Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B
The action of ginkgolide B (GB), the powerful compound of Ginkgo biloba extract, on glycinemediated spontaneous currents in rat spinal sacral dorsal commissural nucleus (SDCN) neurons was examined. IPSCs evoked in spinal cord slices were inhibited in a dose-dependent manner by the addition of GB to...
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Інститут фізіології ім. О.О. Богомольця НАН України
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| Zitieren: | Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B / K. Nonaka, E. Kondratskaya, M. Maeda, T. Yamaga, N. Murayama, M.-C. Shin, N. Akaike // Нейрофизиология. — 2009. — Т. 41, № 4. — С. 301-308. — Бібліогр.: 25 назв. — англ. |
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irk-123456789-683012014-09-21T03:01:46Z Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B Nonaka, K. Kondratskaya, E. Maeda, M. Yamaga, T. Murayama, N. Shin, M.-C. Akaike, N. The action of ginkgolide B (GB), the powerful compound of Ginkgo biloba extract, on glycinemediated spontaneous currents in rat spinal sacral dorsal commissural nucleus (SDCN) neurons was examined. IPSCs evoked in spinal cord slices were inhibited in a dose-dependent manner by the addition of GB to the superfusion solution. The amplitude of eIPSCs was reduced to 61 ± 6.4% by 10 μM GB, with acceleration of the kinetics of the currents indicating the effect of GB on channel pores. Both the amplitude and success ratio (Rsuc) of eIPSC induced by electrical focal stimulation of single glycinergic nerve endings (boutons) also decreased in the presence of 1 μM GB. These data suggest that GB modulates not only post-synaptic glycine receptors but also the pre-synaptic glycine release mac hinery. Вплив гінкголіду В (GB) – діючої сполуки екстракту з гінкго дволопатевого (Ginkgo biloba) на гліцинопосередковані синаптичні струми вивчався на нейронах спінального сакрального дорсального комісурального ядра (SDCN) щурів. Гальмівні постсинаптичні струми (ГПСС), викликані в препаратах зрізів спинного мозку, дозозалежно зменшувалися при аплікації GB. Амплітуда викликаних ГПСС під дією 10 мкМ GB падала до 61 ± 6.4 % з одночасним прискоренням кінетики струмів, що свідчило про наявність впливу на канальні пори. Як амплітуда, так і відносна кількість синаптичних подій (викликаних ГПСС), індукованих електричною фокальною стимуляцією поодиноких гліцинергічних нервових закінчень (бутонів), також зменшувались у присутності 1 мкМ GB. Ці результати свідчать, що GB не тільки модулює постсинаптичні гліцинові рецептори, але й впливає на пре синаптичні механізми вивільнення гліцину. 2009 Article Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B / K. Nonaka, E. Kondratskaya, M. Maeda, T. Yamaga, N. Murayama, M.-C. Shin, N. Akaike // Нейрофизиология. — 2009. — Т. 41, № 4. — С. 301-308. — Бібліогр.: 25 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/68301 612.815.1+577.25 en Нейрофизиология Інститут фізіології ім. О.О. Богомольця НАН України |
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The action of ginkgolide B (GB), the powerful compound of Ginkgo biloba extract, on glycinemediated spontaneous currents in rat spinal sacral dorsal commissural nucleus (SDCN) neurons was examined. IPSCs evoked in spinal cord slices were inhibited in a dose-dependent manner by the addition of GB to the superfusion solution. The amplitude of eIPSCs was reduced to 61 ± 6.4% by 10 μM GB, with acceleration of the kinetics of the currents indicating the effect of GB on channel pores. Both the amplitude and success ratio (Rsuc) of eIPSC induced by electrical focal stimulation of single glycinergic nerve endings (boutons) also decreased in the presence of 1 μM GB. These data suggest that GB modulates not only post-synaptic glycine receptors but also the pre-synaptic glycine release mac hinery. |
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Nonaka, K. Kondratskaya, E. Maeda, M. Yamaga, T. Murayama, N. Shin, M.-C. Akaike, N. |
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Nonaka, K. Kondratskaya, E. Maeda, M. Yamaga, T. Murayama, N. Shin, M.-C. Akaike, N. Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B Нейрофизиология |
| author_facet |
Nonaka, K. Kondratskaya, E. Maeda, M. Yamaga, T. Murayama, N. Shin, M.-C. Akaike, N. |
| author_sort |
Nonaka, K. |
| title |
Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B |
| title_short |
Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B |
| title_full |
Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B |
| title_fullStr |
Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B |
| title_full_unstemmed |
Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B |
| title_sort |
modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide b |
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Інститут фізіології ім. О.О. Богомольця НАН України |
