1Respiratory and Neuroscience Research Groups, Faculty of Medicine, The University of Calgary, Calgary, Alberta T2N 4N1, Canada; and 2Department of Anesthesiology, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan
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ABSTRACT |
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Hamakawa, Toshiro,
Zhong-Ping Feng,
Nikita Grigoriv,
Takuya Inoue,
Mayumi Takasaki,
Sheldon Roth,
Ken Lukowiak,
Shabih U. Hasan, and
Naweed I. Syed.
Sevoflurane Induced Suppression of Inhibitory Synaptic
Transmission Between Soma-Soma Paired Lymnaea Neurons.
J. Neurophysiol. 82: 2812-2819, 1999.
The
cellular and synaptic mechanisms by which general anesthetics affect
cell-cell communications in the nervous system remain poorly defined.
In this study, we sought to determine how clinically relevant
concentrations of sevoflurane affected inhibitory synaptic transmission
between identified Lymnaea neurons in vitro. Inhibitory synapses were reconstructed in cell culture, between the somata of two
functionally well-characterized neurons, right pedal dorsal 1 (RPeD1,
the giant dopaminergic neuron) and visceral dorsal 4 (VD4). Clinically
relevant concentrations of sevoflurane (1-4%) were tested for their
effects on synaptic transmission and the intrinsic membrane properties
of soma-soma paired cells. RPeD1- induced inhibitory postsynaptic
potentials (IPSPs) in VD4 were completely and reversibly blocked by
sevoflurane (4%). Sevoflurane also suppressed action potentials in
both RPeD1 and VD4 cells. To determine whether the anesthetic-induced
synaptic depression involved postsynaptic transmitter receptors,
dopamine was pressure applied to VD4, either in the presence or absence
of sevoflurane. Dopamine (10]5 M) activated a
voltage-insensitive K+ current in VD4. The same
K+ current was also altered by sevoflurane; however, the
effects of two compounds were nonadditive. Because transmitter release from RPeD1 requires Ca2+ influx through voltage-gated
Ca2+ channels, we next tested whether the
anesthetic-induced synaptic depression involved these channels.
Individually isolated RPeD1 somata were whole cell voltage clamped, and
Ca2+ currents were analyzed in control and various
anesthetic conditions. Clinically relevant concentrations of
sevoflurane did not significantly affect voltage-activated
Ca2+ channels in RPeD1. Taken together, this study provides
the first direct evidence that sevoflurane-induced synaptic depression
involves both pre- and postsynaptic ion channels.
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INTRODUCTION |
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Inhalation anesthetics such as halothane,
isoflurane, and sevoflurane induce unconsciousness (general anesthesia)
in animals and are therefore extensively used during surgical
procedures (Krnjevic 1991). Anesthetics are generally
thought to perturb neuronal communication in the nervous system by
either interfering with postsynaptic transmitter receptors and
presynaptic transmitter release or alter various ion channel
conductances (see Bazil and Minneman 1989
;
Dilger et al. 1994
; Franks and Lieb 1988
;
Hirota and Roth 1997
; Pocock and Richards
1991
; Puil and El-Beheiry 1990
; Richards
1983
; Spencer et al. 1995
, 1996
;
Weight et al. 1992
). However, the precise cellular and
synaptic mechanisms underlying anesthetic action have not been fully
defined because synaptic transmission between defined sets of pre- and
postsynaptic neurons in vertebrates is often difficult to study directly.
Due to their relatively simpler nervous systems, various molluscan
species have been used extensively to determine how anesthetics affect
cell-cell communication in the nervous system. For instance, identified
neurons from Lymnaea (Franks and Lieb
1988, 1991a
; Girdlestone et al.
1989a
,b
; Spencer et al. 1995
,
1996
), Helix (Judge and Norman
1982
), and Aplysia (Arimura and Ikemoto
1986
) have been used to determine how halothane, isoflurane,
and enflurane affect synaptic transmission in the nervous system.
Girdlestone et al. (1989a
,b
) found that clinically
relevant concentrations of halothane (1-2%) induced complete
"anesthesia" (suppression of whole body reflexes) in
Lymnaea. Using isolated ganglionic preparations; they
subsequently showed that both halothane and isoflurane suppressed
chemical, but not electrical, synaptic transmission between identified
Lymnaea neurons (Girdlestone et al.
