1 (VR1), a marker of nociceptors (Ichikawa et al., 2004; Ma, 2002), and that the diameter of these neurons was ⬍30 ␮m; these neurons were classified into C- and A␦-type neurons (Takeda et al., 2005a). VR1 immunoreactivity in small-diameter TRG neurons was also reported by Liu and Simon (1996). Since in the rat DRG neurons, there is a positive correlation between the neuronal cell size and conduction velocity for A␦- and C-afferents (Harper and Lawson, 1985), it can be speculated that FG-labeled small-diameter TRG neurons recorded in this study were nociceptive TRG neurons. Decrease in A-type potassium conductance in the TRG neurons after TMJ inflammation There are at least two types of transient outward currents in the TRG neurons (Akins and McCleskey, 1993; Gold et al., 1996; Viana et al., 2002), based on the inactivation time course and the sensitivity to 4-AP. The first component (IA) has a fast inactivation with a relatively lower sensitivity to 4-AP (⬎2 mM), while second component (ID) is ␣-DTX sensitive current (Storm, 1988) with a slower inactivation and a higher sensitivity to 4-AP (0.1 ␮M) (Viana et al., 2002). In the present study, we analyzed the current sensitivity to 4-AP (4 mM). Thus, K⫹ currents recorded belong to the IA category. Kv 1.4 and Kv 4.2 subunits are known contributors to IA current (Mathie et al., 1998), and Kv 4.2 subunits that form DTX insensitive Atype currents play an important role in determining the excitability of superior cervical ganglion cells (Malin and Nerbonne, 2000). In the CNS, the Kv 1.4 subunit is targeted to axons and terminals, while Kv 4.2 is mainly distributed in the dendrite and somata (Sheng et al., 1992; Cooper et al., 1998). From their localizations, it is possible that Kv 1.4 and Kv 4.2 may regulate a synaptic transmission mechanism for presynaptic and postsynaptic sites, respectively. In the sensory neurons, Rasband et al. (2001) indicate that Kv 1.4 channels may contribute directly regulating conduction in C-fibers. More recent reports also suggest that Kv 4.3 channels are expressed in the small DRG neurons and encode A-type K⫹ channels (Winkelman et al., 2005). Thus, it is possible that Kv 1.4, 4.2 and 4.3 channels may be involved in forming functional A-type Kv channels in TRG neurons. The results of this study were in agreement with previous studies demonstrating that peripheral inflammation can alter the excitability of nociceptive afferent sensory neurons (Stewart et al., 2003; Yoshimura and de Groat 1999). Indeed, we found that the mean A-current densities

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Fig. 5. Effect of 4-AP on the firing rate of FG-labeled TTX-R TRG neurons from control rats. (A) Typical effect of 4-AP (0.5 mM) application on the IA. (B) Percentage inhibition of IA by 4-AP (0.5 mM). * P⬍0.05, vs. control. (C) Example of TTX-R action potentials (TTX 1 ␮M) elicited at the threshold and two-times threshold on the FG-labeled TRG neurons from control rats before and after 4-AP (0.5 mM) application. 4-AP application reduced the threshold current while the number of action potential firings was increased by the two-times threshold. (D) Comparison of mean threshold current before and after 4-AP (0.5 mM) (n⫽5). (E) Comparison of mean firing rate before and after 4-AP (0.5 mM) (n⫽5). * P⬍0.05.

were significantly depressed in rats after TMJ inflammation. Furthermore, reduction in the peak IA density was associated with a hyperpolarization shift in the inactivation curve with no significant change in the shift in the activation curve. Under TMJ inflammation conditions, an inactivation curve shift in a hyperpolarization direction would cause fewer activated IA channels to be activated near the resting membrane potentials, leading to a further increase in excitability of TRG neurons, including a repetitive firing of TRG neurons innervating the TMJ. In agree-

ment with these previous findings (Moore et al., 2002; Yoshimura and de Groat 1999), we obtained the following results: 1) TRG neurons in inflamed rats have a lower current threshold for action potential activation compared with those in control rats and exhibit enhanced spike discharge in response to depolarizing pulse stimulation; 2) application of 4-AP (0.5 mM) to control neurons could mimic the changes in the threshold and firing rates seen after TMJ inflammation. These results are consistent with a leftward shift in the inactivation curve and a decrease in IA

