Departments of 1Oral Physiology,
2Orthodontics, and 3Oral
Anatomy,
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.
The trigeminal motor nucleus receives a dense
serotonergic input (Kolta et al. 1993 In addition to the depolarization, 5-HT decreases the postspike
medium-duration afterhyperpolarization (mAHP) in juvenile guinea pig
motoneurons (Hsiao et al. 1997 A calcium-activated potassium current is involved in generating
the mAHP in trigeminal motoneurons (Chandler et al.
1994 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 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
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; 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.
), 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.
; 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.
, 1998
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). 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).
View larger version (68K):
[in a new window]
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 M) (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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M
(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 M
from 10.2 ± 0.8 M
, 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.
|
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 M
, 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).
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).
|
|
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.
|
|
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.
|
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,
). 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
).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|