Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
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ABSTRACT |
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Katsurabayashi, Shutaro,
Hisahiko Kubota,
Zhi Ming Wang,
Jeong Seop Rhee, and
Norio Akaike.
cAMP-Dependent Presynaptic Regulation of Spontaneous Glycinergic
IPSCs in Mechanically Dissociated Rat Spinal Cord Neurons.
J. Neurophysiol. 85: 332-340, 2001.
Spontaneous miniature glycinergic inhibitory postsynaptic currents
(mIPSCs) in mechanically dissociated rat sacral dorsal commissural
nucleus (SDCN) neurons attached with intact glycinergic presynaptic
nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were
investigated using nystatin-perforated patch and conventional whole
cell recording modes under the voltage-clamp conditions. Trans-ACPD
(tACPD) reversibly reduced the mIPSC frequency without
affecting the mean amplitude. The effect was mimicked by a specific
metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1'S,
2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII
subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory
effects on mIPSC frequency were blocked by the specific antagonists for
mGluRII, -methyl-1-(2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine
and (RS)-
-cyclopropyl-4-phosphonophenylglycine. In the slice
preparation, eIPSC amplitude and mIPSC frequency were decreased
reversibly by L-CCG-I
(10
6 M) and L-AP4
(10
6 M). In
K+-free or K+-free external
solution with Ba2+ and Cs+,
Ca2+-free or Cd2+ external
solution, the inhibitory effect of tACPD on mIPSC frequency was
unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic
glycine release, and prevented the inhibitory action of tACPD on mIPSC
frequency. Sp-cAMP, however, did not prevent the inhibitory action of
tACPD on mIPSC frequency. It was concluded that the activation of
mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway.
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INTRODUCTION |
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Glutamate is one of major
excitatory neurotransmitters in the CNS. The receptors are classified
as ionotropic glutamate receptors (iGluRs) and metabotropic glutamate
receptors (mGluRs) (Nakanishi 1992). The iGluRs mediate
fast synaptic transmission through ligand-gated ion channels, and the
mGluRs are coupled to second-messenger systems that are responsible for
slower synaptic events. Recently eight mGluR subtypes have been cloned
(mGluR1-mGluR8) (Pin and Duvosin 1995
; Riedel
1996
). The mGluRs have been further divided into three main
groups according to their sequence homology, signal transduction
mechanism, and agonist selectivity. The mGluR subgroup I includes
mGluR1 and mGluR5, which are coupled to phosphoinositide hydrolysis
(Abe et al. 1992
). Subgroup II (mGluRII) contains mGluR2 and mGluR3, and subgroup III (mGluRIII) includes mGluR4,
mGluR6-mGluR8. Both mGluRII and mGluRIII are negatively coupled to
adenylyl cyclase (AC) (Tanabe et al. 1992
, 1993
). The
modulations of these mGluRs on synaptic transmission enhance or
suppress the release of inhibitory and excitatory neurotransmitters,
such as GABA and glutamate, from nerve terminals (Glitsch et al.
1996
; Poncer et al. 1995
). Several reports show
that these actions involve multiple mechanisms. For example, mGluRs
inhibit Ca2+ channels or activate the
K+ channels of presynaptic terminals
(Cochilla and Alford 1998
; Takahashi et al.
1996
). Also, the mGluRs have direct effects on the vesicle
releasing mechanism (Poncer et al. 1995
) and elicit Ca2+ release from presynaptic
Ca2+ stores (Peng 1996
).
Most mGluRs, with exception of mGluR6 and mGluR8, are distributed
throughout the spinal cord (Valerio 1997; Watkins
and Evans 1981
). These mGluRs have dual effects on the synaptic
transmission in the spinal cord, potentiating or suppressing the
release of excitatory or inhibitory neurotransmitters (Bond and
Lodge 1995
; Cao et al. 1997
; Jane et al.
1996
; King and Liu 1996
). The sacral dorsal
commissural nucleus (SDCN) of spinal cord surrounding the central canal
receives abundant afferent inputs from both the visceral and somatic
organs. The convergence of the visceral and somatic inputs on neurons
in the SDCN has been demonstrated electrophysiologically and
anatomically (Honda 1985
). Also, the SDCN is known to be
involved in nociceptive, analgesic transmission and autonomic functions of visceral and somatic inputs (Ding et al. 1994
;
Honda 1985
; Vizzard et al. 1995
). In the
SDCN area, glycine is one of the major inhibitory neurotransmitters
(Basbaum 1988
; Yoshimura and Nishi 1995
).
