Brain Research 954 (2002) 202–211www.elsevier.com/ locate/bres

Research report

I nvolvement of 5-HT receptors in serotonergic effects on spike7

afterpotentials in presumed jaw-closing motoneurons of ratsa , b c d a*Tomio Inoue , Satsuki Itoh , Satoshi Wakisaka , Satoshi Ogawa , Mitsuru Saito ,

eToshifumi MorimotoaDepartment of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555, Japan

bDepartment of Orthodontics, Faculty of Dentistry, Osaka University, Osaka 565-0871, JapancDepartment of Oral Anatomy, Faculty of Dentistry, Osaka University, Osaka 565-0871, Japan

dDepartment of Removable Prosthodontics, Faculty of Dentistry, Osaka University, Osaka 565-0871, JapaneDepartment of Oral Physiology, Faculty of Dentistry, Osaka University, Osaka 565-0871, Japan

Accepted 12 July 2002

Abstract

Intracellular recordings were obtained from rat presumed jaw-closing motoneurons in slice preparations to investigate the involvementof the serotonin (5-HT ) receptors in serotonergic inhibition of the postspike medium-duration afterhyperpolarization (mAHP) and7 7

enhancement of the afterdepolarization (ADP). 5-HT-induced suppression of the mAHP and enhancement of the ADP were mimicked byapplication of the 5-HT receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and antagonized by the 5-HT1A / 7 2 / 6 / 7

receptor antagonist clozapine, whereas the 5-HT receptor agonista-methyl-5-hydroxytryptamine (a-methyl-5-HT) did not affect the2

mAHP and ADP. 8-OH-DPAT-induced attenuation of the mAHP and enhancement of the ADP were also antagonized by clozapine andanother 5-HT receptor antagonist ritanserin, whereas the 5-HT receptor antagonist pindolol failed to block the 8-OH-DPAT-2 / 6 / 7 1A

induced effects on the mAHP and ADP. 8-OH-DPAT-induced suppression of the mAHP and enhancement of the ADP were alsoantagonized by a protein kinase A (PKA) inhibitor H89, whereas 8-OH-DPAT could inhibit the mAHP and enhance the ADP in thepresence of a protein kinase C (PKC) inhibitor chelerythrine. The 8-OH-DPAT-induced suppression of the mAHP was enhanced under

21raised [Ca ] and this enhancement was reduced by chelerythrine. It is suggested that the 5-HT receptors are involved in 5-HT-inducedo 7

attenuation of the mAHP and enhancement of the ADP through activation of PKA, and the attenuation of mAHP through the 5-HT721receptors is enhanced under raised [Ca ] by PKC activation.o

2002 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters; Modulators; Transporters; and Receptors

Topic: Serotonin receptors

Keywords: 5-HT ; Trigeminal motoneuron; Serotonin; Afterhyperpolarization; Afterdepolarization; Clozapine7

1 . Introduction have previously shown that serotonin (5-HT) inhibits themAHP and enhances the ADP through cAMP-dependent

In trigeminal motoneurons, action potentials are fol- PKA activation, leading to an increase in the excitability oflowed by afterdepolarization (ADP) and postspike rat jaw-closing motoneurons (JCMNs) [12]. It has beenmedium-duration afterhyperpolarization (mAHP), both of proposed that 5-HT-induced attenuation of the AHP iswhich are important factors for affecting firing of trigemi- mediated by the 5-HT receptors in neonatal rat hypo-1A

nal motoneurons [11,12,15]. The trigeminal motor nucleus glossal motoneurons [5,28] or by the 5-HT receptors4

receives a dense serotonergic input [16,22,25], and we through a cAMP/PKA cascade in hippocampal neurons[2,29]. However, neither 5-HT antagonist NAN-190 nor1A

5-HT antagonist SB 203186 affected 5-HT-induced at-4*Corresponding author. Tel.:181-3-3784-8159; fax:181-3-3784-tenuation of the mAHP and enhancement of the ADP in8161.

