Serotonergic Modulation of the Hyperpolarizing Spike Afterpotential in Rat Jaw-Closing Motoneurons by PKA and PKC

Tomio Inoue,1 Satsuki Itoh,2 Masayuki Kobayashi,1 Youngnam Kang,4 Ryuji Matsuo,1 Satoshi Wakisaka,3 and Toshifumi Morimoto1

Departments of  1Oral Physiology,  2Orthodontics, and  3Oral Anatomy, Faculty of Dentistry, Osaka University, Osaka 565-0871; and  4Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8315, Japan


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Inoue, Tomio, Satsuki Itoh, Masayuki Kobayashi, Youngnam Kang, Ryuji Matsuo, Satoshi Wakisaka, and Toshifumi Morimoto. Serotonergic Modulation of the Hyperpolarizing Spike Afterpotential in Rat Jaw-Closing Motoneurons by PKA and PKC. J. Neurophysiol. 82: 626-637, 1999. Intracellular recordings were obtained from rat jaw-closing motoneurons (JCMNs) in slice preparations to investigate the effects of serotonin (5-HT) on the postspike medium-duration afterhyperpolarization (mAHP) and an involvement of protein kinases in the effects. Application of 50 µM 5-HT caused membrane depolarization and increased input resistance in the most cells without affecting the mAHP, whereas not only membrane depolarization and an increase in input resistance, but also the suppression of the mAHP amplitude was induced by higher dose of 5-HT (100 or 200 µM). On the other hand, when the mAHP amplitude was increased by raising [Ca2+]o from 2 to 6 mM, 5-HT-induced attenuation of the mAHP amplitude was enhanced, and even 50 µM 5-HT reduced the mAHP amplitude. This 5-HT-induced suppression of the mAHP could be mimicked by application of membrane-permeable cAMP analogue 8-Bromo-cAMP, potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724 and antagonized by protein kinase A (PKA) inhibitor H89. The enhancement of the mAHP attenuation induced by 50 µM 5-HT under raised [Ca2+]o was blocked by a protein kinase C (PKC) inhibitor chelerythrine, suggesting an involvement of PKC in this enhancement. On the other hand, the attenuation of the mAHP induced by PKC activator phorbol 12-myristate 13-acetate was blocked almost completely by H89, suggesting that the PKC action on the mAHP requires PKA activation. Neither 5-HT1A antagonist NAN-190 or 5-HT4 antagonist SB 203186 blocked 5-HT-induced attenuation of the mAHP. We conclude that 5-HT induces dose-dependent attenuation of the mAHP amplitude through cAMP-dependent activation of PKA and that PKC-dependent PKA activation is also likely to be involved in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca2+]o. Because the slope of the linear relationship between firing frequency and injected current was increased only when the mAHP amplitude was decreased by 5-HT, it is suggested that the relation between incoming synaptic inputs and firing output in JCMNs varies according to serotonergic effects on JCMNs and calcium-dependent modulation of its effects.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The trigeminal motor nucleus receives a dense serotonergic input (Kolta et al. 1993; Nagase et al. 1997; Saha et al. 1991; Takeuchi et al. 1983) and contains serotonergic receptors (Kolta et al. 1993). In the brain stem slice preparations, 5-HT depolarizes trigeminal motoneurons and increases neuronal excitability (Hsiao et al. 1997; Inoue et al. 1995; Trueblood et al. 1996), as previously shown in facial (Aghajanian and Rasmussen 1989; Larkman et al. 1989), hypoglossal (Berger et al. 1992), and spinal (Elliott and Wallis 1992; Lindsay and Feldman 1993; Takahashi and Berger 1990; Wang and Dun 1990; White and Fung 1989) motoneurons. Trigeminal motoneurons innervate the jaw-closing or -opening muscles. Iontophoretically applied serotonin (5-HT) facilitates spike discharge induced by glutamate in both jaw-closing motoneurons (JCMNs) and jaw-opening motoneurons (JOMNs) and also facilitates spike discharge of both motoneurons during cortically induced rhythmic jaw movements (Katakura and Chandler 1990; Kurasawa et al. 1990). However, an immunohistochemical study showed that JCMNs are contacted by a larger number of 5-HT-immunoreactive boutons than JOMNs, suggesting that JCMNs receive a more dense 5-HT innervation (Nagase et al. 1997). Furthermore the incidence of the facilitation by iontophoretically applied 5-HT is higher in JCMNs than JOMNs (Kurasawa et al. 1990). Thus 5-HT may modulate the excitabilities of JCMNs more strongly than those of JOMNs.

In addition to the depolarization, 5-HT decreases the postspike medium-duration afterhyperpolarization (mAHP) in juvenile guinea pig motoneurons (Hsiao et al. 1997), spinal motoneurons of adult cats (White and Fung 1989) and 2- to 3-wk-old rats (Wu et al. 1991), and hypoglossal motoneurons of neonatal rats (Berger et al. 1992). Because AHP is an important factor of controlling neuronal discharge (Hille 1992), the attenuation of AHP by 5-HT likely results in an enhancement of motoneuronal excitability by increasing input-output gain of motoneurons (Berger et al. 1992; Hsiao et al. 1997). However, 5-HT has little effect on the AHP in juvenile rat hypoglossal (Talley et al. 1997) and adult rat facial (Larkman and Kelly 1992) motoneurons. Thus the effects of 5-HT on the AHP are still controversial.

A calcium-activated potassium current is involved in generating the mAHP in trigeminal motoneurons (Chandler et al. 1994; Kobayashi et al. 1997) as well as other cranial motoneurons (Nishimura et al. 1989; Sah and McLachlan 1992; Viana et al. 1993). Bayliss et al. (1995) proposed that 5-HT suppresses high-voltage-activated calcium channels with a resultant decrease in the calcium entry necessary for activation of the potassium currents responsible for the AHP. In contrast, in hippocampal neurons, 5-HT suppressed the AHP without prominent reduction of the amplitude, time course, or threshold voltage of the calcium spike, suggesting that 5-HT suppressed the calcium-activated potassium channels directly (Andrade and Nicoll 1987). It has been reported that 5-HT suppresses the AHP by activation of protein kinase A (PKA) through an increase in intracellular cAMP in hippocampal neurons (Pedarzani and Storm 1993; Torres et al. 1995). Protein kinase C (PKC) also was reported to be involved in inhibition of AHP in hippocampal neurons (Baraban et al. 1985) and enteric neurons (Pan et al. 1997). However, it is not clear in trigeminal motoneurons whether cAMP/PKA or PKC cascades lead to direct suppression of calcium-activated potassium channels responsible for the mAHP or that suppression of calcium entry through voltage-activated calcium channels mediates the attenuation of the AHP.