However, the releasing mechanism of glycine from glycinergic
presynaptic terminals projecting to the SDCN neurons is poorly
understood. The intracellular signaling mechanisms underlying the mGluR
modulation of glycine release are also not known.
In the present study therefore, we investigated the mechanism of the mGluR modulation of the spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs). The preparations made from mechanically dissociated rat SDCN neurons attached with native presynaptic boutons were used that combine the advantages of a simple, reduced system while maintaining native synaptic functions. The activation of metabotropic glutamate receptors on the presynaptic glycinergic terminals reduces transmitter release by interfering with cAMP/PKA pathway.
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METHODS |
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Mechanical dissociation
Ten- to 14-day-old Wistar rats were decapitated under
pentobarbital anesthesia. The spinal cord was quickly removed from the vertebral canal and was sliced at a thickness of 400 µm with a microslicer (DTK-1000, Dosaka, Kyoto, Japan). The slices were kept in
the incubation medium saturated with 95% O2-5%
CO2 at room temperature (22-25°C) for 1 h.
Thereafter the slices were transferred into a 35-mm culture dish
(Primaria3801, Becton Dickinson, NJ) and the SDCN of the spinal
cord was identified under a binocular microscope (SMZ-1, Nikon, Tokyo).
A fire-polished glass pipette was touched lightly onto the surface of
the SDCN region and was vibrated horizontally at 3-5 Hz for ~2 min
by apparatus developed by our laboratory. Then the slices were removed
from the dish. The mechanically dissociated SDCN neurons adhered to the
bottom of the dish within 10 min. These neurons, which were dissociated without using any enzymes, retained their original morphological features, including proximal dendritic processes.
Slice preparation
Thirteen- to 15-day-old Wistar rats were decapitated under pentobarbital anesthesia. The spinal cord was quickly removed from the skull and was sliced at a thickness of 230 µm with a microslicer (VT-1000S, Leica, Germany) in cold Na+-free (including sucrose) medium. Then the slices were kept in incubation medium saturated well with 95% O2-5% CO2 at 30-35°C for ~1 h. Thereafter the slices were transferred into a recording chamber, and the SDCN of the spinal cord was identified under an upright microscope (Axioscope, Zeiss, Germany). The bath solution was perfused at 8-10 ml/min.
All experiments conformed the Guiding Principles for the Care and Use of Animals approved by the Council of the physiological Society of Japan, and all efforts were made to minimize the number of animals used and their suffering.
Electrical measurements
Electrical measurements were performed in the dissociated
neurons attached with synaptic boutons using the
nystatin-perforated-patch recording mode and in the slice preparation
using the whole cell patch recording mode under voltage-clamp
conditions. The nystatin-perforated-patch technique but not whole cell
patch technique was applied to dissociated neuron. When the
nystatin-perforated-patch recording mode was applied to the dissociated
neurons, the technique allows a long time recordings not by like
conventional whole cell patch recording mode. However, the conventional
whole cell patch recording mode was used for slice preparation. In this
preparation, either nystatin- or whole cell patch technique is enough
to record cell response for 3 h because the conventional whole cell
patch technique was much easier and convenient to record the responses
from slice preparation. Patch pipettes were made from borosilicate
glass tubes (1.5 mm OD, 0.9 mm ID; G-1.5, Narishige, Tokyo) in two
stages on a vertical pipette puller (PB-7, Narishige). The resistance of the recording electrode was 5-7 M
. The neurons were visualized with phase-contrast equipment on an inverted microscope (Diapot, Nikon). The current and voltage were measured with a patch-clamp amplifier (EPC-7, List-Electronic, Germany), monitored on both an
oscilloscope (Tektronix 5111A, Sony, Tokyo) and a pen recorder (Recti-Horiz 8K, Nippondenki San-ei, Tokyo), and stored on videotapes (PCM-501 ES, Sony). The membrane currents were filtered at 1 kHz (E-3201A Decade Filter, NF Electronic Instruments, Tokyo), and data
were digitized at 4 kHz. The evoked obtained were performed by applying
short (100 µs) voltage pulses at 0.1 Hz through glass pipette
(
: 10 µm), which was placed around central canal and filled with the incubation solution, using the PULSE program on a
Macintosh computer (HEKA). The signals were filtered at 3 kHz and
digitized at 10 kHz.