E-mail address: inouet@dent.showa-u.ac.jp(T. Inoue). JCMNs [12].

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03286-9

203T. Inoue et al. / Brain Research 954 (2002) 202–211

The 5-HT receptors were reported to activate adenylyl were transferred to an interface-type chamber. The record-7

cyclase [10], and activation of the 5-HT receptors was ing chamber was continuously perfused with N-ACSF at a7

recently reported to decrease the AHP amplitude in rat rate of 1–1.5 ml /min, and humidified 95% O –5% CO2 2

CA3 hippocampal pyramidal cells [3]. In the present study, flowed over the slice. All experiments were performed atwe investigated whether the 5-HT receptors are involved 3261 8C. Intracellular recordings were obtained with glass7

in 5-HT-induced attenuation of the mAHP and enhance- microelectrodes (1.2 mm O.D., 20–40 MV) (Sutter Instru-ment of the ADP of presumed jaw-closing motoneurons ments) filled with 2 M KCl and 0.05 M Tris buffer (pH(pJCMNs) in juvenile rat slice preparations containing the 7.6). The trigeminal motor nucleus was visible in the slicetrigeminal motor nucleus by the use of the intracellular through the stereomicroscope (Leica, Wild M8) with a ringrecording method. We suggest that activation of the 5-HT light unit (Volpi, Intralux 6000). Following adequate7

receptors induces attenuation of the mAHP amplitude and adjustment of a ring polarizer set with a glass analyzer inan increase in the ADP amplitude through activation of the ring light unit, the electrode was confidently inserted

21PKA. When [Ca ] was increased from 2 to 6 mM, the into the dorsolateral portion of the nucleus in the slice [12]o

attenuation of the mAHP amplitude was enhanced through where motoneurons innervating jaw-closing muscles arePKC activation. Preliminary results of this study were located [19,21]. Since 93% of the neurons recorded fromreported previously in abstract forms [13,14]. the dorsolateral portion of the trigeminal motor nucleus

were located within a cluster of jaw-closing motoneuronsin the previous study, we presumed that neurons recorded

2 . Materials and methods from the dorsolateral portion of the trigeminal motornucleus are jaw-closing motoneurons (pJCMNs) (see Ref.

2 .1. Slice preparation [12]). Membrane potentials were recorded using an Axo-clamp 2B amplifier (Axon Instruments) in either bridge or

All experiments were performed with the approval of discontinuous current-clamp (DCC) mode. During DCCthe Osaka University Faculty of Dentistry Intramural recordings, 2–5 kHz sampling rate was employed at a 30%Animal Care and Use Committee. Transverse brainstem duty cycle. The head stage output was monitored on aslices (450mm) including the trigeminal motor nucleus separate oscilloscope to ensure proper capacitance adjust-were prepared from 86 juvenile Sprague–Dawley rats (3–6 ment and adequate settling of the microelectrode. When theweeks old) as previously described [12]. Animals were membrane potential changed during or after application ofanesthetized with ketamine HCl (150 mg/kg i.m.) and a drug, the membrane potential was clamped manually tochlorpromazine HCl (12.5 mg/kg i.m.). Following decapi- the resting potential level in control by injecting constanttation, the brain was excised rapidly and slices were cut on hyperpolarizing or depolarizing current to evaluate thea microslicer (DTK-1500, Dosaka) in cold modified artifi- effects of the drug, except for measuring a change in thecial cerebrospinal fluid (M-ACSF). Slices were then membrane potential after drug application. The membraneincubated in a holding chamber containing normal artificial potential and current were digitized and stored on acerebrospinal fluid (N-ACSF). The composition of the computer hard disk using software (Clampex, Axon Instru-N-ACSF was (in mM) 130 NaCl, 3 KCl, 2 MgCl , 1.25 ments) through an A–D converter and analyzed with the2