In the present study, the effects of 5-HT on the mAHP evoked in JCMNs were investigated in juvenile rat slice preparation containing the trigeminal motor nucleus by the use of the intracellular recording method. We demonstrate that 5-HT induces a dose-dependent attenuation of the mAHP amplitude through an increase in intracellular cAMP and activation of PKA. When the mAHP amplitude was increased by raising [Ca2+]o from 2 to 6 mM, 5-HT-induced attenuation of the mAHP amplitude was enhanced. PKC-dependent activation of PKA is likely to be involved in the enhancement under raised [Ca2+]o. Preliminary results of this study were reported previously in abstract forms (Inoue et al. 1997, 1998).


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Animal preparation

Transverse brain stem slices (450 µm) including the trigeminal motor nucleus were prepared from 87 juvenile Sprague-Dawley rats (3- to 6-wk old) as previously described (Kobayashi et al. 1997). To identify the recorded cell as a jaw-closing motoneuron histologically, we employed the fluorescence double-labeling technique (cf. Viana et al. 1990) in the first series of the experiments (37 of the 87 animals). The 37 animals were anesthetized with ketamine HCl (150 mg/kg im) and chlorpromazine HCl (12.5 mg/kg im), and both sides of the masseter nerve were dissected. About 0.5 µl of 10% solution of dextran-tetramethylrhodamine-lysine (DRL; 10,000 MW, Molecular Probes) in saline was injected into the major trunks of the masseter nerve using a Picospritzer II (General Valve) from a broken micropipette. After 2-5 days, the animals were reanesthetized for slice preparation. The trigeminal motor nucleus was visible in the slice through the stereomicroscope (Leica, Wild M8) with a ring light unit (Volpi, Intralux 6000). The tip of a recording electrode was also visible by adequate adjustment of a ring polarizer set with a glass analyzer in the ring light unit. Thus the electrode was inserted confidently into the dorsolateral portion of the nucleus in the slice. Biocytin (Sigma), which was filled in the recording microelectrode, was injected into the recorded neurons (n = 52 neurons). After recordings, the slices were processed for histological observation as previously described (Kobayashi et al. 1997). Biocytin-injected neurons were visualized with fluorescein isothiocyanate-conjugated streptavidin (FITC; Amersham). Twenty of the 52 neurons were also labeled by DRL (Fig. 1), indicating that these neurons were masseter motoneurons (MAMNs). Twenty-two of the 52 neurons were not labeled by DRL but were located within a cluster of neurons labeled by DRL (CLMNs). The other 10 neurons that were not labeled by DRL and were not located in the cluster of DRL-labeled neurons were not analyzed. Except for the early stage of the first series of experiments, 14 MAMNs (52%) and 11 (41%) CLMNs were obtained from 27 biocytin-injected neurons and the other two neurons were located in the vicinity of the cluster. Therefore in the second series of experiments, recordings were also made from dorsolateral portion of the nucleus but no histological examination was carried out to identify the recorded neurons (n = 69 neurons). It has been shown that motoneurons innervating the masseter or temporal muscles are located in the dorsolateral portion of the trigeminal motor nucleus in rats, whereas motoneurons innervating the anterior digastric muscle are located in the ventromedial portion (Limwongse and DeSantis 1977; Mizuno et al. 1975; Sasamoto 1979). Furthermore interneurons are very few in the trigeminal motor nucleus (Sessle 1977). Thus CLMNs and neurons recorded in the dorsolateral portion of the nucleus (DLMNs) most likely innervate the jaw-closing muscles. Therefore we presumed that MAMNs, CLMNs, and DLMNs are jaw-closing motoneurons (JCMNs).



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Fig. 1. Photomicrographs of masseter motoneurons labeled retrogradely with dextran-tetramethylrhodamine-lysine (DRL; A) and of a motoneuron labeled by intracellular injection of biocytin (B). Arrows in A and B indicate a double-labeled masseter motoneuron (MAMN). Bar indicates 100 µm.

Slice preparation

Animals were reanesthetized with ketamine HCl and chlorpromazine HCl, and slices were cut on a microslicer (DTK-1500, Dosaka) in cold modified artificial cerebrospinal fluid (M-ACSF). Slices then were incubated in a holding chamber containing normal artificial cerebrospinal fluid (N-ACSF). The composition of the N-ACSF was (in mM) 130 NaCl, 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, and 10 D-glucose. The M-ACSF was made of N-ACSF by replacing 130 mM NaCl with 260 mM sucrose. In some experiments, the Ca2+ concentration was raised to 6 mM. Tetrodotoxin (TTX, 1 µM; Wako), methysergide (20 µM; Novartis), 5-hydroxytryptamine creatinine sulfate (5-HT, 20-200 µM; Sigma), 8-Bromo-cAMP (1 mM; RBI), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 15-25 µM; RBI), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89, 10-20 µM; Seikagaku), phorbol 12-myristate 13-acetate (PMA, 2-5 µM; Sigma), 1,2-dimethoxy-12-methyl-[1,3]benzodioxolo[5,6-c]phenanthridinium chloride (chelerythrine, 10 µM; RBI), (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT, 25-50 µM; RBI), 1-[2-methoxyphenyl]-4-[4-(2-phtalimido)butyl]-piperazine (NAN-190, 10 µM; Sigma) and 1-piperidinylethyl 1H-indole-3-carboxylate (SB 203186, 10 µM; Tocris) were added directly to the perfusate. In some experiments, microdroplets of 5-HT (10-20 mM dissolved in N-ACSF) were applied to the surface of a slice by pressure-ejection using a Picospritzer II from a broken micropipette positioned upstream of the recording electrode.

Recording

After 2-10 h incubation in the holding chamber, slices were transferred to an interface-type chamber. The recording chamber was perfused continuously with N-ACSF at a rate of 1-1.5 ml/min and humidified 95% O2-5% CO2 flowed over the slice. All experiments were performed at 32 ± 1 °C. Intracellular recordings were obtained with glass microelectrodes (1.2-1.5 mm OD, 20-50 MOmega ) (Sutter Instruments). The microelectrode was filled with 1% biocytin in 1 M KCl and 0.05 M Tris buffer (pH 7.6) for intracellular staining in the first series of the experiments or filled with 2 M KCl and 0.05 M Tris buffer (pH 7.6) in the second series of experiments. Membrane potentials were recorded using an Axoclamp 2B amplifier (Axon Instruments) in either bridge or discontinuous current-clamp (DCC) mode. During DCC recordings, a 2- to 5-kHz sampling rate was employed at a 30% duty cycle. The head stage output was monitored on a separate oscilloscope to ensure proper capacitance adjustment and adequate settling of the microelectrode. When membrane potential changed during or after application of a drug, the membrane potential was clamped manually to the resting potential level in control by injecting constant hyperpolarizing or depolarizing current to evaluate the effects of the drug, except for measuring a change in the membrane potential after the drug application and evaluating the effects of microdroplet application of 5-HT. Membrane potential and current were digitized and stored on a computer hard disk using software (Clampex, Axon Instruments) through A-D converter, and analyzed with the use of Clampfit (Axon Instruments) and Excel (Microsoft) software.