Data analysis
Events were counted and analyzed using DETECTiVENT (Ankri
et al. 1994) and IGOR PRO software (Wavemetrics, Lake Oswego,
OR), and the time to peak and decay time of individual mIPSCs were analyzed using pCLAMP software (Axon Instruments). Analysis of mIPSCs
was performed with cumulative probability plots. Cumulative amplitude
histograms were compared using the Kolmogorov-Smirnov test for
significant difference (P < 0.05). Numerical values
are provided as means ± SE. Differences in amplitude and
frequency distribution were tested by paired two-tailed
t-test.
Solutions
The ionic composition of the incubation medium was (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 24 NaHCO3, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose. The pH of the incubation medium was adjusted to 7.4 with 95% O2-5% CO2. The ionic composition of the external standard solution was (in mM) 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES. Ca2+-free external solution contained (in mM) 150 NaCl, 5 KCl, 3 MgCl2, 10 glucose, 10 HEPES, and 2 EGTA. The ionic composition of the Na+-free medium was (in mM) 230 sucrose, 2.5 KCl, 1.25 Na2HPO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 30 glucose. The composition of K+-free external solution was (in mM) 147 NaCl, 5 CsCl, 5 BaCl2, 1 MgCl2, 10 glucose, and 10 HEPES. These external solutions were adjusted pH 7.2 with Tris-OH.
In recording mIPSCs, these solutions routinely contained 3 × 107 M tetrodotoxin (TTX)
to block voltage-dependent Na+ channels, 3 × 10
6 M bicuculline to
block the GABAA response, and
10
6 M
6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and
10
5 M
DL
2-amino-5-phosphovaleric acid (DL-AP5) to
block glutamatergic responses. The ionic composition of the internal
(patch pipette) solution for the nystatin-perforated-patch recording
was (in mM) 20 N-methyl-D-glucamine
methanesulfonate, 20 Cs-methanesulfonate, 5 MgCl2, 100 CsCl, and 10 HEPES. The pH of internal
solution was adjusted to 7.2 with Tris-OH. Nystatin was dissolved in
acidified methanol at 10 mg/ml. The stock solution was diluted with
internal solution just before use to a final concentration of 100-200
µg/ml. The ionic composition of the internal (patch pipette) solution for the whole cell patch recording was (in mM) 43 CsCl, 92 Cs-methanesulfonate, 5 TEA-Cl, 2 EGTA, 4 ATP-Mg, and 10 HEPES. The pH
of internal solution was adjusted to 7.2 with Tris-OH.
Drugs
Drugs used in the present study were AP5, bicuculline, CNQX,
EGTA, nystatin, forskolin, 1,9-dideoxy-forskolin, 8Br-cAMP, Sp-cAMP, Rp-cAMP, and -bungarotoxin (Sigma); ATP (Yamasa, Japan); TTX and
strychnine (Wako Pure Chemicals, Japan); and
2-amino-4-phosphonobutyrate (L-AP4), (2S, 1'S, 2'S)-2-(carboxycyclo
propyl) glycine (L-CCG-I),
-methyl-1- (2S, 1'S, 2'S)-2-(carboxycyclo
propyl) glycine (MCCG), and
(RS)-
-cyclopropyl-4-phosphonophenylglycine (CPPG) (Tocris, UK).
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RESULTS |
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Spontaneous glycinergic mIPSCs
The spontaneous postsynaptic currents were recorded from the
acutely dissociated rat SDCN neurons attached with the native presynaptic nerve endings, namely "synaptic bouton preparation." The recording utilized the nystatin-perforated-patch recording mode at
a holding potential (VH) of 60 mV.