NaH PO , 26 NaHCO , 2 CaCl , and 10D-glucose. The use of Clampfit (Axon Instruments) and Excel (Microsoft)2 4 3 2

M-ACSF was made of N-ACSF by replacing 130 mM software. The mAHP and ADP amplitude were measuredNaCl with 260 mM sucrose. In some experiments, the from the resting potential to the most negative and positive

21Ca concentration was raised to 6 mM. 5-Hydroxy- peak, respectively (see Ref. [15]).tryptamine creatinine sulphate (5-HT, 20–200mM; Data obtained from each neuron except for the mem-Sigma), 8-hydroxy-2-(di-n-propylamino)tetralin hydro- brane potential are the mean of five to eight trials. Data arebromide (8-OH-DPAT, 25–200mM; RBI), a-methyl-5- presented as mean6S.E. Comparisons of data before andhydroxytryptamine maleate (a-methyl-5-HT, 50–200mM; during drug application within groups were based onRBI), clozapine (20mM; RBI), ritanserin (10mM; RBI), Student’s pairedt-tests or two-way analysis of variancepindolol (10mM; RBI), H89 (10–20mM; Seikagaku) and (ANOVA). Differences of % change of the ADP or mAHPchelerythrine (10mM; RBI) were added directly to the amplitude caused by drug application between groups wereperfusate. To evaluate effects of the 5-HT receptor antago- analyzed by Student’st-tests, one-way or two-waynists or protein kinase inhibitors, slices were incubated ANOVA. ANOVA was followed by post hoc Newman–with one of these drugs for 1.5–2.5 h and then intracellular Keuls multiple comparison tests when justified. Prob-recordings were performed in the presence of the same abilities,0.05 were considered significant.dose of the drug as used during preincubation.

2 .2. Recording 3 . Results

After 2–10 h incubation in the holding chamber, slices Since no selective ligand for the 5-HT receptors was7

T. Inoue et al. / Brain Research 954 (2002) 202–211204

Fig. 1. Effects of clozapine on 5-HT-induced attenuation of mAHP and enhancement of ADP. A: superimposed traces of a pJCMN before and after bathapplication of 200mM 5-HT. Inhibition of mAHP and enhancement of ADP (inset) were observed. B: superimposed traces of a pJCMN before and afterapplication of 200mM 5-HT in the presence of 20mM clozapine. Clozapine antagonized 5-HT-induced effects of mAHP and ADP. C: summary of theeffects of 200mM 5-HT, and addition of 200mM 5-HT and 20mM clozapine on mAHP and ADP.Error bars: standard error associated with each group.‡: P,0.01 versus control. * and **:P,0.05 andP,0.01 between groups, respectively. Each trace in A and B is the average of five records. Dotted linesin A and B indicate resting membrane potential in control.

commercially available, we tested several agonists and of 5-HT on the mAHP and ADP. 8-OH-DPAT dose-antagonists for 5-HT receptor subtypes to evaluate the dependently reduced the mAHP amplitude of pJCMNsinvolvement of the 5-HT receptors in serotonergic effects (Fig. 2D). Two-way ANOVA showed that 8-OH-DPAT7

on the mAHP and ADP. To investigate whether a blockade significantly suppressed the mAHP (n528, P,0.001) andof the 5-HT receptor activity reduces 5-HT-induced the post hoc Newman–Keuls test revealed that a high dose7

attenuation of the mAHP, we first examined the effects of (100 or 200mM) of 8-OH-DPAT significantly inhibited theclozapine, the 5-HT receptor antagonist. As we previ- mAHP (Fig. 2B, D and Table 2), whereas a low dose (252 / 6 / 7