Data obtained from each neuron except for resting membrane potential, rheobase and firing frequency are the mean of 5-8 trials. Data are presented as means ± SE. Comparisons of data before and during the drug application within groups were based on Student's paired t-tests. Differences of data between groups were analyzed by Student's t-tests, one-way or two-way ANOVA. ANOVA was followed by post hoc Newman-Keuls multiple comparison tests when justified. Probabilities <0.05 were considered significant.


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INTRODUCTION
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DISCUSSION
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General properties

The present study was based on the recordings from 111 JCMNs (20 MAMNs, 22 CLMNs, and 69 DLMNs), which had stable resting potentials more negative than -55 mV (-67.3 ± 0.6 mV; n = 111) and displayed action potentials (measured from resting membrane potential to the spike peak) >70 mV (98.3 ± 0.7 mV; n = 111). Input resistance was 9.6 ± 0.3 MOmega (n = 111), which was calculated from the relationship between injected current intensity (-0.2 to -0.5 nA) and the voltage response. Such values were similar to those previously reported (Kobayashi et al. 1997) and were not significantly different among MAMNs, CLMNs, and DLMNs. Furthermore the effects of 50 µM 5-HT applied in normal perfusate (2 mM Ca2+) on resting membrane potential, input resistance, rheobase, and characteristics of spike afterpotentials were not significantly different between MAMNs (n = 8) and CLMNs (n = 8), although the effects on DLMNs were not examined under this condition. Thus MAMNs, CLMNs, and DLMNs were not distinguished for further analysis.

Subthreshold membrane properties

The effects of bath (20-200 µM) or microdroplet (10-20 mM) application of 5-HT in normal perfusate (2 mM Ca2+) were tested in 47 JCMNs (16 MAMNs, 21 CLMNs, and 10 DLMNs). As has been reported in cranial and spinal motoneurons (reviewed by White et al. 1996), bath (50 µM) or microdroplet (20 mM) application of 5-HT produced a slow depolarization (4.1 ± 0.6 mV, P < 0.001, n = 31) and increased input resistance by 31% (13.1 ± 1.0 MOmega from 10.2 ± 0.8 MOmega , P < 0.001, n = 31) (Fig. 2A, left). These effects persisted in the presence of tetrodotoxin (n = 5). The nonselective 5-HT antagonist methysergide (20 µM) antagonized the effects of 5-HT on input resistance and resting membrane potential (Fig. 2A, right; n = 3). Those results suggest that 5-HT produced those changes through direct activation of 5-HT receptors in JCMNs.



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Fig. 2. Effects of 5-HT on membrane potential and spike afterpotentials in normal perfusate (2 mM Ca2+). A: membrane depolarization and an increase in input resistance were elicited in a MAMN by microdroplet application of serotonin (5-HT) in control (left). Perfusion with 20 µM methysergide for 20 min abolished the response to 5-HT (right). B: superimposed traces of action potentials (truncated) of a motoneuron within a cluster of neurons labeled by DRL (CLMN) before (Control) and after (5-HT) application of 50 µM 5-HT. medium-duration afterhyperpolarization (mAHP) amplitude was not altered by 5-HT. Inset: afterdepolarization (ADP) was not altered, either. C: superimposed traces of a MAMN before and after application of 200 µM 5-HT. Marked suppression of mAHP was observed. Inset: enhancement of ADP also was observed. Each trace in B and C is the average of 5 records. · · · , resting membrane potential in control.

Effects of 5-HT on spike afterpotentials

NORMAL PERFUSATE. The effects of 5-HT on spike afterpotentials were examined in 31 JCMNs (11 MAMNs, 11 CLMNs, and 9 DLMNs) in normal perfusate. As we previously reported (Kobayashi et al. 1997), an afterdepolarization (ADP) and a medium-duration afterhyperpolarization (mAHP) followed single action potentials which were elicited by an injection of brief (2-3 ms) depolarizing current pulses. In 5 of the 31 neurons a prominent ADP was not observed, and the ADP was not analyzed in the five neurons. As described in the preceding text, 50 µM 5-HT induced depolarization in 16 JCMNs tested (8 MAMNs and 8 CLMNs; 3.5 ± 0.7 mV, n = 16, P < 0.001) and increased input resistance by 28.7 ± 7.1% (9.4 ± 0.7 to 12.0 ± 1.0 MOmega , n = 16, P < 0.005). However, this dose of 5-HT had relatively minor effects on the afterpotentials. An exemplary response to 50 µM 5-HT obtained from a CLMN is shown in Fig. 2B. The traces before and after application of 50 µM 5-HT were superimposed, and each trace was obtained by averaging five records before or after 5-HT application by triggering with the peak of the action potential. Application of 50 µM 5-HT did not change the mAHP (5.0 ± 0.4 to 5.2 ± 0.6 mV, n = 16, P > 0.2) and ADP (9.0 ± 1.2 to 8.4 ± 1.4 mV, n = 12, P > 0.1) amplitude in the 16 JCMNs (Fig. 2B).

Then the effects of higher dose of 5-HT (100 or 200 µM) were examined in 18 JCMNs (5 MAMNs, 4 CLMNs, and 9 DLMNs). Compared with the effect of 50 µM 5-HT, 200 µM 5-HT invariably decreased the mAHP amplitude by 31.2 ± 6.6% (3.6 ± 0.2 to 2.5 ± 0.3 mV, n = 10, P < 0.001) and increased the ADP amplitude by 23.3 ± 6.0% (9.1 ± 0.9 to 11.1 ± 1.1 mV, n = 10, P < 0.005; Fig. 2C). Application of 100 µM 5-HT also significantly reduced mAHP by 13.2 ± 5.4% (5.2 ± 0.6 to 4.5 ± 0.6 mV, n = 8, P < 0.05) although the effect was less than those of 200 µM 5-HT. This dose of 5-HT was not effective on the ADP amplitude (6.2 ± 0.3 to 6.1 ± 0.4 mV, n = 6, P > 0.4). In contrast to the alteration of spike afterpotentials, no significant change was observed in spike height or spike half-amplitude duration during application of 5-HT.