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current densities, resulting in the enhanced excitability of A␦-and C-TRG neurons innervating the TMJ. Furthermore, changes in voltage-gated Na⫹ and K⫹ channels are important factors in inflammatory pain, mainly due to changes in nociceptive neurons that exhibit the hyperexcitability characterized by a decreased threshold for activation and an increased firing rate (Yoshimura and de Groat, 1999; Moore et al., 2002; Stewart et al., 2003; Dang et al., 2004; Beyak and Vanner, 2005). In particular, inflammatory bowel disease has been associated with a suppression of voltage-gated K⫹ currents (IA and IK) as well as an augmentation of tetrodotoxin resistant Na⫹ currents (TTX-R-INa), suggesting that multiple ionic channels contribute to the potentiation of excitability of the peripheral nociceptive neurons (Stewart et al., 2003). In contrast, it was reported recently that inflammation of TMJ caused no significant changes in TTX-R-Na⫹ conductance in TRG neurons retrogradely labeled with TMJ afferents. They also indicated that decreases in the rates of afterhyperpolarization decay were observed in the action potentials of inflamed TRG neurons (Flake and Gold, 2005). The latter effect was consistent with our results in that a decrease in the both the IA density and the action potential threshold was seen in inflamed TRG neurons. IA has been linked to the firing rate of action potentials as well as the action potential duration, whereas IK is related to the duration of action potentials (Kocsis et al., 1982; Honmou et al., 1994; Waddell and Lawson 1990; Hille, 2001). In this study, we found no significant differences in IK current density or the action potential half duration between control and inflamed rats. This finding is supported by the recent demonstration of no significant increases in action potential duration in inflamed TMJ neurons (Flake and Gold, 2005). In this study, we found that the half duration of the first action potential was slightly increased by inflammation, but no significant differences were observed between control and inflamed TRG neurons. It can therefore be suggested that a decrease in the IA caused a reduction in the action potential threshold seen during TMJ inflammation. This suggestion was further confirmed by the current-clamp data. Recent evidence demonstrated that ID contributes to the modification of neuronal function in adult rat TTX-R TRG neurons (Yoshida and Matsumoto, 2005), suggesting that TMJ inflammation may modify ID currents. In the presence of ␣-DTX (0.5 ␮M), no significant differences in the mean decreased IA density were observed between control and inflamed rats. Although we cannot completely rule out the possibility that the decreased IA may be involved in the decreased ID, it is likely that ID does not completely account for the changes in current density that occur after TMJ inflammation. Functional significance of enhanced excitability in TRG neurons due to suppressing IA after TMJ inflammation We have previously reported that TMJ inflammation significantly increases both the number of neurofilament 200positive and -negative TRG neurons that express SP

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(Takeda et al., 2005b), suggesting that both myelinated (A␦-fiber type) and unmyelinated (C-fiber type) neurons may contribute to SP synthesis/release from TRG neuron cell bodies (Takeda et al., 2005b). In fact, the upregulation of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) in inflammatory conditions has been implicated in the regulation of Kv protein expression (Obata et al., 2004; Braun et al., 2004; Groth and Aanonsen, 2002). For example, peripheral inflammation increases the expression of BDNF preferentially in DRG neurons that contain SP (Groth and Aanonsen, 2002), and both BDNF and NT3 regulate some Kv subunits that mediate the transient potassium current (Park et al., 2003). Taken together, it can be speculated that persistent exposure to inflammatory mediators may alter the properties of A-type potassium channels, such as phosphorylation-related events. Under inflammatory conditions, the sustained depolarization of nerve terminals caused by inflammatory mediators would prolong firing, resulting in the increase in SP released from small TRG neuronal soma within the trigeminal ganglia. These mechanisms may account for the emergence of trigeminal inflammatory allodynia in the TMJ disorder. Acknowledgments—This study was supported by a grant from the Ministry of Education, Science and Culture of Japan (No.15591980).

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(Accepted 19 November 2005) (Available online 4 January 2006)