ously reported [12], 5-HT (200mM) by itself reduced the or 50mM) of 8-OH-DPAT did not significantly affect themAHP amplitude by 31.266.6% (n510) and increased the mAHP (Fig. 2A, D and Table 2). The percent reduction ofADP amplitude by 23.366.0% (n56) in rat JCMNs (Fig. the mAHP induced by 200mM 8-OH-DPAT was sig-1A). By contrast, in the presence of 20mM clozapine, 200 nificantly larger than that induced by 100mM 8-OH-DPATmM 5-HT did not affect either mAHP (P.0.3) or ADP (P,0.001, one-way ANOVA;P,0.001, Newman–Keulsamplitude (P.0.3) in pJCMNs (Fig. 1B, Table 1). Rela- test). 8-OH-DPAT also significantly affected the ADPtive changes of both mAHP and ADP amplitude after (n520, P,0.01, two-way ANOVA). A 100 or 200mMco-application of 5-HT and clozapine were significantly 8-OH-DPAT significantly enhanced the ADP (Fig. 2B, Esmaller than those after application of 5-HT by itself and Table 2), whereas 50mM 8-OH-DPAT did not affect(mAHP: P,0.01, ADP:P,0.05; Fig. 1C). the ADP (Fig. 2A, E and Table 2). In contrast to the

Subsequently, we examined whether activation of the effects of 8-OH-DPAT on the mAHP, % increase induced5-HT receptors by application of 25–200mM 8-OH- by 100 mM 8-OH-DPAT was not different from that7

DPAT, the 5-HT receptor agonist, mimics the effects induced by 200mM 8-OH-DPAT (P.0.3, one-way1A / 7

Table 1Effects of serotonin antagonists on 5-HT-induced or 8-OH-DPAT-induced change of the mAHP and ADP in pJCMNs

Antagonist Agonist mAHP amplitude (mV) ADP amplitude (mV)

Before After % Change n Before After % Change n

Clozapine 5-HT 4.561.0 4.461.0 21.066.9 5 9.861.5 9.661.3 0.466.0 520 mM 200 mMClozapine 8-OH-DPAT 3.660.4 3.760.6 0.767.4 6 5.861.4 6.561.2 29.1615.0 620 mM 200 mMRitanserin 8-OH-DPAT 3.860.6 3.260.5* 211.764.4 9 11.462.4 11.261.9 13.9611.6 920 mM 200 mMPindolol 8-OH-DPAT 4.560.6 2.860.6** 236.965.0 6 9.862.0 12.661.9** 39.0614.3 620 mM 200 mM

Values are means6S.E. Differences are significant at: *P,0.05 and **P,0.01.

205T. Inoue et al. / Brain Research 954 (2002) 202–211

Fig. 2. Effects of the 5-HT receptor agonists on mAHP and ADP. A, B and C: superimposed traces of pJCMNs before and after application of 50mM (A)and 200mM (B) 8-OH-DPAT, and 200mM a-methyl-5-HT (C). 200mM 8-OH-DPAT inhibited mAHP and enhanced ADP, whereas 200mMa-methyl-5-HT affected neither mAHP nor ADP. D and E: summary of the effects of 5-HT, 8-OH-DPAT anda-methyl-5-HT on mAHP (D) and ADP (E).Error bars: standard error associated with each group. * and **:P,0.05 andP,0.01 versus control, respectively. Each trace in A, B and C is the averageof five records. Dotted lines in A, B and C indicate resting membrane potential in control.

ANOVA). We then compared the effects of 8-OH-DPAT on and enhancement of the ADP induced by 8-OH-DPAT arethe mAHP and ADP with those of 5-HT. The percent of mediated by activation of the 5-HT receptors, we com-7

change of the mAHP and ADP induced by 8-OH-DPAT pared the effects of antagonists of the 5-HT receptors onwas not significantly different from that induced by 5-HT the 8-OH-DPAT-induced effects of the mAHP and ADP. In(mAHP: P.0.05; ADP: P.0.2, two-way ANOVA, Fig. the presence of 20mM clozapine, 200mM 8-OH-DPAT2D and E). Since clozapine has activity at the 5-HT had no significant effects on either mAHP (P.0.3) or ADP2