RAISED [CA2+]o ENHANCED 5-HT-INDUCED ATTENUATION OF THE MAHP. Because both mAHP and ADP are calcium-dependent in rat trigeminal motoneurons (Kobayashi et al. 1997), raising [Ca2+]o results in enhancements of the mAHP and ADP probably due to an increase in Ca2+ influx. To make the effects of 5-HT on the mAHP and ADP more evident, we examined the effects of 5-HT under raised [Ca2+]o from 2 to 6 mM in 22 JCMNs (4 MAMNs, 1 CLMN, and 17 DLMNs). Slices were held under raised [Ca2+]o for 1-2 h and then intracellular recordings were performed. Before 5-HT application, those neurons under raised [Ca2+]o showed significantly larger amplitude of both ADP and mAHP compared with neurons incubated in normal perfusate (Table 1). The effects of 5-HT under raised [Ca2+]o were different from those under normal [Ca2+]o. Under raised [Ca2+]o, 50 µM 5-HT invariably decreased the mAHP amplitude by 19.4 ± 3.4% (6.0 ± 0.8 to 5.0 ± 0.8 mV, P < 0.001, n = 9) and increased the ADP amplitude by 15.5 ± 8.0% (11.8 ± 1.8 to 13.1 ± 1.7 mV, n = 6, P < 0.05; Fig. 3A). Effects of 200 µM 5-HT under raised [Ca2+]o were examined in 13 JCMNs. In 12 of 13 neurons, 200 µM 5-HT under this condition further decreased the mAHP amplitude by 40.4 ± 6.8% (5.0 ± 0.6 to 3.2 ± 0.6 mV, P < 0.001, n = 12) and increased the ADP amplitude by 10.1 ± 3.0% (14.5 ± 1.4 to 16.0 ± 1.5 mV, P < 0.01, n = 10; Fig. 3B).


                              
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Table 1. Effect of [Ca2+]o on electrical properties of JCMNs



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Fig. 3. Effects of 5-HT on spike afterpotentials under raised [Ca2+]o from 2 to 6 mM. A: superimposed traces of a motoneuron recorded in the dorsolateral portion of the trigeminal motor nucleus (DLMN) before and after application of 50 µM 5-HT under raised [Ca2+]o. In contrast to 5-HT application to normal perfusate, attenuation of mAHP and enhancement of ADP (inset) were observed. B: superimposed traces of a DLMN before and after application of 200 µM 5-HT. Application of 200 µM 5-HT under raised [Ca2+]o further suppressed mAHP. Enhancement of ADP (inset) also were observed. C and D: comparison of the effects of 5-HT on afterpotentials under normal and raised [Ca2+]o. Error bars: SE associated with each group. Responses of mAHP to 50 and 200 µM 5-HT under normal [Ca2+]o were assessed in 16 and 10 neurons, respectively. Responses of mAHP to 50 and 200 µM 5-HT under raised [Ca2+]o from 2 to 6 mM were assessed in 9 and 12 neurons, respectively. Responses of ADP to 50 and 200 µM 5-HT under normal [Ca2+]o were assessed in 12 and 6 neurons, respectively. Responses of ADP to 50 and 200 µM 5-HT under raised [Ca2+]o were assessed in 6 and 11 neurons, respectively. dagger  and ddager : P < 0.05 and P < 0.01 vs. control, respectively. * and **: P < 0.05 and P < 0.01 between groups, respectively. Each trace in A and B is the average of 5 records. · · · , resting membrane potential in control.

The effects of 5-HT under normal and raised [Ca2+]o are summarized in Fig. 3 C and D. The effects of 5-HT on the mAHP and ADP amplitude were different when [Ca2+]o and/or 5-HT concentration were altered. Two-way ANOVA showed that percent reduction in the mAHP amplitude from control after 200 µM 5-HT application was larger than those after 50 µM 5-HT (P < 0.001; Fig. 3C). Percent reduction in the mAHP amplitude under raised [Ca2+]o was also larger than that under normal [Ca2+]o (P < 0.05, 2-way ANOVA) and post hoc Newman-Keuls test revealed that the reduction after 50 µM 5-HT application under raised [Ca2+]o were larger than that under normal [Ca2+]o (P < 0.05; Fig. 3C). On the other hand, no significant differences were observed between the effects of 5-HT on the ADP amplitude under normal and raised [Ca2+]o (P > 0.1, 2-way ANOVA) and between those effects of 50 and 200 µM 5-HT (P > 0.5, 2-way ANOVA; Fig. 3D). However, a significant interaction was found between the factors of [Ca2+]o and concentration of 5-HT (P < 0.05, 2-way ANOVA), and post hoc Newman-Keuls test showed that the effect of 200 µM 5-HT on the ADP amplitude was significantly larger than that of 50 µM 5-HT under normal [Ca2+]o (P < 0.05). The effects of 5-HT on the resting membrane potential and input resistance under normal [Ca2+]o were not different from those under raised [Ca2+]o (P > 0.3, 2-way ANOVA).

INVOLVEMENT OF CAMP/PKA AND PKC CASCADES. To examine whether activation of PKA is involved in 5-HT-induced attenuation of the mAHP in rat JCMNs, the following experiments were performed under normal [Ca2+]o. First, effects of 8-Bromo-cAMP (1 mM), a membrane permeable cAMP analogue was examined in four DLMNs in the normal perfusate. Similar to the effects of 200 µM 5-HT, 8-Bromo-cAMP invariably decreased the mAHP amplitude by 39.3 ± 8.1% (3.1 ± 0.2 to 1.9 ± 0.3 mV, n = 4, P < 0.01) and increased the ADP amplitude by 9.8 ± 3.0% (14.1 ± 2.3 to 15.3 ± 2.2 mV, n = 4, P < 0.01; Fig. 4A). Application of 8-Bromo-cAMP also increased the spike amplitude slightly (from 103.3 ± 1.3 to 104.4 ± 1.0 mV, n = 4, P < 0.05) and induce depolarization by 2.3 ± 0.5 mV (from -68.8 ± 1.9 mV, n = 4, P < 0.01), however, no significant change was observed in spike half-amplitude or input resistance. Second, the effects of Ro 20-1724, a cAMP-specific phosphodiesterase inhibitor were examined in five DLMNs. The mAHP amplitude was not altered during application of 15-25 µM Ro 20-1724 for 25-30 min (3.1 ± 0.6 to 3.3 ± 0.6 mV, n = 5, P > 0.05; Fig. 4Ba). However, when the same dose of Ro 20-1724 was coapplied with 25-50 µM 5-HT, which did not alter the mAHP amplitude by itself, the mAHP amplitude was reduced by 28.8 ± 5.7% (3.3 ± 0.6 to 2.4 ± 0.6 mV, n = 5, P < 0.005; Fig. 4Bb). In contrast, the ADP amplitude was not altered by this dose of 5-HT in the presence of Ro 20-1724 (14.1 ± 0.9 to 14.0 ± 1.2 mV, n = 4, P > 0.1). Third, we examined the effects of H89, a selective inhibitor of PKA in five DLMNs. Slices were preincubated with H89 (10-20 µM) for 1.5-2.5 h and then intracellular recordings were performed. In the presence of H89, 200 µM 5-HT reduced the mAHP amplitude to only a small degree (4.2 ± 0.5 to 3.9 ± 0.6 mV, n = 5, P < 0.05; Fig. 4C), and percent reduction in the mAHP amplitude after coapplication of 200 µM 5-HT and H89 was about one fourth of the percent reduction after 200 µM 5-HT application by itself (8.1 ± 3.3% vs. 31.2 ± 6.6%, P < 0.05; Fig. 4D). In the presence of H89, 200 µM 5-HT had no effect on the ADP amplitude, either (5.0 ± 2.2 to 5.2 ± 1.9 mV, n = 5, P > 0.3). These results suggest that activation of PKA through an increase in intracellular cAMP is involved in 5-HT-induced attenuation of the mAHP.