Strychnine abolished the spontaneous inhibitory currents in a
reversible and a dose-dependent manner in the presence of 3 × 10
7 M TTX,
10
5 M
2-amino-5-phosphonovaleric acid,
10
6 M CNQX, and
10
6 M bicuculline (Fig.
1A). Since it is reported that
strychnine is also a potent competitive antagonist of nicotinic
acetylcholine receptors (nAChRs) having
7 subunits in the rat
hippocampal neurons (Matsubayashi et al. 1998
), we
examined whether SDCN neurons have nicotinic components or not. Thus
the effect of
-bungarotoxin (
-BTx)
(10
7 M), a selective
antagonist of
7-containing nAChRs, was tested on the spontaneous
postsynaptic current. The
-BTx did not alter the frequency or
amplitude of the mIPSCs, indicating that nicotinic component does not
exist (data not shown).
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Representative spontaneous glycinergic mIPSCs were also investigated at
various VHs (Fig. 1B). The
average current amplitude of the mIPSCs during the 3-min sampling
period was plotted against to each
VHs. The reversal potential of mIPSCs
estimated from the current-voltage (I-V) relationship was
about 10 mV. The value was almost identical to the theoretical
Cl
equilibrium potential
(ECl) of
10.4 mV calculated from Nernst equation using 161 mM
[Cl
]o and 110 mM
[Cl
]i (Fig.
1Bb). These data clearly indicate that the spontaneous miniature postsynaptic currents are glycinergic mIPSCs.
Inhibitory effect of tACPD on glycinergic mIPSCs
The effect of trans-ACPD (tACPD), a metabotropic glutamate
agonist, on glycinergic mIPSCs was examined. In the present study, SDCN
neurons were subdivided to three groups according to the cell size:
i.e., small (<15 µm), middle (15-30 µm), and large (>30 µm).
Among 92 dissociated SCDN neurons in the presence of 105 M tACPD, the mIPSC
frequency decreased in 69 neurons, which were middle- and large-size
neurons. The tACPD increased the mIPSC frequency in 13 small neurons.
There was no change in 10 neurons, which were small- and middle-size
neurons. In the following experiments, therefore we used the
middle-size neurons.
The tACPD (105 M)
reversibly suppressed mIPSC frequency but had no effect on the
distribution of the current amplitudes (Fig. 2, A and C),
indicating that the probability of glycine release decreased. In fact,
the mIPSC frequency significantly decreased to 72.7 ± 1.7% of
control (P < 0.01, n = 69) in the
presence of tACPD, whereas the mean mIPSC amplitude was 100.8 ± 1.3% of control (n = 69; Fig. 2B). Figure
2C indicates the superimposed amplitude histograms of mIPSCs
with (
) or without (
) tACPD for 3 min. The amplitude distribution
of the mIPSCs was skewed toward large-amplitude events. There was a
clear decrease in the number of mIPSC events at all amplitudes in the
presence of tACPD. However, no significant difference in the
distribution of mIPSC amplitudes was detected between control and
tACPD-treated neurons (P = 0.4328 by Kolmogorov-Smirnov test, number of events 297 for control and 187 for tACPD). Figure 2C, inset, shows the superimposed typical mIPSCs with or
without tACPD. The decay time,
, was measured and averaged for 38 individual events in each neuron for control and tACPD-treated neurons.
Both decay times of each group were fitted by a single-exponential function. The means of
values for control and tACPD-treated neurons
were 19.6 ± 1.3 and 18.8 ± 1.1 ms (n = 9),
respectively. There was no significant difference in the time to peak
and
values between control and tACPD-treated neurons. These results suggest that mGluR exists in the glycinergic nerve terminal and exerts
an inhibitory effect on the presynaptic glycine release process without
affecting the sensitivity of postsynaptic glycine receptors.
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Presynaptic mechanism of tACPD in glycinergic nerve terminal
Three kinds of inhibitory mechanisms could account for the effect
of tACPD on glycine release. First, tACPD might suppress Ca2+ influxes through voltage-dependent
Ca2+ channels (VDCCs) in the nerve terminal,
resulting in decrease in exocytosis (Morishige et al.
1996; Takahashi et al. 1996
). Second, tACPD
might open K+ channels, thus hyperpolarizing the
presynaptic membrane and reducing synaptic release (Cochilla and
Alford 1998
). Third, the agonist might activate
intracellular messenger pathways that inhibit the release mechanism in
presynaptic terminals (Trudeau et al. 1996a
,b
).