receptors, we examined the effects ofa-methyl-5-HT, the (P.0.2) amplitude (Fig. 3A, Table 1). When 20mM5-HT agonist. In contrast to the effect of 8-OH-DPAT, ritanserin, another 5-HT receptor antagonist, was co-2 2 / 6 / 7

a-methyl-5-HT (50–200mM) altered neither mAHP (P. applied with 200mM 8-OH-DPAT, the mAHP amplitude0.4, two-way ANOVA) nor ADP (P.0.3, two-way was decreased only by 12% (P,0.05), and the ADPANOVA) (Fig. 2C, D, E and Table 2), although application amplitude was not significantly altered (P.0.4) (Fig. 3Bof even 50mM a-methyl-5-HT induced membrane depo- and D, Table 1). Percent reduction of the mAHP amplitudelarization by 2.260.8 mV (n56, P,0.05) and an increase after 8-OH-DPAT application was significantly smaller inin input resistance by 1.960.8 MV (n56, P,0.05). the presence of clozapine (P,0.001, one-way ANOVA;

To further investigate whether attenuation of the mAHP P,0.001, Newman–Keuls test) or ritanserin (P,0.001,

T. Inoue et al. / Brain Research 954 (2002) 202–211206

Table 2Effects of serotonin agonists on the mAHP and ADP of pJCMNs

mAHP amplitude (mV) ADP amplitude (mV)

Drug Before After % Change n Before After % Change n

8-OH-DPAT 5.361.1 4.960.9 27.561.8 3 6.4 6.4 0.3 225 mM8-OH-DPAT 5.461.1 5.160.7 26.162.7 8 7.462.1 8.162.6 6.465.2 450 mM8-OH-DPAT 4.560.6 3.660.5** 219.662.0 7 9.461.4 11.161.3* 23.769.6 7100 mM8-OH-DPAT 3.860.5 2.160.4** 246.064.7 10 15.160.9 17.060.9* 13.765.6 7200 mMa-Methyl-5-HT 6.161.3 6.461.4 3.964.1 6 7.562.7 7.262.9 210.368.7 350 mMa-Methyl-5-HT 5.360.5 5.060.6 25.665.4 5 10.260.7 10.960.6 6.965.1 4100 mMa-Methyl-5-HT 4.960.6 5.460.7 11.364.7 6 10.261.8 10.861.9 6.769.1 5200 mM

Values are means6S.E. Differences are significant at: *P,0.05 and **P,0.01.

Newman–Keuls test) than that of the mAHP after 8-OH- chelerythrine, 8-OH-DPAT significantly reduced theDPAT application by itself (Fig. 3D). In contrast to the mAHP amplitude (P,0.01) and increased the ADP am-effects of clozapine and ritanserin, pindolol, the 5-HT plitude (P,0.05) (Fig. 4B, Table 3). Percent decrease of1A

receptor antagonist, did not antagonize the effects of 8- the mAHP after co-application of chelerythrine and 8-OH-OH-DPAT. In the presence of 20mM pindolol, 200mM DPAT was significantly larger than that after co-applica-8-OH-DPAT significantly decreased the mAHP amplitude tion of H89 and 8-OH-DPAT (P,0.01, Newman–Keuls(P,0.005) and significantly increased the ADP amplitude test), although it was slightly smaller than % decrease of(P,0.005) (Fig. 3C and D, Table 1). Percent reduction of the mAHP by application of 8-OH-DPAT by itself (P,

the mAHP amplitude after co-application of pindolol and 0.05, Newman–Keuls test) (Fig. 4C, Table 2 and 3). On200 mM 8-OH-DPAT was not different from the % the other hand, % increase of the ADP amplitude was notreduction after 200mM 8-OH-DPAT application by itself significantly different among applications of 8-OH-DPAT(P.0.2, Newman–Keuls test, Fig. 3D). However, % by itself, co-application of H89 and 8-OH-DPAT, andincrease of the ADP amplitude was not significantly co-application of chelerythrine and 8-OH-DPAT (P.0.1,different among 5-HT antagonists (P.0.4, one-way one-way ANOVA). These results suggest that 8-OH-DPATANOVA). These results suggest that 5-HT receptor acti- could inhibit the mAHP and enhance the ADP even after7

vation is likely involved in 5-HT-induced attenuation of the blockade of PKC activity. Therefore, it is most likelythe mAHP and enhancement of the ADP. that activation of PKA is involved in the effects of 8-OH-