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Fig. 4. Involvement of a cAMP/protein kinase A (PKA) cascade in 5-HT-induced attenuation of mAHP. A: superimposed traces of a DLMN before and after bath application of 1 mM 8-Bromo-cAMP under normal [Ca2+]o. Marked suppression of mAHP and enhancement of ADP (inset) were observed. Ba: superimposed traces of a DLMN before and after application of 15 µM Ro 20-1724 under normal [Ca2+]o. Neither mAHP nor ADP (inset) was altered during Ro 20-1724 application. Bb: same neuron in Ba. Application of 50 µM 5-HT in addition to 15 µM Ro 20-1724 attenuated mAHP under normal [Ca2+]o. C: superimposed traces of a DLMN before and after bath application of 200 µM 5-HT in the presence of 10 µM H89 under normal [Ca2+]o. H89 antagonized 5-HT-induced attenuation of mAHP. D: summary data of the effects of 8-Bromo-cAMP (n = 4), Ro 20-1724 (n = 5), the addition of 50 µM 5-HT to Ro 20-1724 (n = 5), 200 µM 5-HT (n = 10, same data in Fig. 3C) and 200 µM 5-HT in the presence of H89 (n = 5) on mAHP. Error bars: SE associated with each group. dagger  and ddager : P < 0.05 and P < 0.01 vs. control, respectively. * and **: P < 0.05 and P < 0.01 between groups, respectively. Each trace in A-C is the average of 5 records. · · ·, resting membrane potential in control.

Then we examined whether activation of PKC is also involved in 5-HT-induced attenuation of the mAHP or not. Bath application of PMA (2-5 µM), a PKC activator, invariably reduced the mAHP amplitude by 30.5 ± 4.7% (3.9 ± 0.3 to 2.7 ± 0.3 mV, P < 0.005; Fig. 5A) under normal [Ca2+]o in four DLMNs tested, suggesting that PKC might reduce the mAHP. In contrast the ADP amplitude was not altered by PMA (8.6 ± 1.0 to 8.9 ± 1.2 mV, n = 4, P > 0.2; Fig. 5A). Because PKC is activated Ca2+-dependently (Hug and Sarre 1993; Nishizuka 1995), it might be possible that a transient increase in [Ca2+]i generated by an action potential activates more PKC under raised [Ca2+]o and results in an enhancement of 5-HT-induced attenuation of the mAHP. To examine this possibility, we investigated the effects of chelerythrine, a selective inhibitor of PKC, on the attenuation of the mAHP induced by 50 µM 5-HT under raised [Ca2+]o in five DLMNs. In slices preincubated with chelerythrine (10 µM) for 1.5-2.5 h under raised [Ca2+]o, 50 µM 5-HT had no significant effect on the mAHP amplitude under this condition (6.6 ± 0.8 to 6.3 ± 1.0 mV, n = 5, P > 0.1; Fig. 5C). The difference between percent decreases of the mAHP amplitude after 50 µM 5-HT application under raised [Ca2+]o in the presence and absence of chelerythrine was significant (6.6 ± 4.6% vs. 19.4 ± 3.4%, P < 0.05; Fig. 5D). These results suggest that activation of PKC might be involved in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca2+]o.



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Fig. 5. Contribution of protein kinase C (PKC) to 5-HT-induced attenuation of mAHP. A: superimposed traces of a DLMN before and after bath application of 5 µM phorbol 12-myristate 13-acetate (PMA) under normal [Ca2+]o. PMA reduced mAHP amplitude, whereas PMA did not affect ADP (inset). B: superimposed traces of a DLMN before and after application of 5 µM PMA in the presence of 10 µM H89 under normal [Ca2+]o. H89 almost completely blocked PMA-induced attenuation of mAHP. C: superimposed traces of a DLMN before and after application of 50 µM 5-HT in the presence of 10 µM chelerythrine under raised [Ca2+]o. Chelerythrine blocked mAHP attenuation induced by 50 µM 5-HT under raised [Ca2+]o. D: summary data of the effects of PMA (n = 4), PMA in the presence of H89 (n = 5), 50 µM 5-HT under raised [Ca2+]o (n = 9, same data shown in Fig. 3C) and 50 µM 5-HT under raised [Ca2+]o in the presence of chelerythrine (n = 5) on mAHP. Error bars: SE associated with each group. dagger  and ddager : P < 0.05 and P < 0.01 vs. control, respectively. * and **: P < 0.05 and P < 0.01 between groups, respectively. Each trace in A-C is the average of 5 records. · · · , resting membrane potential in control.

Thus both PKA and PKC are likely to be involved in the 5-HT effects on the mAHP. Cross-talk between cAMP/PKA and PKC cascades might occur, as demonstrated in several cellular systems (Cooper et al. 1995; Pieroni et al. 1993). To examine whether PKA activation is required for the effects of PKC on the mAHP, the effects of PMA (5 µM) on the attenuation of the mAHP in the presence of H89 (10-20 µM) were investigated under normal [Ca2+]o in five DLMNs. In slices preincubated with H89 for 1.5-2.5 h, PMA did not induce any significant attenuation of the mAHP (3.6 ± 0.8 to 3.5 ± 0.8 mV, n = 5, P > 0.2; compare Fig. 5, A and B). There was a significant difference in percent reduction of the mAHP amplitude after PMA application between in the presence and absence of H89 (30.5 ± 4.7% vs. 2.1 ± 2.2%; P < 0.005; Fig. 5D). Because the Ki value of H89 for PKC that were obtained from purified enzyme assay was reported to be 31.7 µM (Chijiwa et al. 1990), it is possible that 10-20 µM H89 might block PMA-induced attenuation of the mAHP by inhibiting PKC activity. However, the Ki value of H89 for PKC in in vivo experiments is likely to be higher than that obtained from enzyme assay. In fact, 30 µM H89 had no effects on PKC activity in PC12D pheochromocytome cells (Chijiwa et al. 1990), and hypoxia-induced inhibition of whole cell Na+ current in dissociated hippocampal neurons was not altered by inclusion of 30 µM H89 in the patch electrode but was greatly attenuated by inclusion of PKC inhibitors such as calphostin C or PKCi in the electrode (O'Reilly et al. 1997). In the present study, percent reduction of the mAHP amplitude after PMA application in the presence of 10 µM H89 was <2.5% (n = 3). Thus it is less likely that H89 inhibits PKC activity, leading to abolishing the effects of PMA on the mAHP. Therefore it can be assumed that PKC-induced attenuation of the mAHP is mediated by PKA activation.