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2009 |
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http://dspace.nbuv.gov.ua/handle/123456789/68301 |
| citation_txt |
Modulation of glycinergic transmission in the rat spinal dorsal commissural nucleus by ginkgolide B / K. Nonaka, E. Kondratskaya, M. Maeda, T. Yamaga, N. Murayama, M.-C. Shin, N. Akaike // Нейрофизиология. — 2009. — Т. 41, № 4. — С. 301-308. — Бібліогр.: 25 назв. — англ. |
| series |
Нейрофизиология |
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НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4 301
UDC 612.815.1+577.25
K. NONAKA,1,2 E. KONDRATSKAYA,1, 4 M. MAEDA,1,3 T. YAMAGA,1
N. MURAYAMA,2 M. -C. SHIN,1 and N. AKAIKE1
MODULATION OF GLYCINERGIC TRANSMISSION IN THE RAT SPINAL DORSAL
COMMISSURAL NUCLEUS BY GINKGOLIDE B
Received 01.08.09
The action of ginkgolide B (GB), the powerful compound of Ginkgo biloba extract, on glycine-
mediated spontaneous currents in rat spinal sacral dorsal commissural nucleus (SDCN) neurons
was examined. IPSCs evoked in spinal cord slices were inhibited in a dose-dependent manner
by the addition of GB to the superfusion solution. The amplitude of eIPSCs was reduced
to 61 ± 6.4% by 10 µM GB, with acceleration of the kinetics of the currents indicating the
effect of GB on channel pores. Both the amplitude and success ratio (Rsuc) of eIPSC induced
by electrical focal stimulation of single glycinergic nerve endings (boutons) also decreased
in the presence of 1 µM GB. These data suggest that GB modulates not only post-synaptic
glycine receptors but also the pre-synaptic glycine release machinery.
Keywords: spinal glycine receptor, evoked inhibitory postsynaptic current (eIPSC),
presynaptic bouton, focal stimulation, ginkgolide B.
1 Kumamoto Health Science University, Kumamoto, Japan.
2 Graduate School of Science and Technology, Kumamoto University,
Kumamoto, Japan.
3 Kurume University, Kurume, Japan.
4 Bogomolets Institute of Physiology, National Academy of Sciences of
Ukraine, Kyiv, Ukraine.
Correspondence should be addressed to E. Kondratskaya
(e-mail: elena_kondratskaya@yahoo.com).
INTRODUCTION
Glycine and γ-aminobutyric acid (GABA) are
considered to assemble fast synaptic inhibitory
transmission within neuronal networks in the CNS [1].
The primary impact of glycine on the control of both
motor and sensory pathways has been confirmed to be
mediated throughout the brainstem and spinal cord [2].
A pharmacological influence of glycinergic inhibition
by a glycine transporter produced a profound anti-
allodynia effect in a peripheral nerve partial ligation
model in mice [3], whereas disinhibitory mechanisms
may contribute to abnormal pain occurring after
peripheral nerve injury [4]. Therefore, ligands for
both GABA and glycine receptors have important
implications in relative disease states and nociception,
since these agents can potentially be used to relieve
the pain [5, 6], whereas antagonists cause neuronal
excitation. Such antagonists are often used to
discriminate between receptor subtypes with respect
to their brain region and/or cellular localization.
Remarkably, the functional alliance between the
above-mentioned inhibitory transmitter systems
has been proved for the intraspinal circuitry. The
respective statements include the synaptic co-
localization of special receptors, the existence of
functional transporters, and co-release of both active
substances due to input stimulation [7]. Therefore, the
development of selective pharmacological tools is a
promising outline to control glycinergic transmission
and modulate the neuronal excitability, especially in
the spinal cord, since the latter was reported to serve as
an intermediary “relay” formation for pain processing
and suppression mechanisms, where the role of glycine
receptors is beyond doubts.
Strychnine-sensitive glycine receptors share the
topology, transmembrane arrangement, and pentameric
subunit assembly with members of the ligand-gated
ion channel receptor superfamily [8]. The general
similarities of receptor structure might predict the
common influence of both glycine and GABA receptor
types by the rank of modulators, as has been found for
picrotoxin and strychnine [9].
A terpenic trilactone, ginkgolide B (GB), the
powerful constituent from Ginkgo biloba extract
demonstrated previously to be a platelet-activity
factor (PAF) antagonist [10], is a blocker of glycine-
gated channels with a somewhat higher potency for β
subunit-composed receptors, in particular α2β hetero-
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4302
oligomers [11]. The efficacy of GB as an open-channel
blocker was mainly investigated in hippocampal and
cortex glycine receptors [12], i.e., in the brain regions
never evidenced to compose pure glycinergic synapses,
where glycine receptors are rarely expressed. The
impact of inhibitory glycinergic transmission in these
structures, if at all, seems to be a minor to GABA-
ergic.