We, therefore examined the possible contribution of presynaptic VDCCs in the mediation of tACPD action on mIPSC. The removal of Ca2+ from external solution significantly decreased mIPSC frequency without affecting the distribution of their current amplitudes. Figure 3Aa shows a typical response of mIPSCs before, during, and after the application of tACPD in Ca2+-free external solution. tACPD decreased the mIPSCs frequency in a reversible manner without affecting the distribution of the current amplitudes (Fig. 3Ab). The inhibition of mIPSC frequency by tACPD was 77.8 ± 4.3% of control even in the Ca2+-free solution (P < 0.05, n = 5).
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Moreover, the application of 100 µM Cd2+, which completely blocks VDCCs, also failed to remove the inhibitory effect of tACPD on mIPSCs frequency (Fig. 3B). The inhibition was almost the same as that in Ca2+-free solution (78.3 ± 4.5% of control, n = 5). The data suggest that the inhibitory effect of mGluRs in the presence of tACPD is independent of Ca2+ influxes passing through VDCC.
It was investigated that the possible role of presynaptic K+ channels in the mediation of tACPD action on mIPSC frequency. Figure 4A shows the relationship between external K+ concentration ([K+]o) and spontaneous IPSCs frequency with or without TTX in the standard solution. The removal of K+ from the control solution slightly increased mIPSC frequency (P < 0.05, n = 5; Fig. 4A) but did not affect the distribution of the current amplitudes (data not shown).
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Figure 4Ba shows a typical response of mIPSCs before, during, and after the application of tACPD in K+-free external solution, containing both Ba2+ and Cs+ to block the K+ conductance. Even in this K+-channel-blocking solution, tACPD reversibly suppressed mIPSC frequency to 78.6 ± 6.8% of control (P < 0.05, n = 6) but had no effect on the distribution of current amplitudes (Fig. 4B, b and c). The results suggest that the inhibitory effect of tACPD on mIPSC frequency is not due to the increase of presynaptic K+ conductance.
The last possibility is a presynaptic inhibition mediated through an
intracellular signaling mechanism. The mGluRs could alter synaptic
transmission by coupling to phosphoinositide hydrolysis or to adenylyl
cyclase (AC) pathways via heterotrimeric G protein (Tanabe et
al. 1992, 1993
). Forskolin
(10
5 M), a membrane
permeable AC activator, nearly doubled the mIPSC frequency (195.5 ± 16.3% of control, P < 0.01, n = 9)
without affecting the mean mIPSC amplitude (111.8 ± 8.4% of
control, n = 9, Fig.
5A, a and b). After
application of forskolin, it took 5 min for the frequency to become
stable (Fig. 5Ab). The result indicates that forskolin is
acting at a glycinergic presynaptic site (Fig. 5, A and
B). The data also suggest that either cAMP or PKA might be
related to the inhibition of glycine release from the presynaptic
terminals.
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Forskolin (105 M)
completely prevented tACPD action on the mIPSCs frequency (Fig.
6A, a and b).
Before the treatment of forskolin, tACPD reduced mIPSC frequency
without affecting the mean mIPSC amplitude. During the treatment of
forskolin in the same neuron, the inhibitory effect of tACPD on mIPSC
frequency disappeared (102.4 ± 8.7% to control,
n = 7) without altering the current amplitude
(106.8 ± 3.4% to control, n = 7; Fig. 6,
Ac and B). Recent reports indicate that forskolin
enhances glutamate release by not only activating AC but also by
promoting a Ca2+ influx mechanism (Hoshi
et al. 1988
; Lonart et al. 1998
; Wagoner and Pallotta 1988
). Thus it was examined whether
1-9-dideoxy-forskolin, an inactive form of forskolin, has an
inhibitory effect on glycine release from the nerve terminals. The
inactive form itself potentiated the frequency of mIPSCs (191.0 ± 33.0% of control, n = 5) without altering the current
amplitude distribution. But tACPD still had an inhibitory effect on
mIPSCs frequency (91.4 ± 5.3% of control, n = 5, figure not shown). Therefore these results suggest that this change of
synaptic efficacy by tACPD is elicited via AC-cAMP/PKA pathway.