1989a
,b
). Halothane (1-2%) was also shown to suppress the
peptidergic synaptic transmission between cultured
Lymnaea neurons (Spencer et al. 1995
,
1996
). Similarly, cholinergic synaptic transmission
between identified Aplysia neurons was found to be
blocked by clinically relevant concentrations of enflurane
(Arimura and Ikemoto 1986
). Studies from the
laboratories of Franks and Lieb (1988
,
1991b
) have since identified and characterized various
ion channels that are altered by halothane in cultured
Lymnaea neurons. Together, the above studies have
established the usefulness of several simpler invertebrate preparations
for research on the cellular and synaptic mechanisms by which
anesthetics affect cell- cell interactions in the nervous system.
However, even in the above simple model systems, sevoflurane is yet to
be tested for its actions on synaptic transmission.
In this study, we utilized a recently developed model system
(Feng et al. 1997b), where a specific inhibitory
synapse between individually identifiable pre- [right pedal dorsal 1 (RPeD1)] and postsynaptic [visceral dorsal 4 (VD4)] neurons from the
mollusk Lymnaea stagnalis was reconstructed between the
cell bodies. This soma-soma model has several advantages over
conventionally used neurite-neurite synapses (Feng et al.
1997b
). For example, because the synapses develop between the
cell bodies (in the absence of neurites), they are suitable for direct
electrophysiological analysis. In addition, both the somata and their
synapses can be exposed simultaneously and directly to various
anesthetic agents.
RPeD1 and VD4 are respiratory central pattern generating (CPG) neurons,
and when stimulated electrically (either in vivo or in vitro), RPeD1
produces 1:1 inhibitory postsynaptic potentials (IPSPs) in VD4. This
synaptic transmission is dopaminergic and similar to that seen in the
intact brain (Syed and Winlow 1991a). The soma-soma
synapse between RPeD1 and VD4 provided us with an excellent opportunity
to test both pre- and postsynaptic mechanisms by which clinically
relevant concentrations of sevoflurane (1-4%) affected inhibitory
synaptic transmission in the nervous system.
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METHODS |
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Animals
Laboratory-raised stocks of the fresh water snail Lymnaea
stagnalis were maintained at room temperature (18-20°C) in
well-aerated artificial pond water and fed lettuce (Ridgway et
al. 1991). Snails with a shell length of 20-25 mm (approximate
age 4-6 mo) were used in all experiments.
Cell culture
Animals were dissected under sterile cell culture
conditions as described earlier (Syed et al. 1990). The
isolated central ring ganglia were washed several times (6-7 washes of
10 min each) with antibiotic saline (Gentamycin, 50 µg/ml). These
were subsequently enzyme treated (Trypsin, Sigma type III) for 20-40
min and pinned to the bottom of a dissection dish (Syed et al.
1990
). Fine forceps were used to remove the connective sheath
surrounding the somata. A fire-polished glass pipette attached to a
microsyringe (Gilmont) was used to extract individual neurons from the
intact ganglion. The isolated cells were plated on
poly-L-lysine-coated tissue culture dishes (Falcon 3001),
containing 3 ml of either defined medium (DM) or brain conditioned
medium (CM) (see Ridgway et al. 1991
). Soma-soma
synapses were prepared by juxtaposing freshly isolated somata of
identified neurons (see Feng et al. 1997
for details).
The paired neurons were left undisturbed overnight at room temperature.
Electrophysiology
SHARP ELECTRODE RECORDINGS.
Conventional intracellular recording techniques were used (Syed
and Winlow 1991b). Specifically, glass microelectrodes (1.5 mm
ID, WPI) were pulled on a vertical electrode puller (Kopf, 700C) and
filled with a saturated solution (3 M) of K2SO4
(resistance 20-40 M
). Neurons were viewed under a Zeiss (Axiovert
135 or Telaval 31) inverted microscope and impaled using Narishige
micromanipulators (model MO-103). The intracellular signals were
amplified via a preamplifier (NeuroData, IR-283), displayed on a
storage oscilloscope (Tektronix R5103N), and recorded on a Gould chart
recorder (Recorder 2200 s). All experiments were performed at room
temperature (18-22°C).
WHOLE CELL PATCH-CLAMP RECORDINGS.