In the current study, we investigated the action of GB
on glycine-mediated currents in spinal neurons of the
rat sacral dorsal commissural nucleus (SDCN). Neurons
from the SDCN are known to receive glycinergic,
GABA-ergic, and mixed synaptic inputs [13, 14]
and are implicated in pain perception [15, 16]. In an
attempt to clarify the physiological relevance for GB
as a channel blocker due to diverse “vivid” conditions
for signal driving, we have examined the effect of GB
on spontaneous and evoked inhibitory postsynaptic
currents (sIPSCs and eIPSCs, respectively) both in
spinal cord slices and in acutely isolated neurons with
functional synaptic boutons remaining [13, 17]. Thus,
we have compared here the effects of GB on sIPSCs
in slices and single neurons, as well as on electrically
induced eIPSCs in slices and with a focal single-bouton
stimulation technique, i.e., in the situations reflecting
the action of ginkgolides on glycine receptors within
different transmission modes.
METHODSs
Surgical procedures. All experiments were performed
in accordance with the Guiding Principles for Care
and Use of Animals in the Field of Physiological
Sciences of the Physiological Society of Japan and
approved by the local Animal Experiment Committee
in the Kumamoto Health Science University (Japan).
Wistar rats (10-12 days old) were decapitated under
pentobarbital anesthesia (50 mg/kg, i.p.). A segment of
the lumbosacral (L5-S4) spinal cord was dissected and
transversely sliced in ice-cold artificial cerebrospinal
fluid at a thickness of 350-370 µm using a microslicer
(VT1000S; Leica, Germany). Slices were kept in
an incubation medium at room temperature (25°C).
The incubation medium consisted of (mM): NaCl,
124; KCl, 5; KH2PO4, 1.2; NaHCO3, 24; CaCl2, 2.4;
MgSO4⋅7H2O, 1.3; and glucose 10; the saline was
saturated with 95% O2 and 5% CO2 (pH 7.4).
Slice preparation and isolation of neurons. Slices
were transferred to a recording chamber (volume 300 µl)
that was continuously perfused with a normal
external solution (mM): NaCl, 119; KCl, 2.5; CaCl2,
2.5; MgSO4⋅7H2O, 1.3; NaH2PO4, 1; NaHCO3, 26.2;
and glucose, 11, equilibrated with 95% O2+5% CO2
and delivered with a gravity-fed perfusion system
at a flow rate of 2-3 ml/min. Neurons of the SDCN
(Fig. 1A) were visually identified with an infrared-
differential interference contrast (IR-DIC) video
microscope (Leica DM SDK; IR-1000 (DAGE-
MTI, USA) and captured with CoolSNAP ES2 (CCD
ICX285). The patch pipette solution for the whole-
cell patch recording in slice preparations consisted of
(mM): Cs methanesulfonate, 145; TEA-Cl, 5; CsCl, 5;
EGTA, 2, HEPES, 10 (pH 7.2). For suppression of the
postsynaptic GABAA responses, most experiments were
performed in an internal solution without ATP, where
the GABAA response was eliminated by superfusion
with an ATP-free internal solution [21, 22].
For mechanical dissociation, slices were transferred
into a culture dish (Primaria 3801; Becton Dickinson,
USA). The region of the SDCN (Fig. 1A) was identified
under a binocular microscope. The details of mechanical
dissociation have previously been described [23, 24].
Single SDCN neurons were acutely dissociated from
slices using a manufactured vibrating cell-isolating
setup (S-I L cell Isolator; KT Labo, Japan). Then, SDCN
neurons were identified under microscopic visual
control. The external solution used for dissociated
neurons consisted of (mM): NaCl, 150; KCl, 5;
CaCl2, 2; MgCl2, 1; glucose 10, and HEPES 10; pH
was adjusted to 7.4 with Tris-OH. The composition of
a pipette-filling solution for single-cell experiments
was as follows (mM): CsF, 135; TEA-Cl, 5; CsCl, 5;
EGTA, 2 and HEPES, 10, but without ATP; pH of the
solution was adjusted to 7.2 with Tris-OH.
Electrophysiological recordings. All electrical
measurements were performed using the conventional
whole-cell patch recording mode at a holding potential
(VH) of 0 mV using a patch-clamp amplifier (CEZ-
2300; Nihon Kohden, Japan). Patch electrodes were
fabricated from borosilicate glass capillaries of a 1.5 mm
outer diameter (Model GD-1.5, Narishige Scientific
Instruments Lab, Japan) with a programmable puller
(P-97; Sutter Instruments, USA). The tip resistance
of the electrodes filled with the solution was 6-8 MΩ
for slice preparations and 4-5 MΩ for dissociated
neurons. Current and voltage values were continuously
monitored on an oscilloscope and a pen recorder. The
membrane currents were filtered at 1 kHz (E-3201A
Decade Filter; NF Electronic Instruments, Japan),
digitized at 4 ⋅ 10–3 sec–1, and stored on a computer
equipped with pCLAMP 8.02 (Axon Instruments,
K. NONAKA, E. KONDRATSKAYA, M. MAEDA, et al.
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4 303
USA). To monitor the access resistance, 10 mV
hyperpolarizing step pulses (30 msec long) were
periodically delivered.