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The effect of cAMP analogues was further examined to test the direct action of cAMP/PKA pathway. Both 8-Br-cAMP, membrane permeable cAMP activator and Sp-cAMP, membrane permeable PKA activator, increased the frequency of glycine release (126.8 ± 6.9 and 133.1 ± 5.4% of control, respectively, n = 5). 8-Br-cAMP blocked completely the inhibitory action of tACPD (132.0 ± 2.0% of control, n = 5). Sp-cAMP, however, did not block the inhibitory effect of tACPD (96.0 ± 1.7% of control, n = 5). Rp-cAMP, a membrane permeable PKA inhibitor, facilitated the mIPSCs frequency (127.5 ± 4.02% to control, n = 5) without changing the current amplitude distribution (98.9 ± 2.57% to control, n = 5). Rp-cAMP, however, did not block the inhibitory effect of tACPD (96.7 ± 2.91% of control, n = 5; Fig. 6B). Thus these results could be suggested that the inhibitory action of tACPD related to cAMP but not PKA.
Subtype of mGluRs
To determine which subtypes of mGluRs participated in the
inhibition of mIPSCs, the effects of mGluR agonists and antagonists were examined. Figure 7Aa
shows the mIPSCs before, during, and after the application of a
mGluRIII specific agonist, L-AP4
(105 M). L-AP4 mimicked
the action of tACPD, and the inhibitory effect of L-AP4 disappeared in
the presence of 10
4 M
CPPG, a mGluRIII specific antagonist (Conn and Pin
1997
). The L-AP4 application decreased reversibly the mIPSC
frequency to 74.1 ± 4.2% of control (P < 0.01, n = 12) without affecting the mean amplitude (Fig.
7B). CPPG at
10
4 M itself did not
affect cumulative distributions of mIPSCs (data not shown).
mGluRII-specific agonist,
10
5 M L-CCG-I, also
reduced mIPSC frequency (75.0 ± 4.6% to control) without
affecting the amplitude distribution in the same cells (Fig.
7B). The inhibitory effect of L-CCG-I was blocked fully by
3 × 10
4 M MCCG, a
specific mGluRII antagonist. These results indicate that the inhibitory
effect of tACPD on the mIPSC frequency is mediated by at least
mGluRII and/or mGluRIII in these glycinergic nerve terminals.
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We also examined the effect of mGluR antagonists on tACPD action. CPPG
(104 M) reduced the
inhibitory effect of tACPD (from 73.0 ± 3.5 to 82.5 ± 2.0%
of control, n = 4), and 3 × 10
4 M MCCG also relieved
the tACPD action in the same cells (to 79.0 ± 3.2% of control,
n = 4; Fig. 7C). However, each antagonist
did not completely block the inhibitory action of tACPD. The percent of
inhibition by L-AP4 and L-CCG-I varied considerably among neurons. Some
cells were only sensitive to one agonist, suggesting that the nerve
endings of SDCN neurons have only one type of mGluRs. However, most
cells were sensitive to both L-AP4 and L-CCG-I. Thus these data suggest
that the mGluR subtypes such as mGluRII and mGluRIII in the glycinergic
nerve terminals projecting on SDCN neuron exist heterogeneously.
We also examined the existence of mGluRII and/or mGluRIII on
glycinergic nerve terminal in the slice preparation using the whole
cell patch recording technique at VH
of 60 mV.
SDCN neurons had also been subdivided to three groups according to the
cell size as that observed in dissociated SDCN neurons. Therefore the
experiment was performed on the same middle size neurons. Figure
8Aa shows that the glycinergic
eIPSC before, during, and after the application of
106 M L-CCG-I and
10
6 M L-AP4,
respectively. The application of L-CCG-I decreased reversibly the eIPSC
amplitude (47.9 ± 3.9% of control, n = 6). After
wash out, when the eIPSC amplitude recovered on control baseline, L-AP4 was applied. L-AP4 also decreased reversibly the eIPSC amplitude (50.0 ± 4.4% of control, n = 6, Fig.
8Ab).
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Figure 8Ba shows effects of
106 M L-CCG-I and L-AP4
on glycinergic mIPSCs of the slice preparation in the presence of TTX. Both L-CCG-I and L-AP4 decreased reversibly the mIPSC frequency (47.9 ± 5.9, 54.6 ± 6.4% of control, respectively,
n = 5) without affecting its current amplitude (Fig.
8Bb).