Whole cell voltage-clamp (membrane rupture) or current-clamp recordings
were made using an Axopatch 1D amplifier (Axon Instruments, Foster
City, CA). Patch electrodes (1-2 M) were pulled from PG150T-7.5 glass tubing (Warner Instrument Hamden, CT), on a vertical pipette puller (Kopf 750, Tujunga, CA). To study K+
currents, pipettes were filled with filtered pipette solution containing (pH 7.4; in mM): 29 KCl, 1/11
Ca2+/BAPTA buffer, and 10 HEPES, supplemented
with 2 ATP-Mg and 0.1 GTP-Tris. To study Ca2+
currents, pipettes were filled with filtered (0.22 µm filter) cesium
pipette solution consisting of (in mM) 29 CsCl, 1/11 Ca/BAPTA buffer, 1 tetraethylammonium (TEA Cl), 10 HEPES, 2 ATP-Mg, and 0.1 GTP-Tris, and
the pH was adjusted to 7.4 (with CsOH). Potassium currents and membrane
potentials were recorded in standard Lymnaea saline
consisting of (in mM) 51.3 NaCl, 1.7 KCl, 1.5 MgCl2, and 4.1 CaCl2. To
record Ca+2 currents, the bath solution consisted
of (in mM) 52 TEA Cl, 1 MgCl2, 4 CaCl2, 1 4-aminopyridine (4-AP), and 5 HEPES (pH 7.4). After obtaining a gigaohm-seal, the series resistance
was compensated (<0.8 M
) using the membrane test function
(pClamp-7, Axon Instruments). The Ca2+ current measured in
this study was filtered at 1 kHz using a 4-pole Bessel filter and
digitized at a sampling frequency of 2 kHz. The voltage command
generation and data acquisition were carried out using a Dell (XPS
R350) computer equipped with a Digidata 1200 interface (Axon
Instruments) in conjunction with pClamp-7 software (Axon Instruments).
Gas chromatography
Sevoflurane (Maruishi Pharmaceutical, Japan) was vaporized
in 100% O2 using a sevoflurane Type-s MK
III-VII (Acoma) vaporizer and bubbled for at least 15 min into the
reservoirs containing Lymnaea saline (see Girdlestone
et al. 1989a). To minimize gas loss over time, all anesthetic
solutions were prepared fresh in sealed glass reservoirs. To determine
the final anesthetic concentration, 1 ml samples (from the outlet of
the experimental dish) were collected, and standards were prepared in
2-ml sealed vials, to which a 10-µl internal standard (enflurane) was
added. Sevoflurane samples and standards were analyzed using a HP 5890A
Gas Chromatograph, equipped with flame-ionizing detector (FID). The
column used was a DB-1 (30 m × 0.53 mm × 5.0 µm film J&W
Scientific), and the carrier gas (helium) flow was adjusted to 6.0 ml/min. The oven program were set to run at 40°C for 5.2 min, then
switched to 125°C at a rate of 30°C/min. Sample injections of 1.0 µl were split 1:3. The chromatograph acquisition and analysis was
performed with a HP 3396A Integrator. The above program produced
retention times of 2.4 min for sevoflurane and 3.4 min for the internal
standard enflurane. Analysis of perfusate, collected from the outlet of perfusion system, allowed us to establish molar concentrations of
anesthetic in the vicinity of neurons. For example, the lowest concentration (1%) used in this study corresponded to 0.3 mM; whereas
the highest sevoflurane concentration (7%) was estimated to be 2.5 mM.
Therefore 1% sevoflurane used in this study corresponded to an aqueous
equivalent of mammalian minimal alveolar concentration (MAC)
(see Franks and Lieb 1997
).
Anesthetic delivery
To minimize gas loss, Teflon tubing was used throughout the perfusion system, and sevoflurane solution was delivered directly to the somata using a computer-controlled pressurized perfusion system (Multi-Flo RVA-6 Norscan Instruments Winnipeg, Canada). This perfusion system was connected to the micromanifold, with dead volume <2 µl and allowed us to switch between two solutions in <200 ms.
Statistics
All parametric data were presented as means ± SD. Differences between mean values from each experimental group were tested using either a Student's t-test (for 2 groups) or repeated measures ANOVA (for multiple comparisons). Differences among groups were considered significant if P was <0.05 (P < 0.05).