The SDCN neurons receive glutamatergic,
glycinergic, GABA-ergic, and mixed synaptic inputs.
To isolate pharmacologically glycine-mediated
events from GABA-ergic ones, an ATP-free internal
solution and 3 µM bicuculline were used. In addition,
to suppress the possible activation of glutamate
receptors, 5 µM NBQX and 5 µM D-AP5 were
routinely added to the external solution. The SDCN
region in slices was visualized using video-enhanced
infrared microscopy, and individual SDCN neurons
were chosen for recording due to their morphology.
Spontaneous sIPSCs and evoked ones (eIPSCs) were
recorded in a whole-cell recording mode at a VH of
0 mV under voltage-clamp conditions. In order to
achieve eIPSCs from SDCN neurons, the stimulation
electrode was positioned near the central canal region.
Short negative pulses (5-10 μA, 100 μsec long) were
applied through a glass pipette filled with the standard
external solution via a stimulus isolation unit.
Synaptic bouton preparation. Both sIPSCs
and eIPSCs were recorded from synaptic bouton
preparations. In recordings of eIPSCs at single
glycinergic synapses, the electrical focal stimulation
pipette was placed close to the surface of a dissociated
SDCN neuron, from which whole-cell recording was
made. Then, negative pulses (100 µsec long) were
delivered every 3 or 5 sec. The stimulating pipette
having about a 0.5 μm diameter was filled with the
standard external solution and moved along the surface
of the somatic membrane and proximal dendrites until
an eIPSC was elicited in an “all-or-none” fashion,
indicating that the stimulating pipette (electrode)
was positioned just above the bouton. The current
appeared in an “all or none” fashion when the stimulus
strength was increased or when the stimulation pipette
was moved. The current was highly sensitive to
0.3 µM tetrodotoxin (TTX) [23]. The success ratio
(Rsuc) was measured as a ratio of the number of eIPSCS/
the number of focal stimulations with or without GB.
For example, 24 eIPSCS/30 focal stimulations gives
Rsuc = 0.8. The eIPCSs appeared like in an “all or
none” fashion, but the amplitude histogram of eIPSCs
recorded in the normal external solution with 2 mM
Ca2+ could be fitted by Poisson process.
Data analysis. Glycinergic IPSCs were counted and
analyzed in preset epochs before, during, and after
each test condition using the MiniAnalysis Program
(Synaptosoft, USA). Briefly, the events were initially
screened automatically using an amplitude threshold
of 10 pA and then visually accepted or rejected based
on their 10 to 90% rise and 90 to 37% decay times. The
interevent intervals and amplitudes of sIPSCs were
examined by constructing and comparing cumulative
probability distributions under different conditions
using the Kolmogorov–Smirnov (K–S) test (Stat View
software, SAS Institute, USA). The average values
of both frequency and amplitude of synaptic events
during the control period (3-5 min) were calculated,
and the frequency and amplitude of all events in
the course of drug application were normalized to
these values. The effects of drugs were quantified as
percentage changes in the frequency and amplitude
of synaptic events and compared with the individual
control. Possible differences between the amplitude
and frequency distributions were tested by Student’s
paired two-tailed t-test using their absolute values. On
the other hand, the interevent intervals and amplitudes
of a large number of synaptic events obtained from
the same neuron were examined by constructing
cumulative probability distributions and compared
using the K–S test within the MiniAnalysis Program.
The current amplitude of eIPSCs was analyzed using
pCLAMP 8.0. Numerical values are reported as means ±
± s.e.m. A possible significance of the differences
in the amplitude was tested by Student’s paired two-
tailed t-test using the absolute values of the parameter
(not normalized ones). The differences with P < 0.05
were considered to be significant.
Drugs exposure. The following drugs were used
in this study: TTX, bicuculline methiobromide,
strychnine, and GB (Sigma, USA). All drugs were
stored frozen (–20°C) as concentrated stock solutions
and dissolved in the extracellular solution to the
appropriate concentration just before recording. All
test solutions containing drugs were applied by a
standard gravity bath perfusion system at a flow rate
of 2 to 3 ml/min for slice preparations and by a “Y-
tube system” for rapid solution exchange within
20 msec in experiments on dissociated neurons [25].