As we previously reported, 5-HT-induced attenuation of DPAT on the mAHP and ADP.the mAHP amplitude is mediated by cAMP-dependent We also reported that 5-HT-induced attenuation of the

21activation of PKA [12]. Therefore, we examined whether mAHP in JCMNs was enhanced under raised [Ca ] , ando

activation of the 5-HT receptors results in suppression of activation of PKC is likely involved in this enhancement7

the mAHP through cAMP/PKA cascade. In the presence [12]. Therefore, we investigated whether the mAHP at-of H89 (10 mM), a selective inhibitor of PKA, 200mM tenuation induced by activation of the 5-HT receptors is7

218-OH-DPAT altered neither the mAHP (P.0.07) nor ADP enhanced through activation of PKC under raised [Ca ]o21(P.0.5) (Fig. 4A, Table 3). Percent change of mAHP after from 2 to 6 mM. Slices were held under raised [Ca ] foro

co-application of H89 and 8-OH-DPAT was about one- 1–2 h and then intracellular recordings were performed.sixth of the % change after 8-OH-DPAT application by Under this condition 8-OH-DPAT (50 or 100mM) de-itself (P,0.001, one-way ANOVA;P,0.001, Newman– creased the mAHP amplitude (P,0.001, two-wayKeuls test) (Fig. 4C, Tables 2 and 3). Since theK value of ANOVA) and even 50mM 8-OH-DPAT invariably at-i

H89 for PKC which was obtained from purified enzyme tenuated the mAHP amplitude by about 17% (P,0.001,assay was reported to be 31.7mM [6], it is possible that 10 Newman–Keuls test) (Fig. 5A, Table 3). Furthermore,mM H89 might block 8-OH-DPAT-induced attenuation of two-way ANOVA revealed that % decrease of the mAHP

21the mAHP by inhibiting PKC activity. To further examine after 8-OH-DPAT application under raised [Ca ] waso21the involvement of PKA in 8-OH-DPAT-induced attenua- larger than the % decrease under normal [Ca ] (P,o

tion of the mAHP, the effect of 8-OH-DPAT was tested 0.005); the Newman–Keuls test showed that % decrease of21when PKC activity was blocked by chelerythrine, a the mAHP after 50mM 8-OH-DPAT under raised [Ca ]o21selective inhibitor of PKC. Even in the presence of 10mM was significantly larger than that under normal [Ca ]o

207T. Inoue et al. / Brain Research 954 (2002) 202–211

Fig. 3. Effects of 5-HT receptor antagonists on 8-OH-DPAT-induced effects on mAHP and ADP. A, B and C: superimposed traces of pJCMNs before andafter application of 200mM 8-OH-DPAT in the presence of 20mM clozapine (A), 20mM ritanserin (B) and 20mM pindolol (C). D: summary of theeffects of 8-OH-DPAT, the addition of clozapine to 8-OH-DPAT, the addition of ritanserin to 8-OH-DPAT and the addition of pindolol to 8-OH-DPAT.Error bars: standard error associated with each group. † and ‡:P,0.05 andP,0.01 versus control, respectively. **:P,0.01 between groups. Each tracein A, B and C is the average of five records. Dotted lines in A, B and C indicate resting membrane potential in control.