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RESULTS |
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Sevoflurane suppressed inhibitory synaptic transmission between RPeD1 and VD4 in a concentration-dependent manner
To test sevoflurane (1-4%) for its action on inhibitory synaptic
transmission, synapses between the somata of RPeD1 and VD4 were
reconstructed in vitro (Fig.
1A). Following 12-18 h of
soma-soma pairing, simultaneous intracellular recordings were made from both cells, and a chemical synapse was demonstrated
electrophysiologically (Fig. 1B). Specifically, current
induced action potentials in RPeD1 produced 1:1 inhibitory postsynaptic
potentials (IPSPs) in VD4 (n = 37, Fig. 1B).
These synapses were similar to those seen in vivo (Syed and
Winlow 1991a). To determine whether sevoflurane affected the
amplitude of RPeD1-induced IPSPs in VD4, synapses were tested either in
the absence or presence of the anesthetic (Fig. 1C). Single
action potentials were induced in RPeD1 via current injection, and the
average IPSPs amplitude was analyzed in VD4, either in the absence or
presence of sevoflurane (Fig. 1C). Sevoflurane in a
dose-dependent (Fig. 1D) and reversible manner (Fig.
1C), blocked RPeD1-induced IPSPs in VD4 (n = 6). It is important to mention that because both neurons were
hyperpolarized by sevoflurane below the threshold for their spiking
activity, greater currents were often required to elicit action
potentials in the presence of this anesthetic.
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Sevoflurane blocked action potentials in both RPeD1 and VD4
To deduce the possible sites of sevoflurane actions, we began our analyses by examining effects on the intrinsic membrane properties of both RPeD1 and VD4. Specifically, individually isolated neurons RPeD1 and VD4 were maintained in vitro for 12-24 h. Current-clamp recordings were made from individual somata, and sevoflurane (4%) was applied directly to the cell body via a fast perfusion system. As shown in Fig. 2, sevoflurane completely blocked an induced (previously quiescent cells were depolarized just above the threshold for firing) train of action potentials in both RPeD1 and VD4. Throughout the course of anesthetic delivery, both cells remained hyperpolarized below their threshold for the activation of action potentials. Normal spiking activity, however, resumed immediately after wash out with normal saline.
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To further examine the potential synaptic sites for anesthetic actions, we sought to determine whether sevoflurane modulated the postsynaptic dopamine response in VD4.
Dopamine-induced effects on postsynaptic K+ conductance were mimicked by sevoflurane
RPeD1 is known to contain and release dopamine. Its synaptic
transmission with VD4 is dopaminergic (Magoski et al.
1995). Moreover, RPeD1-induced effects on VD4 activity are
mimicked by exogenous dopamine and involve changes in
K+ channel activity (Barnes et al.
1994
). To test the hypothesis that sevoflurane affected the
dopamine-activated, postsynaptic K+ current in
VD4, we made current-clamp and voltage-clamp recordings from
individually cultured cells. Exogenous dopamine (10 µm) application hyperpolarized VD4 from its resting membrane potential of
53 to
72
mV (69.8 ± 1.65; mean ± SD, n = 4). These
effects were mimicked by sevoflurane (4%; n = 9, Fig.
3A). When applied in the
presence of sevoflurane (4%), dopamine produced a further hyperpolarization in VD4. The net response was, however, similar to
that produced by dopamine alone (n = 5, Fig.
3A). The dopamine-induced hyperpolarizing response in VD4
was, however, fully matched by 7% (higher than clinical range)
sevoflurane. Together, these data suggest that both dopamine and
sevoflurane-induced hyperpolarizing responses in VD4 are highly
reproducible and reversible and do not desensitize rapidly. Moreover,
sevoflurane and dopamine-induced hyperpolarizing responses in VD4 were
nonadditive.
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We next sought to determine whether both dopamine and
sevoflurane-induced hyperpolarizing effects in VD4 involved the same K+ channel. To test this possibility,
voltage-clamp recordings were made from VD4 cells, and
K+ conductance was analyzed (Fig. 3B).
When held near its resting membrane potential (50 mV), dopamine (10 µm) application alone initiated a 92-pA outward current in VD4.