RESULTS
First, we examined the effect of GB on sIPSCs recorded
in spinal cord slices. Bath application of 1.0 µM GB
exerted negligible effects on the sIPSC amplitude
and frequency (data not illustrated). At a higher
GB concentration (10 µM), strong inhibition was
observed, with the mean IPSC amplitude decreasing
MODULATION OF GLYCINERGIC TRANSMISSION IN THE RAT SPINAL DORSAL COMMISSURAL NUCLEUS
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4304
to 61 ± 6.4%, while the frequency decreased to 78 ±
± 9% (n = 4).
The amplitude of eIPSCs was randomly distributed
varying between 50 and 350 pA. When 1.0 µM GB
dissolved in the standard extracellular solution was
applied, a slight decrease in the eIPSC amplitude
was observed without affecting the Rsuc (Fig. 1B, C).
However, application of GB at a higher concentration
(10 µM) significantly decreased the eIPSC amplitude
(n = 4, P < 0.05) and also decreased slightly the Rsuc
(P > 0.05), as shown in Fig. 1 B, C. Moreover, 10
µM GB profoundly changed the inactivation kinetics
of evoked glycinergic currents being indicative of a
channel mode of the inhibitory action, as reported by
Kondratskaya et al. (2004). In the responses shown
in Fig. 1D, the half-decay time was 33 msec for the
control and changed to 20 msec in the presence of
10 µM GB.
Spontaneous IPSCs were also observed in dissociated
SDCN neurons (synaptic bouton preparations) at the
Fig. 1. Effects of ginkgolide B (GB) on glycinergic evoked inhibitory postsynaptic currents (eIPSCs) recorded from neurons of the sacral
dorsal commissural nucleus (SDCN) in rat spinal cord slices.
A) Schematic illustration of the SDCN region (shown by an ellipse on the cord section) and positions of recording (Rec) and stimulation
(Stim) electrodes. B) Amplitudes of representative recordings of eIPSCs (pA) before and during application of GB (1 and 10 µM) shown
by dashed bars above the diagram. C) Summary of the GB effect on the normalized amplitude and success ratio (Rsuc) (%) of eIPSCs; n = 5,
* P < 0.05 C. D) Original traces of eIPSCs recorded before and during application of 10 µM GB, and currents normalized by peak before
and after GB application (1 and 2, respectively).
Р и с. 1. Вплив гінкголіду Б (GB) на викликані гліцинергічні гальмівні постсинаптичні струми, зареєстровані в нейронах сакрального
дорсального комісурального ядра у зрізах спинного мозку щура.
100
50
0
1.0
0.5
0
Control 1 µM GB 10 µM GB
ns
ns
ns
%
1
2
a
b
1
2
50 msec
50 msec
100 pA
Rec
Stim 300
200
100
0
0 200 400 600 sec
1 µM GB 10 µM GB
NBQX, D-APV, BIC
1
2
B
C
A
D
pA
*
K. NONAKA, E. KONDRATSKAYA, M. MAEDA, et al.
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4 305
holding potential (VH) of 0 mV in a whole-cell patch
recording configuration (Fig. 2A). In the experiments,
contamination by glutamatergic sEPSCs and GABA-
ergic sIPSCs was removed pharmacologically. All
sIPSCs could be completely inhibited by a competitive
glycine receptor antagonist, strychnine (1 µM),
indicating that the recorded sIPSCs are mediated by
strychinine-sensitive glycine receptors. Both the
amplitude and frequency of sIPSCs were decreased by
GB (1 µM), suggesting that dissociated preparations
are more sensitive than slice ones (Fig. 2A, 2, right
panel).
Glycinergic eIPSCs evoked by electrical focal
simulation of single glycinergic boutons using
“synaptic bouton” preparations are also shown in
Fig. 2A [13, 17]. Strychinine (1 µM) reversibly
inhibited these eIPSCs even if the current strength of
focal stimulation was increased. By recording eIPSCs
at the V
H
between +30 mV and –110 mV, the mean
reversal potential estimated from the corresponding
I-V relationship was –73.1 mV (n = 4, Fig. 2B). This
value was close to the ECl (–69.9 mV) calculated from
the Nernst equation using the [Cl-]i = 10 mM and
[Cl-]o = 161 mM values for internal and external
Fig. 2. Glycinergic eIPSCs and sIPSCs observed in dissociated SDCN neurons (in a synaptic bouton preparation).
In A: panel a) Typical IPSCs evoked by focal stimulation (eIPSPs) of a single glycinergic bouton before and after application of 1 µM
strychinine, Stry (shown by downward arrow). Many spontaneous IPSPs (sIPSPs) were recorded simultaneously from the same neuron.