21(P,0.01) (Fig. 5C). Application of 50 or 100mM 8-OH- mAHP amplitude under raised [Ca ] (P.0.4) (Fig. 6A,o21DPAT under raised [Ca ] significantly increased the Table 3), and % decrease after co-application of 8-OH-o

ADP amplitude (P,0.001, two-way ANOVA; 50mM: DPAT and chelerythrine was significantly smaller than thatP,0.005, 100mM: P,0.001, Newman–Keuls test, Fig. after 8-OH-DPAT application by itself (P,0.05) (Fig. 6B,5A, B and Table 3). However, two-way ANOVA did not Tables 2 and 3). Therefore, it is most likely that PKCshow a significant difference between % increase of the activation is involved in the enhancement of 8-OH-DPAT-

21ADP after 8-OH-DPAT application under normal and induced attenuation of the mAHP under raised [Ca ] .o21raised [Ca ] (P.0.08). These results suggest that theo

mAHP attenuation induced by the 5-HT receptor activa-721tion is enhanced under raised [Ca ] . Subsequently, we 4 . Discussiono

examined whether this enhancement of the mAHP attenua-tion is mediated by activation of PKC. In the presence of Our previous study has shown that 5-HT inhibits the10 mM chelerythrine, 8-OH-DPAT did not decrease the mAHP and enhances the ADP through PKA activation in

T. Inoue et al. / Brain Research 954 (2002) 202–211208

Fig. 4. Involvement of PKA activation in 8-OH-DPAT-induced effects on mAHP and ADP. A: superimposed traces of a pJCMN before and afterapplication of 200mM 8-OH-DPAT in the presence of 10mM H89. H89 antagonized 8-OH-DPAT-induced attenuation of mAHP and enhancement of ADP.B: superimposed traces of a pJCMN before and after application of 200mM 8-OH-DPAT in the presence of 10mM chelerythrine. 8-OH-DPAT couldinhibit mAHP and enhance ADP in the presence of chelerythrine. C: summary of the effects of addition of H89 or chelerythrine to 8-OH-DPAT.Errorbars: standard error associated with each group. † and ‡:P,0.05 andP,0.01 versus control, respectively. * and **:P,0.05 andP,0.01 betweengroups, respectively. Each trace in A–C is the average of five records. Dotted lines in A–C indicate resting membrane potential in control.

rat JCMNs [12]. The inhibition of the mAHP and enhance- for 8-OH-DPAT-induced attenuation of the mAHP arement of the ADP were most likely made through activation different from the mechanisms for 8-OH-DPAT-inducedof the 5-HT receptors because 8-OH-DPAT attenuated the enhancement of the ADP.7

21mAHP and increased the ADP amplitude through PKA In rat trigeminal motoneurons, a Ca influx through21activation, and the effects of 8-OH-DPAT on the mAHP N-type Ca channels activates apamin-sensitive calcium-

and ADP were antagonized by clozapine or ritanserin but dependent potassium channels (SK channels) responsiblenot by pindolol. Because suppression of the mAHP by for generating the mAHP [15]. Thus, activation of theapamin increases the ADP amplitude, the early phase of 5-HT receptors might lead to inhibition of SK channels7

21the mAHP is likely to somewhat overlap the ADP [15]. and/or N-type Ca channels through PKA activationThus, it is possible that activation of the 5-HT receptors although phosphorylation of SK channels is more likely to7

might indirectly increase the ADP amplitude by inhibiting be responsible for inhibition of the mAHP because cal-the mAHP. However, 8-OH-DPAT suppressed the mAHP cium-dependent potassium channels are important targetsin a dose-dependent manner, whereas the enhancement of for modulation by phosphorylation [18]. On the other

21 21the ADP by 8-OH-DPAT was not dose-dependent sug- hand, a Ca influx through T- and P-type Ca channelsgesting that 8-OH-DPAT differentially affected the mAHP contributes to generation of the ADP in the rat trigeminaland ADP. Therefore, it is most likely that the mechanisms motoneurons [15]. Because activation of PKA has been

Table 3Effects of serotonin antagonists on 5-HT-induced or 8-OH-DPAT-induced change of the mAHP and ADP in pJCMNs