These results indicate that the inhibitory effects of mGluR on eIPSC amplitude and mIPSC frequency are mediated by the function of both mGluRII and mGluRIII in these glycinergic nerve terminals. Thus these data suggest that the mGluR subtypes such as mGluRII and mGluRIII in the glycinergic nerve terminals projecting on SDCN neuron also exist heterogeneously in the slice preparation.
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DISCUSSION |
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Multisynapses on the dissociated single neuron
In the present study, the slice preparation and the mechanically
dissociated neurons without treating any enzymes were used. The
"synaptic bouton preparation" preserves native synaptic boutons on
isolated SDCN cell bodies. Using pharmacological isolation of the
glycinergic IPSCs, the functional modulation of glycine release
mediated by mGluRs. The amplitude distribution of the mIPSCs was skewed
toward large amplitude events (Fig. 2), indicating that the dissociated
single neuron has multiple intact functional synapses. The data are
consistent with recordings from slice preparation (Zhou and
Hablitz 1997). TTX reversibly decreased mIPSC frequency to
45.5% of control (P < 0.01, n = 52)
but had no effect on the distribution of their current amplitudes. The
decrease of mIPSCs frequency was not accompanied by a change in the
current amplitude, indicating that the probability of glycine release
decreases in the presence of TTX. In addition, TTX-sensitive
Na+ channels that contribute to spontaneous
glycine release in this isolated preparation evidently exist quite near
to the nerve terminals.
Inhibitory mechanism of tACPD
It is reported that activation of K+
conductance and/or reduction of voltage-dependent
Ca2+ currents induce presynaptic inhibition
(Cochilla and Alford 1998; Takahashi et al.
1996
). Figure 4A shows the effect of extracellular K+ concentration
([K+]o) in the standard
solution. Both [K+]o-free
and 10 mM [K+]o solution
slightly depolarized the presynaptic terminals by different mechanisms.
In [K+]o-free solution,
the Na pump could be inhibited, resulting in depolarization
(Akaike et al. 1992
) while 10 mM
[K+]o solution
depolarized cell membrane as predicted from the Goldman-Hodgkin-Katz equation. Consequently, both conditions could activate VDCC
(Akaike et al. 1992
; Haage et al. 1998
).
Interestingly, although
[K+]o-free and 10 mM
[K+]o solutions increased
the frequency of mIPSCs, the values of frequency change normalized at 5 mM [K+]o were not altered
by TTX. The data suggest that TTX-sensitive Na+
channels in or near the presynaptic terminal might contribute little to
spontaneous glycine release induced by
[K+]o. Postsynaptically,
tACPD indirectly activates a Ca2+-activated
K+ currents
(IKCa) via IP3,
Ca/CaM pathway coupled to a GTP-binding protein (G protein) in rat
hippocampal neurons (Shirasaki et al. 1994
). In the
present experiment, using an internal solution with Cs+ instead of K+, the
activation of postsynaptic K+ channels could be
excluded. In any case, the quantitative and kinetic analysis showed
that tACPD suppressed only mIPSC frequency, but not the current
amplitude, and that tACPD did not change time to peak or the decay time
course of individual mIPSCs. These results indicate that tACPD acts
selectively on the presynaptic glycine release process without altering
postsynaptic glycine receptor sensitivity.
The VDCCs in the active zone of synapses are found in dense aggregates
that have an important role in the triggering or the modulation of
transmitter release in presynaptic nerve terminals (Haage et al.
1998; Stanley 1997
; Takahashi et al.
1996
). VDCC subtypes such as N, P, and/or Q types participate
in the inhibitory modulation of excitatory synaptic transmission in
nerve endings of CNS (Huang et al. 1996
; Poncer
et al. 1997
). In our experiment, Ca2+
influxes via VDCC at the glycinergic presynaptic terminals did not
contribute to the inhibitory effect of tACPD on mIPSCs since the tACPD
inhibition of mIPSC frequency did not alter in either Ca2+-free or Cd2+-external solution.
The modulation of transmitter release by a
Ca2+-independent mechanism has been also reported
recently (Thomson et al. 1993; Trudeau et al.