Focal stimulation was given at every 5 sec. Arrow 1 shows an eIPSP before Stry application; arrow 2 shows the absence of an eIPSP upon
Stry application (the respective fragments 1 and 2 in dotted frames are presented at an expanded sweep scale). Inset at the right) Schematic
illustration of a “synaptic bouton” preparation with positions of recording and stimulation electrodes. Panel b is analogous to panel B in
Fig. 1; application of Stry reversibly inhibits both eIPSCs and sIPSCs. B) Current-voltage (I-V) relationship for eIPSCs induced by focal
stimulation of a single glycinergic bouton. The mean amplitudes (I, pA) are plotted vs the holding potential V
H
(V, mV). Insets 1-3 show typical
eIPSCs at different V
H
values. The reversion potential Er and chloride equilibrium potential ECl are indicated.
Р и с. 2. Гліцинергічні викликані та спонтанні гальмівні постсинаптичні струми в дисоційованих нейронах сакрального дорсального
комісурального ядра (препарат синаптичного з’єднання).
a
B
A
b
1
1
pA
300
200
100
0
0 100 200 300 400 500 600 sec
1 µM Stry
1 µM Stry
1 sec
20 pA
20 pA
20 msec
artifact
2
2
20 pA
50 msec
10 mV– 40 mV
1
2
3
– 110 V
pA
Er
ECl
–120 –100 –60 –40 –20 0 20 40 mV
–100
100
200
300
MODULATION OF GLYCINERGIC TRANSMISSION IN THE RAT SPINAL DORSAL COMMISSURAL NUCLEUS
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4306
solutions, respectively. These results indicate that
eIPSC is a Cl- current passing through glycine-gated
chloride channels.
The effect of GB (1 and 10 µM) on eIPSCs elicited
at every 5 sec was examined by focal stimulation of
a single glycinergic bouton. Application of 1 µM GB
slightly decreased the eIPSC amplitude, as shown in
Fig. 3A, B. At the same time, the Rsuc for eIPSCs was
also somewhat decreased. Gingkolide B applied in
the concentration of 10 µM significantly decreased
both the eIPSC amplitude and Rsuc (not illustrated).
In addition, as is seen in Fig. 3C, the inactivation
kinetics of eIPSC was accelerated in the presence of
GB, indicating channel-blocker features for the GB
effect.
DISCUSSION
Our data confirmed the inhibitory action of GB
on glycine receptors previously reported for the
active compound of Ginkgo biloba extract (GB) on
glycine currents in spinal cord neurons. The details
of the effect of the potent blocker GB on glycinergic
transmission within the SDCN neuronal circuitry are
reported at first. An additional new finding of this our
Fig. 3. The effect of ginkgolide B (GB) on eIPSCs in a single glycinergic synapse.
A) Amplitudes of eIPSCs (pA) induced by repeated stimulation (abscissa, numbers of stimuli) and recorded before and during application
of 1 µM GB and during the recovery. Focal stimulation was given every 5 sec. Analysis during GB application (filled circles) was made for
2 min 30 sec. Inset at the right is the same as in Fig.2A. B) Summarized results of the action 1 µM GB on the normalized eIPSC amplitude
(n = 4, P < 0.05) and Rsuc (%, n = 4, P < 0.05). * P < 0.05. C) Original traces of eIPSCs with or without the action of 1 µM GB. Currents
normalized by the peak value are shown in the control (1) and after 1 µM GB application (2).
Р и с. 3. Вплив гінкголіду Б на викликані гальмівні постсинаптичні струми в поодинокому гліцинергічному синапсі.
pA
1000
1 µM GB
800
600
400
200
0
100 200 300
1
2
A
B С
1.0
0.5
0
Control 1 µM GB
100
50
0
%
1
a
2
1
2
20 msec
10 msec
200 pA
b
K. NONAKA, E. KONDRATSKAYA, M. MAEDA, et al.
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4 307
study is the evidence for GB action as a modulator of
the presynaptic glycine release machinery.
Recently, the potent antagonistic activity of
ginkgolides with respect to inhibitory glycine-activated
receptors was reported [24]. The analysis of receptor-
drug relationships for a wild type and selected point
mutations of the channel-forming subunit of glycine
receptors has revealed the use-dependent channel
blocking potency for GB, as well as its selectivity to
β subunit-containing glycine receptors [11, 18]. The
latter point is indicative of the GB-induced blockade
of postsynaptic glycine receptors. In the present
study, we have combined the experiments on rat
spinal cord slices and acutely isolated SDCN neurons
to examine the efficacy of GB as a receptor blocker
within the framework of synaptic, extrasynaptic, and
mixed patterns of native glycine receptor-mediated
transmission.
It is well documented that the inhibitory postsynaptic
events in spinal cord slices are mediated by co-release
of GABA and glycine within the same synaptic space
and, in turn, by activation of both GABA and glycine
receptors [19]. Glycinergic postsynaptic events were
isolated, as described earlier [23]. The action of 1 µM
GB was examined on glycinergic sIPSCs recorded
from neurons in slices and acutely isolated units. We
found that 1 µM GB influences sIPSCs in slices with a
low efficacy but with an increased potency when used
in the concentration of 10 µM.