RECEPTOR PHARMACOLOGY. Because 5-HT-induced attenuation of the AHP was reported to be mediated by 5-HT1A receptors in neonatal rat hypoglossal motoneurons (Bayliss et al. 1995; Talley et al. 1997) or by 5-HT4 receptors in hippocampal neurons (Andrade and Chaput 1991; Torres et al. 1994), we tested whether those receptors mediate 5-HT-induced attenuation of the mAHP in JCMNs. The effects of a 5-HT1A agonist 8-OH-DPAT (25-50 µM) and a 5-HT1A antagonist NAN-190 (10 µM) were examined under normal [Ca2+]o in six and four DLMNs, respectively. Although 8-OH-DPAT slightly reduced the mAHP amplitude by 6.9 ± 0.8% (4.7 ± 0.8 to 4.3 ± 0.7 mV, n = 6, P < 0.01; Fig. 6A), in slices preincubated with NAN-190 for 1.5-2.5 h, 200 µM 5-HT could reduce the mAHP amplitude by 41.1 ± 12.6% (4.2 ± 1.0 to 2.7 ± 1.0 mV, n = 4, P < 0.01) and increase the ADP amplitude by 41.0 ± 12.8% (9.7 ± 2.8 to 12.2 ± 3.0 mV, n = 4, P < 0.05; Fig. 6B). Then we examined the effects of SB 203186 (10 µM), a 5-HT4 antagonist under normal [Ca2+]o in four DLMNs. In slices preincubated with SB 203186 for 1.5-2.5 h, 200 µM 5-HT could reduce the mAHP amplitude by 45.6 ± 9.7% (4.9 ± 1.1 to 3.0 ± 1.1 mV, n = 4, P < 0.001) and increase the ADP amplitude by 36.0 ± 5.5% (9.1 ± 1.7 to 12.2 ± 2.0 mV, n = 4, P < 0.01; Fig. 6C). The effects of 200 µM 5-HT co-applied with NAN-190 or SB 203186 were not significantly different from those of 200 µM 5-HT by itself (P > 0.4, 1-way ANOVA). Those results suggest an involvement of receptor subtype(s) other than 5-HT1A and 5-HT4 in 5-HT-induced attenuation of the mAHP.



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Fig. 6. Effects of 5-HT1A and 5-HT4 antagonists on 5-HT-induced attenuation of mAHP under normal [Ca2+]o. A: superimposed traces of a DLMN before and after bath application of 50 µM (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT). B: superimposed traces of a DLMN before and after application of 200 µM 5-HT in the presence of 10 µM 1-[2-methoxyphenyl]-4-[4-(2-phtalimido)butyl]-piperazine (NAN-190). C: superimposed traces of a DLMN before and after application of 200 µM 5-HT in the presence of 10 µM 1-piperidinylethyl 1H-indole-3-carboxylate (SB 203186). Although 8-OH-DPAT slightly reduced mAHP amplitude, NAN-190 did not block 5-HT-induced attenuation of mAHP. SB 203186 did not block 5-HT-induced attenuation of mAHP, either. Each trace in A-C is the average of 5 records. · · · , resting membrane potential in control.

Effects of 5-HT on repetitive firing properties

The effects of 5-HT on repetitive firing discharge in response to intracellular injection of long (1-2 s) depolarizing current pulses were examined in 19 JCMNs (3 MAMNs, 4 CLMNs, and 12 DLMNs). After application of 50 µM 5-HT under normal [Ca2+]o, rheobase was decreased significantly by 0.4 ± 0.1 nA (1.7 ± 0.3 to 1.3 ± 0.3 nA, n = 9, P < 0.001), and a higher frequency spike train was elicited compared with control (Fig. 7A, compare left and right). The relationship between firing frequency (f) and injected current intensity (I) was plotted in Fig. 7D. Both f-I relationships for the first interspike interval (1st ISI; Fig. 7D, left) and steady-state firing (average firing discharge over the last half of the injected current pulse; Fig. 7D, right) were shifted to the left. However, neither f-I slope of linear region of 1st ISI or steady-state relationship was significantly altered after 50 µM 5-HT under this condition (1st ISI: 19.0 ± 3.5 to 18.8 ± 2.4 Hz/nA, n = 7, Fig. 7D, left; steady-state: 13.5 ± 2.5 to 12.8 ± 1.5 Hz/nA, n = 7, Fig. 7D, right). Similar results have been reported in neonatal rat phrenic (Lindsay and Feldman 1993) and adult rat hypoglossal (Talley et al. 1997) motoneurons. On the other hand, 200 µM 5-HT under raised [Ca2+]o from 2 to 6 mM affected the slope of the linear region of the f-I relationship. Before application of 5-HT the f-I slopes were not very steep for both the 1st ISI and the steady-state firing under raised [Ca2+]o (1st ISI: 12.7 ± 2.5 Hz/nA, n = 6; steady-state: 6.3 ± 0.6 Hz/nA, n = 6; Fig. 7F, left and right, open circle ). As mentioned above, mAHP amplitude of those neurons was large under this condition. Such large mAHP most likely slowed the neuronal firing. After 5-HT application, the mAHP amplitude was remarkably reduced (Fig. 7E) and the f-I relationships were shifted to the left and their slopes became significantly steeper (1st ISI: 20.2 ± 4.7 Hz/nA, P < 0.05; steady-state: 8.0 ± 0.5 Hz/nA, P < 0.05; Fig. 7F, left and right, ). Slopes of the linear region of f-I relationships for both 1st ISI and steady-state also were increased by 50 µM 5-HT under raised [Ca2+]o (n = 3) and 200 µM 5-HT under normal [Ca2+]o (n = 3). In all neurons that were examined on the f-I relationships, percent increases in the f-I slope for 1st ISI and steady-state after 5-HT application were correlated significantly with percent decrease in the mAHP amplitude (1st ISI: R2 = 0.53, n = 19, P <0.05; steady state: R2 = 0.50, n = 19, P <0.05). These results suggest that the 5-HT-induced attenuation of the mAHP amplitude likely leads to an increase in the f-I slope in rat JCMNs as reported in other motoneurons (Berger et al. 1992; Hsiao et al. 1997; Talley et al. 1997).