Similarly, sevoflurane at a concentration of 7% (higher than clinical
range) and 4% activated a 40- and 30-pA outward conductance,
respectively. Dopamine application in the presence of sevoflurane
enhanced this outward current; however, the sum of total outward
conductance activated by both compounds was equivalent to that induced
by dopamine alone (n = 5, Fig. 3B). These
data suggest that both dopamine and sevoflurane may activate the same
K+ channel. Figure 3C shows ramp
I-V relationship between sevoflurane-sensitive current
conductances, demonstrating that the voltage-insensitive outward
current is concentration dependent and has a reversal potential of
69.6 ± 3.1 mV (n = 8). These data are
consistent with those presented in Fig. 3A and suggest that
the current is most likely carried by K+ ions
[according to the Nernst equation, the K+
equilibrium potential for Lymnaea neurons is
71 mV (under
our experimental conditions with 29 mM K inside and 1.7 mM outside the
cell)].
Sevoflurane reduced presynaptic Ca2+ currents in RPeD1
Synaptic transmission between RPeD1 and VD4 requires
Ca2+ influx through high-voltage-activated
(HVA), and L-type-like Ca2+ channels
(Feng et al. 1997a). To test whether sevoflurane-induced suppression of synaptic transmission between RPeD1 and VD4 involved presynaptic Ca2+ channels,
Ca2+ currents were recorded from RPeD1 in a whole
cell voltage-clamp configuration. Specifically, individual somata were
isolated in culture, and macroscopic Ca2+
currents were recorded either in the absence or presence of
sevoflurane. Ca2+ current was evoked by 450-ms
depolarizing pulses applied from a holding potential of
80 to +50 mV,
in increments of 5 mV. As shown in Fig.
4A, inward
Ca2+ current in RPeD1 neurons was reduced
proportionally by increasing concentrations of sevoflurane. At
depolarizing pulses, applied from a holding potential of
80 to +10
mV, significant reductions (27% ±12) in the peak
Ca2+ current (n = 10, P < 0.05) were seen only at the higher (6% or equivalent to 6 × MAC) concentration of anesthetic. At lower
concentrations, however, changes in peak current amplitude were
insignificant. The Ca2+ current inactivation rate
was more sensitive to sevoflurane, and a marked increase was seen at
concentrations as low as 2% (equivalent to 2 × MAC; Fig. 4).
Sevoflurane-induced effects on the HVA Ca2+
currents were reversible, and the current amplitude returned to its
control level after the anesthetic was washed out with normal saline.
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DISCUSSION |
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In the present study, we demonstrated that sevoflurane blocked the
inhibitory synaptic transmission between neurons RPeD1 and VD4. Because
sevoflurane also blocked action potentials in both pre- and
postsynaptic neurons, we postulate that these effects on the intrinsic
membrane properties of the cells can account for the suppression of
synaptic transmission between the cells. Halothane was reported earlier
to suppress action potentials in other, as yet unidentified
Lymnaea neurons (Franks and Lieb 1997) and
together with our data, these studies suggest that the
anesthetic-induced effects on action potential parameters may be common
to various different neuronal cell types. As compared with halothane,
however (Franks and Lieb 1997
), we found that most
Lymnaea neurons (in addition to RPeD1 and VD4, data not
shown), were sensitive to lower concentrations of sevoflurane (2-4%).
These differences in neuronal responsiveness recorded in the above two
studies can be attributed either to the nature of different anesthetic
used or the type of cells examined. Furthermore, because sevoflurane hyperpolarized VD4 toward its reversal potential for K ions, we suggest
that the anesthetic-induced suppression of inhibitory synaptic
transmission may also involve voltage-insensitive K conductances.
The anesthetic-induced suppression of postsynaptic response has also
been shown to involve ligand-gated ion channels (Weight et al.
1992). For instance, Arimura and Ikemota (1986)
showed that enflurane-induced blockade of cholinergic synaptic
transmission between Aplysia neurons involved a suppression
of nicotinic Cl
currents. A similar suppression
of acetylcholine-induced, nicotinic Cl
currents
was observed in identified Lymnaea neurons (Franks
and Lieb 1991a
). In contrast,
-aminobutyric acid
(GABA)-activated Cl
currents in rat dorsal
root ganglia (DRG) neurons were significantly enhanced by halothane,
isoflurane, and enflurane (Nakahiro et al. 1989
). A
similar halothane-induced enhancement of GABA-gated inward current was
seen in cultured hippocampal neurons (Jones et al.