With a purpose to further determine the effect of GB,
we examined its action on glycine-elicited currents
in freshly isolated SDCN neurons. An additional set
of the experiments aimed to observe the effect of the
examined drug on IPSCs evoked by focal stimulation of
single synaptic boutons was performed. Postsynaptic
currents evoked by focal single-bouton stimulations
reflect the features of glycine receptor-mediated
events, since they were insensitive to bicuculline
and reversibly abolished by a competitive antagonist,
strychnine (1 µM). Moreover, the reversal potential
for evoked postsynaptic currents was estimated as
–73.1 mV, which is rather close to the ECl calculated
from Nernst equation for chloride ions (–69 mV).
The kinetic features were compared for eIPSCs in
the control and after GB pre-incubation (10 µM). The
half-decay time (τ1) for control eIPSCs was calculated
as 33 msec, whereas the half-decay time (τ2) for
eIPSCs in the presence of GB (10 min pre-perfusion)
was significantly reduced and comprised 20 msec.
Such acceleration of the current kinetics is indicative
of the channel blocking action of GB, as was reported
earlier [20]. In isolated neurons, 1 µM GB did cause
a slight decrease in the average eIPSC amplitude and
Rsuc, while 10 µM GB led to a significant reduction
of the amplitude and Rsuc, suggesting that GB acts not
only on post-synaptic glycine-gated Cl– channels but
also on the pre-synaptic glycine release machinery, i.e.,
this agent reduces the release probability. The details
of the presynaptic effect of GB remain uncertain and
require further examination.
К. Нонака1,2, O. Кондрацька1, 4, M Маеда1,3, T. Ямага1,
Н. Мураяма2, М. Шін1, Н. Акаїке1
МОДУЛЯЦІЯ ГЛІЦИНЕРГІЧНОЇ ПЕРЕДАЧІ
В НЕЙРОНАХ СПІНАЛЬНОГО ДОРСАЛЬНОГО
КОМІСУРАЛЬНОГО ЯДРА ЩУРА ГІНКГОЛІДОМ В
1 Університет наук про здоров’я, Кумамото (Японія).
2 Коледж наук та технологій при університеті Кумамото
(Японія).
3 Університет Куруме, Куруме (Японія).
4 Інститут фізіології ім. О. О. Богомольця НАН України,
Київ (Україна).
Р е з ю м е
Вплив гінкголіду В (GB) – діючої сполуки екстракту з гінк-
го дволопатевого (Ginkgo biloba) на гліцинопосередкова-
ні синаптичні струми вивчався на нейронах спінального са-
крального дорсального комісурального ядра (SDCN) щурів.
Гальмівні постсинаптичні струми (ГПСС), викликані в пре-
паратах зрізів спинного мозку, дозозалежно зменшували-
ся при аплікації GB. Амплітуда викликаних ГПСС під дією
10 мкМ GB падала до 61 ± 6.4 % з одночасним прискорен-
ням кінетики струмів, що свідчило про наявність впливу на
канальні пори. Як амплітуда, так і відносна кількість си-
наптичних подій (викликаних ГПСС), індукованих елек-
тричною фокальною стимуляцією поодиноких гліцинер-
гічних нервових закінчень (бутонів), також зменшувались
у присутності 1 мкМ GB. Ці результати свідчать, що GB не
тільки модулює постсинаптичні гліцинові рецептори, але й
впливає на пресинаптичні механізми вивільнення гліцину.
REFERENCES
1. E. A. Barnard, “Receptor classes and the transmitter-gated ion
channels,” Trends Biochem. Sci., 17, 368-374 (1992).
2. H. Betz, J. Kuhse, V. Schmieden, et al., “Structure And
functions of inhibitory and excitatory glycine receptors,” Ann.
New York Acad. Sci., 868, 667-676 (1999).
3. K. Morita, N. Motoyama, T. Kitayama, et al., “Spinal
antiallodynia action of glycine transporter inhibitors in
neuropathic pain models in mice,” J. Pharmacol. Exp. Ther.,
326, 633-645 (2008).
MODULATION OF GLYCINERGIC TRANSMISSION IN THE RAT SPINAL DORSAL COMMISSURAL NUCLEUS
НЕЙРОФИЗИОЛОГИЯ / NEUROPHYSIOLOGY.—2009.—T. 41, № 4308
4. H. U. Zeilhofer, “The glycinergic control of spinal pain
processing,” Cell Mol. Life Sci., 62, 2027-2035 (2005).
5. K. Hosl, H. Reinold, R. J. Harvey, et al., “Spinal prostaglandin
E receptors of the Ep2 subtype and the glycine receptor alpha3
subunit, which mediate central inflammatory hyperalgesia, do
not contribute to pain after peripheral nerve injury or formalin
injection,” Pain, 126, 46-53 (2006).