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Fig. 7. Effects of 5-HT on repetitive discharge characteristics. A: repetitive firing of a CLMN induced by 1-s constant current pulse before (left) and after (right) 50 µM 5-HT application to normal perfusate. B: repetitive firing of a DLMN before (left) and after (right) 200 µM 5-HT application under raised [Ca2+]o. C and E: same neurons in A and B, respectively. Compared with neuron in C, marked suppression of mAHP was observed after application of 200 µM 5-HT under raised [Ca2+]o (E). D: frequency-current (f-I) relationship for first interspike interval (1st ISI) (left) and steady-state (right) firing in same neuron in A before and after 5-HT application. F: f-I relationship for 1st ISI (left) and steady-state (right) firing in same neuron in B before and after 5-HT application. Lines in D and F are the slopes of linear regions of f-I relationship and were determined by eye. Note increases in the f-I slopes for 1st ISI and steady state after application of 200 µM 5-HT under raised [Ca2+]o (F, left and right).


    DISCUSSION
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METHODS
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Mechanisms for 5-HT-induced change in the afterpotentials

It has been proposed that the 5-HT-induced suppression of the mAHP may be a property of neonatal but not juvenile or adult motoneurons (Talley et al. 1997). Consistent with this previous observation, 50 µM 5-HT under normal [Ca2+]o (2 mM) caused membrane depolarization and an increase in input resistance but did not reduce the mAHP amplitude in juvenile rat JCMNs. However, we demonstrated that even in juvenile motoneurons 5-HT suppresses the mAHP when it is applied either under raised [Ca2+]o from 2 to 6 mM or at a high concentration (100 or 200 µM).

We previously reported that N-type Ca2+ currents activate apamin-sensitive calcium-activated potassium channels (SK channels) responsible for generating the mAHP in trigeminal motoneurons (Kobayashi et al. 1997). Thus it might be possible that 5-HT decreases Ca2+ influx through N-type Ca2+ channels. High-voltage-activated Ca2+ currents including N-type currents have been reported to be inhibited by 5-HT through activation of 5-HT1A receptors in pyramidal neurons (Foehring 1996), hypothalamic neurons (Koike et al. 1994; Rhee et al. 1996), dorsal (Penington and Kelly 1990), and caudal raphe neurons (Bayliss et al. 1997). In addition to those reports Bayliss et al. (1995) have suggested that 5-HT decreased N- and P-type Ca2+ currents responsible for the mAHP through activation of 5-HT1A receptors in neonatal hypoglossal motoneurons. However, in the present study, inhibition of 5-HT1A receptors by NAN-190 did not alter 5-HT-induced inhibition of the mAHP. Talley et al. (1997) have shown that expression of 5-HT1A receptor greatly decreases in rat hypoglossal motoneurons with maturation and 5-HT did not inhibit the mAHP in juvenile animals. It is also possible that 5-HT1A receptor might be expressed to a small extent in juvenile JCMNs, as was the case with juvenile rat hypoglossal motoneurons. Furthermore a membrane-delimited G-protein pathway has been shown repeatedly to mediate the inhibition of N-type Ca2+ channels by 5-HT in various vertebrate neurons (Anwyl 1991; Foehring 1996; Penington et al. 1991) as well as hypoglossal motoneurons (Bayliss et al. 1995). Suppression of N-type Ca2+ channels by neurotransmitter through the membrane-delimited pathway was shown to be independent of soluble intracellular messengers (Hille 1994). On the other hand, we found that 5-HT-induced attenuation of the mAHP could be mimicked by 8-Bromo-cAMP, potentiated by Ro 20-1724, and blocked by H89, suggesting that a cAMP/PKA cascade is involved in 5-HT-induced attenuation of the mAHP. Therefore it is likely that 5-HT-induced suppression of the mAHP in JCMNs is not mediated mainly by the inhibition of N-type Ca2+ channels through a membrane-delimited G-protein pathway. However, we cannot exclude the possibility that PKA might inhibit N-type Ca2+ channels because PKA was reported to reduce N-type Ca2+ channels indirectly in rat neostriatal neurons (Surmeier et al. 1995).

As calcium-dependent potassium channels are known to be important targets for modulation by protein phosphorylation (Levitan 1994), 5-HT might have reduced the mAHP amplitude through PKA-dependent phosphorylation of SK channels. In CA1 hippocampal pyramidal neurons, activation of 5-HT4 receptors elicits reduction in the slow AHP through a cAMP/PKA cascade (Torres et al. 1995). However, it was suggested in these cells that 5-HT reduces the slow AHP by inhibiting calcium-induced calcium release (CICR) because Ca2+ increase due to CICR triggered by Ca2+ influx may activate K+ channels responsible for the slow AHP (Torres et al. 1996). The mAHP in rat trigeminal motoneurons peaks rapidly in amplitude (<30 ms), lasts 50-100 ms after even a burst of action potentials (Fig. 7, A and B) and is apamin-sensitive (Kobayashi et al. 1997), whereas the slow AHP in CA1 pyramidal neurons has a slow rising phase, lasts several seconds, and is apamin-insensitive (Sah 1996). Furthermore blockade of 5-HT4 receptors by SB 203186 did not alter 5-HT-induced attenuation of the mAHP. Thus the mAHP in JCMNs appears to be generated in a different way from the slow AHP in CA1 pyramidal neurons. It is most likely that inflowing Ca2+ directly gates SK channels leading to the generation of the mAHP (Sah 1996; Schwindt et al. 1992). 5-HT receptor(s) other than 5-HT4 receptors might be involved in the effects on the mAHP, although we could not determine which subtype of 5-HT receptors mediates an increase in cAMP. 5-HT6 and 5-HT7 receptors were reported to activate adenylyl cyclase (Hoyer et al. 1994), and activation of both or either receptor(s) might increase intracellular cAMP and activate PKA, leading to phosphorylation of SK channels.