Inhibitor Agonist mAHP amplitude (mV) ADP amplitude (mV)

Before After % Change n Before After % Change n

Normal RingerH89 8-OH-DPAT 5.360.7 4.960.7 28.063.7 7 8.961.5 8.961.5 0.764.5 710 mM 200 mMChelerythrine 8-OH-DPAT 3.660.7 2.560.5** 228.065.4 8 10.761.8 12.361.5* 21.069.9 610 mM 200 mM

21High Ca Ringer– 8-OH-DPAT 7.060.7 5.860.6** 217.361.2 8 9.662.0 11.261.9** 24.468.8 7

50 mM– 8-OH-DPAT 5.760.6 4.260.4** 227.563.8 7 8.862.0 11.461.8** 43.9615.8 5

100 mMChelerythrine 8-OH-DPAT 7.161.6 7.061.6 23.667.6 5 20.264.4 20.565.0 29.569.9 310 mM 50 mM

Values are means6S.E. Differences are significant at: *P,0.05 and **P,0.01.

209T. Inoue et al. / Brain Research 954 (2002) 202–211

21Fig. 5. Effects of 8-OH-DPAT under raised [Ca ] from 2 to 6 mM. A and B: superimposed traces of pJCMNs before and after application of 50mM (A)o21 21and 100mM (B) 8-OH-DPAT under raised [Ca ] . C: comparison of the effects of 8-OH-DPAT on mAHP under normal and raised [Ca ] .o o

218-OH-DPAT-induced attenuation of mAHP was enhanced under raised [Ca ] .Error bars: standard error associated with each group. †:P,0.01 versuso

control. *: P,0.05 between groups. Each trace in A and B is the average of five records. Dotted lines in A and B indicate resting membrane potential incontrol.

21reported to increase T-type Ca currents in frog adreno- of the 5-HT receptors. A histological study shows that621cortical cells [17] and NIH 3T3 cells [24], and P-type Ca 5-HT receptor-like immunoreactivity was found in6

currents in rat striatal nerve terminals [9], the 5-HT oculomotor, trigeminal motor and facial nuclei [8]. How-7

receptors might be involved in enhancement of T- and/or ever, the potency of 8-OH-DPAT, which has a low affinity21P-type Ca currents. Therefore, the 5-HT receptors for the 5-HT receptors [10], on the inhibition of the7 6

likely have opposite effects on the ion channels which are mAHP is similar to that of 5-HT. Therefore, contributionresponsible for generation of the mAHP and ADP, through of the 5-HT receptors to attenuation of the mAHP might6

PKA activation in a single JCMN. be minor if the 5-HT receptors are involved in mAHP6

Because clozapine has an affinity for the 5-HT re- attenuation.6

ceptors as well as the 5-HT receptors, it is possible that The 5-HT receptors have been shown to couple posi-7 2

the antagonism of the 5-HT-induced effects on the mAHP tively to phospholipase C which may lead to activation ofand ADP by clozapine might be partly due to a blockade PKC [26]. Activation of PKC by application of phorbol

Fig. 6. Contribution of PKC to 8-OH-DPAT-induced attenuation of mAHP. A: superimposed traces of a pJCMN before and after application of 50mM218-OH-DPAT under raised [Ca ] . B: superimposed traces of a pJCMN before and after application of 50mM 8-OH-DPAT in the presence of 10mMo

chelerythrine. Chelerythrine antagonized the effects of 8-OH-DPAT on mAHP. C: summary of the effects of chelerythrine on 8-OH-DPAT-inducedattenuation of mAHP.Error bars: standard error associated with each group. ‡:P,0.01 versus control. *:P,0.05 between groups. Each trace in A and Bis the average of five records. Dotted lines in A and B indicate resting membrane potential in control.

T. Inoue et al. / Brain Research 954 (2002) 202–211210

12-myristate 13-acetate (PMA) inhibited the mAHP in R eferencesJCMNs [12]. Furthermore, PKC is likely to be involved in

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