6. J. W. Lynch and R. J. Callister, “Glycine receptors: a new
therapeutic target in pain pathways,” Curr. Opin. Invest.
Drugs, 7, 48-53 (2006).
7. S. M. Wojcik, S. Katsurabayashi, I. Guillemin, et al., “A
shared vesicular carrier allows synaptic corelease of GABA
and glycine,” Neuron, 50, 575-587 (2006).
8. P. Legendre, “The glycinergic inhibitory synapse,” Cell Mol.
Life Sci., 58, 760-793 (2001).
9. H. Betz, B. Schmitt, C. M. Becker, et al., “Structure and
biology of central nervous system neurotransmitter receptors,”
Biochem. Soc. Trans., 15, 107-108 (1987).
10. P. Braquet, “Proofs of involvement of PAF-acether in various
immune disorders using BN 52021 (ginkgolide B): A powerful
PAF-acether antagonist isolated from Ginkgo biloba L,”
Adv. Prostaglandin Thromboxane Leukot. Res., 16, 179-198
(1986).
11. E. L. Kondratskaya, H. Betz, O. A. Krishtal, and B. Laube,
“The beta subunit increases the ginkgolide B sensitivity of
inhibitory glycine receptors,” Neuropharmacology, 49, 945-
951 (2005).
12. L. Ivic, T. T. Sands, N. Fishkin, K. Nakanishi, et al., “Terpene
trilactones from Ginkgo biloba are antagonists of cortical
glycine and GABA(A) receptors,” J. Biol. Chem., 278, 49279-
49285 (2003).
13. S. Katsurabayashi, H. Kubota, H. Higashi, et al., “Distinct
profiles of refilling of inhibitory neurotransmitters into
presynaptic terminals projecting to spinal neurones in immature
rats,” J. Physiol., 560, 469-478 (2004).
14. L. J. Wu, Y. Li, and T. L. Xu, “ Co-release and interaction of
two inhibitory co-transmitters in rat sacral dorsal commissural
neurons,” NeuroReport, 13, 977-981 (2002).
15. J. Nabekura, T. L. Xu, J. S. Rhee, et al., “Alpha2-adrenoceptor-
mediated enhancement of glycine response in rat sacral dorsal
commissural neurons,” Neuroscience, 89, 29-41 (1999).
16. E. D. Schomburg, E. Jankowska, and F. K. Wiklund,
“Nociceptive input to spinal interneurones in reflex pathways
from group I muscle afferents in cats,” Neurosci. Res., 38,
447-450 (2000).
17. N. Akaike and A. J. Moorhouse, “Techniques: Applications of
the nerve-bouton preparation in neuropharmacology,” Trends
Pharmacol. Sci., 24, 44-47 (2003).
18. N. Harata, J. Wu, H. Ishibashi, et al., “Run-down of the GABAA
response under experimental ischemia in acutely dissociated
CA1 pyramidal neurons of the rat,” J. Physiol, 500, Part 3,
673-688 (1997).
19. T. Shirasaki, K. Aibara, and N. Akaike, “Direct modulation of
GABAA receptor by intracellular ATP in dissociated nucleus
tractus solitarii neurons of rat,” J. Physiol., 449, 551-572
(1992).
20. N. Akaike, N. Murakami, S. Katsurabayashi, et al., “Focal
stimulation of single GABAergic presynaptic boutons on the
rat hippocampal neuron,” Neurosci. Res., 42, 187-195 (2002).
21. J. S. Rhee, Z. M. Wang, J. Nabekura, et al., “ATP facilitates
spontaneous glycinergic IPSC frequency at dissociated rat
dorsal horn interneuron synapses,” J. Physiol., 524, Part 2,
471-483 (2000).
22. R. Hawthorne, B. A. Cromer, M. W. Parker, and J. W. Lynch,
“Molecular determinants of ginkgolide binding in the glycine
receptor pore,” J. Neurochem., 98, 395-407 (2006).
23. I. S. Jang, H. J. Jeong, S. Katsurabayashi, and N. Akaike,
“Functional roles of presynaptic GABA(A) receptors on
glycinergic nerve terminals in the rat spinal cord,” J. Physiol.,
541, 423-434 (2002).
24. E. L. Kondratskaya, P. V. Lishko, S. S. Chatterjee, and
O. A. Krishtal, “BN52021, a platelet activating factor
antagonist, is a selective blocker of glycine-gated chloride
channel,” Neurochem. Int., 40, 647-653 (2002).
25. K. Murase, M. Randic, T. Shirasaki, et al., “Serotonin suppresses
N-methyl-D-aspartate responses in acutely isolated spinal dorsal
horn neurons of the rat,” Brain Res., 525, 84-91 (1990).
K. NONAKA, E. KONDRATSKAYA, M. MAEDA, et al.
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