It has been shown that 5-HT enhances the inward rectifier current activated by membrane hyperpolarization (Ih) in motoneurons (Hsiao et al. 1997; Larkman and Kelly 1992; Takahashi and Berger 1990), and an increase in Ih is suggested to decrease the mAHP amplitude (Schwindt et al. 1988; Spain 1994; Spain et al. 1987). Thus it is possible that an enhancement of Ih by 5-HT might result in a decrease in the mAHP amplitude. However, even in the presence of 5-HT the activation of Ih by the mAHP could be small because the membrane potential of the mAHP peak was only 3-7 mV lower than resting membrane potential (~67 mV). Ih was reported to be activated around -70 mV with a potential for half-maximal activation of -86 to -88 mV in the presence of 5-HT in guinea pig trigeminal motoneurons (Hsiao et al. 1997) and rat facial motoneurons (Larkman and Kelly 1992). Therefore an enhancement of Ih by 5-HT is less likely to be involved in 5-HT-induced suppression of the mAHP under both normal and raised [Ca2+]o.

In accordance with suppression of the mAHP, the ADP amplitude was enhanced when 5-HT was applied under raised [Ca2+]o or applied at a high concentration. However, the ADP and the mAHP were differentially affected by 5-HT when [Ca2+]o was raised. Under raised [Ca2+]o the mAHP was suppressed by 5-HT in a dose-dependent manner, whereas the enhancement of the ADP by 5-HT was not dose-dependent. Furthermore the mAHP was similarly suppressed by 8-Bromo-cAMP and PMA, whereas the ADP was enhanced only by 8-Bromo-cAMP but not by PMA (compare Figs. 4A and 5A). Therefore the increase in the ADP amplitude might not be simply explained by 5-HT-induced suppression of the mAHP alone. 5-HT is most likely to increase Ca2+ currents responsible for the generation of the ADP; however, further studies will be necessary to clarify the mechanisms for this enhancement.

Involvement of PKA and PKC in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca2+]o

Because PKC activation induced by PMA suppressed the mAHP and inhibition of PKC by chelerythrine application blocked the attenuation of the mAHP induced by 50 µM 5-HT under raised [Ca2+]o, PKC activation is likely to be involved in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca2+]o. On the other hand, intracellular Ca2+ was shown to stimulate some isoforms of adenylyl cyclase through calmodulin (Cooper et al. 1995). Thus a transient increase in [Ca2+]i generated by an action potential might have stimulated adenylyl cyclase and also resulted in an enhancement of 5-HT-induced attenuation of the mAHP. However, the contribution of this pathway to the enhancement of the mAHP attenuation should be minor because the percent decrease in the AHP amplitude after application of 50 µM 5-HT under raised [Ca2+]o was small in the presence of chelerythrine.

As mentioned in the preceding text, a cAMP/PKA cascade is involved in 5-HT-induced attenuation of the mAHP. Because H89 almost completely blocked PMA-induced attenuation of the mAHP, PKA activation is likely to be required for PKC action on the mAHP. Furthermore H89 greatly reduced the attenuation of the mAHP induced by 200 µM 5-HT, suggesting that PKA activation appears to be the key component in 5-HT-induced attenuation of the mAHP. Therefore it can be assumed that through activation of PKA, PKC might inhibit Ca2+-activated K+ channels responsible for the mAHP, as suggested in enteric neurons (Pan et al. 1997). PKC has been shown to increase the activity of adenylyl cyclases and the level of cAMP (reviewed by Cooper et al. 1995; Pieroni et al. 1993). If this is the case, a transient increase in [Ca2+]i generated by an action potential under raised [Ca2+]o might stimulate PKC, leading to an increase in PKA activity through elevation of intracellular cAMP when 5-HT was applied. This increased activity of PKA might phosphorylate more Ca2+-activated K+ channels and result in more suppression of the mAHP. Therefore it is likely that PKC-dependent PKA activation might be involved in the enhancement of 5-HT-induced attenuation in the mAHP under raised [Ca2+]o.

Role of 5-HT-induced modulation of afterpotentials for firing patterns

Application of 50 µM 5-HT to normal perfusate shifted the f-I relationships for both 1st ISI and steady-state to the left, however, it did not change the f-I slope of the linear relationships. Such constancy of the slope after 50 µM 5-HT application is probably due to the unchanged mAHP amplitude because the percent increases in the f-I slopes of the linear relationship for 1st ISI and steady-state after 5-HT application were correlated significantly with the percent decrease in the mAHP amplitude. On the other hand, 200 µM 5-HT added to normal perfusate or 5-HT in combination with high Ca2+ perfusate reduced the mAHP amplitude and increased the f-I slope of the linear relationship as well as shifting the relationship to the left. Similar changes in the mAHP and the f-I slope induced by lower dose (10 µM) of 5-HT were reported in juvenile guinea pig trigeminal motoneurons (Hsiao et al. 1997). The discrepancy of the dose of 5-HT necessary to induce those changes between rat and guinea pig trigeminal motoneurons might be related to species of the motoneuron or the activities of 5-HT reuptake mechanism in the slice (Wang and Dun 1990). The increase in the f-I slope indicates that such serotonergic inputs do not only lower the threshold to induce spike firing but also increase input-output gain of JCMNs, suggesting that small changes in incoming synaptic inputs would produce large alterations in spike frequency output of JCMNs. Under physiological condition, such increase in input-output gain of JCMNs might occur only when JCMNs receive serotonergic inputs under raised [Ca2+]i because a low dose (50 µM) of 5-HT by itself could not attenuate the mAHP amplitude. Elevation of [Ca2+]i could be induced via Ca2+ influx through N-methyl-D-aspartate (NMDA) channels (MacDermott et al. 1986), by release of Ca2+ from intracellular stores that are, for example, caused by activation of some types of metabotropic glutamate receptors (Pin and Duvoisin 1995), or by repetitive spike firing at high-frequency. In fact, activation of NMDA receptors was reported to elicit a Ca2+-dependent increase in cAMP in hippocampal neurons (Chetkovich et al. 1991). Thus it is possible that the relation of incoming synaptic inputs and firing output in JCMNs is modulated by combination of serotonergic and other inputs inducing an increase in [Ca2+]i. Activities of both jaw-closing and -opening muscles are influenced by physical properties of food (Weijs and Dantuma 1981); however, the jaw-closing muscles are likely to be facilitated more readily by peripheral sensory inputs during chewing than the jaw-opening muscles (Hidaka et al. 1997; Inoue et al. 1989). Such serotonergic modulation of activities of JCMNs could contribute to adequate regulation of masticatory force for the properties of food.


    ACKNOWLEDGMENTS

We thank Prof. S. H. Chandler for critically reading this manuscript. We also thank M. Saito for software development.

This study was supported by Grants-in-Aid for Scientific Research 09470402, 09832008, and 09877350 from the Japanese Ministry of Education, Science, and Culture.


    FOOTNOTES

Address reprint requests to: T. Inoue, Dept. of Oral Physiology, Faculty of Dentistry, Osaka University, Yamadaoka 1-8, Suita, Osaka 565-0871, Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 July 1998; accepted in final form 13 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society