1996a
,b
). Some studies revealed that the increase of cAMP/PKA
directly activated presynaptic transmission in rat CNS neurons
(Capogna et al. 1995
; Chavez-Noriega and Stevens
1994
; Chen and Regher 1997
; Trudeau et
al. 1996a
,b
). In the present experiment, the effect of tACPD
was blocked by forskolin, which activates the AC cascade leading to
cAMP activation of PKA (Zhang et al. 1997
). tACPD,
however, decreased glycine release in the presence of Sp-cAMP, a cAMP
analogue that directly activated PKA. On the other hand, 8-Br-cAMP
blocked the inhibitory effect of tACPD. Evidently, the cAMP receptor
responsible for the increase in transmitter release is much more
sensitive to 8-Br-cAMP than to Sp-cAMP. Thus cAMP itself contributes to
presynaptic glycine release and the presynaptic inhibitory effect of
tACPD could be mediated by decreasing the intracellular cAMP
concentration ([cAMP]i) level. Therefore
present results suggest that the inhibitory action of mGluR is mediated
by the decrease of [cAMP]i but not the
reduction of PKA in the nerve terminals.
Distribution of mGluR subtypes
The present study suggests that both mGluRII and mGluRIII exist in the presynaptic terminals of majority of SDCN neurons (Figs. 7 and 8). tACPD, L-AP4, and L-CCG-I reversibly suppressed the mIPSC frequency but had no effect on the distribution of the current amplitude in the dissociated neuron and also suppressed both mIPSC frequency and eIPSC amplitude in the slice preparation. The mean inhibition induced by tACPD, L-AP4, and L-CCG-I was almost the same, and yet mGluR antagonists alone could not block the inhibitory action of tACPD. Thus it was considered the possibility of three models about the various distributions of their mGluRs in the synaptic terminals. First, the postsynaptic neuron may have several presynaptic terminals in which each terminal has only one kind of mGluR subtypes (mGluRII or mGluRIII). Second, one kind of mGluR subtype may have an effect on each terminal, but the terminals with different subtypes are variously distributed on postsynaptic neurons. And last, the mGluR subtypes may coexist in the same presynaptic nerve terminal. In the present experiments, a few cells responded to only one of mGluR subtypes. However, the distribution of the magnitudes of the inhibition by each of the agonists varied considerably among the middle size cells tested, suggesting that most of SDCN neurons may have a heterogeneous distribution of presynaptic mGluR subtypes.
The first and third hypotheses could be supported by the slice experiment because both mGluRII and III agonists modulated the evoked glycine release in all neurons tested. These results suggest that if only the nerve ending coexisting of both mGluR subtypes was stimulated, the evoked glycine release could be modulated by both of agonists. On the other hand, if a few nerve endings in spite of existence of one kind of different mGluR subtype in each terminal were stimulated, the evoked glycine release could be also modulated by both of agonists.
Physiological role
SDCN receives abundant inputs from the raphe, locus coeruleus, and
hypothalamus. The innervation of the sensory afferents to the SDCN
region is involved in nociceptive transmission. The SDCN neurons could
be subdivided into three regions according to the effective somatic
and/or visceral inputs (Honda 1985). The size of neurons
responsive to somatic stimulation (SOM neurons) was between 15 and 50 µm, while the neurons responsive to visceral (VISC neurons) or to
both somatic and visceral stimulation (SOMVISC neurons) was <15 µm
(Honda 1985
). Thus the middle-size neurons used in the
present experiments might be visceral neuron or somatic visceral neurons.
In the present study, tACPD induced presynaptic inhibition in the glycinergic nerve terminal of the dissociated SDCN neurons. The inhibition of glycine release might result in the depolarization of the postsynaptic VISC or SOMVISC neurons. Such a change of synaptic efficacy might enhance the sensation of pain from the visceral and somatic organs, resulting in hyperalgesia.
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ACKNOWLEDGMENTS |
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The authors thank Dr. M. C. Andresen and Dr. M. Brodwick for advice and critical reading of the manuscript.
This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan to N. Akaike (10044301 and 10470009), and by Kyushu University Interdisciplinary Programs in Education and Projects in Research Development and by National Natural Science Foundation of China to Z. M. Wang (39625011 and 39800044).
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FOOTNOTES |
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Address for reprint requests: N. Akaike (E-mail: akaike{at}mailserver.med.kyushu-u.ac.jp).
Received 5 June 2000; accepted in final form 27 September 2000.
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REFERENCES |
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