1992
). In the present study, we demonstrated that a
dopamine-sensitive, voltage-independent potassium conductance was
enhanced by sevoflurane. Because this enhancement brought the resting
membrane potential closer to the K+ equilibrium
potential, we propose that the anesthetic-induced modulation of
intrinsic membrane properties may be sufficient to account for the
suppression of RPeD1-induced IPSPs in VD4. Consistent with this idea
are our unpublished observations that RPeD1-induced IPSPs in VD4
persisted in the presence of anesthetic, when the postsynaptic membrane
potential was adjusted closer to its spike threshold (data not shown).
We believe that the sevoflurane-modulated potassium current reported in
this study is most likely analogous to the Ik(An) that was first identified and characterized in Lymnaea
neurons by Franks and Lieb (1991b). These
workers were the first to show that Ik(An) was cell type
specific and was activated by both general anesthetics and synaptic
inputs (Lopes et al. 1998
). Specifically, they found
that both halothane and dopamine activated the same K+
current in the intact ganglia. In cell culture, however, the anesthetic-sensitive neurons did not respond to exogenous dopamine (Franks and Lieb 1997
). Based on these results, they
concluded that dopaminergic responses seen in the intact ganglia were
most likely mediated indirectly via other neurons. Because in our
cultures, VD4 exhibited robust postsynaptic response to dopamine, in a
manner seen in vivo (Magoski et al. 1995
), we postulate
that dopamine and sevoflurane-induced effects most likely involved the
same K+ channel, although the underlying mechanisms remain
to be determined.
Presynaptic transmitter release requires Ca2+ influx
through voltage-gated, N-Type Ca2+ channels (Reuter
1996). Because most anesthetics are believed to suppress
transmitter release, it therefore seemed logical that the
anesthetic-induced synaptic depression might also involve these
Ca2+ channels. Because synaptic transmission between RPeD1
and VD4 requires Ca2+ influx through voltage-gated
Ca2+ currents (Feng et al. 1997a
), we asked
whether anesthetic-induced synaptic depression between these cells
involved Ca2+ currents. Our data (at room temperature)
demonstrated that the sevoflurane-induced modulation of
Ca2+ current amplitude was insignificant. These data are
consistent with other earlier studies, in which the voltage-gated
Ca2+ currents were found to be relatively insensitive to
clinically relevant concentration of volatile anesthetics (see
Franks and Lieb 1993
; Kress and Tas
1993
). In the present study, sevoflurane exerted more
pronounced effects on the rate of Ca2+ current
inactivation. Together, these data suggest that, although sevoflurane
did not exert significant effects on whole cell total Ca current, these
may nevertheless be sufficient to suppress transmitter release from RPeD1.
In summary, the data presented in this study demonstrate that clinically relevant concentrations of sevoflurane block inhibitory synaptic transmission between soma-soma paired Lymnaea neurons. The anesthetic-induced synaptic depression is both reversible and concentration dependent. We propose that sevoflurane may either block transmitter release by suppressing presynaptic Ca currents, or activate the dopamine-sensitive K conductance in the postsynaptic cell. The latter will hyperpolarize the membrane potential toward the reversal for K conductance and thus block the RPeD1-induced IPSPs in VD4. Although sevoflurane did not significantly suppress Ca2+ currents in RPeD1, these data do not, however, rule out the possibility that the resulting changes may indeed be sufficient to perturb Ca2+-dependent exocytosis.
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ACKNOWLEDGMENTS |
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The authors acknowledge excellent technical support by W. Zaidi and R. Bennington and thank Dr. Gaynor Spencer for critical comments on an earlier draft of this manuscript.
This work was supported by the Medical Research Council (MRC; Canada) and a University Research Grant (URG) to N. Grigori. Z.-P. Feng is a recipient of MRC-ALA studentship, and N. I. Syed is an Alberta Heritage Foundation for Medical Research senior scholar.
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FOOTNOTES |
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Address for reprint requests: N. I. Syed, Dept. of Cell Biology and Anatomy, Faculty of Medicine, The University of Calgary, 3330-Hospital Drive, NW, Calgary, Alberta T2 N 4N1, Canada.
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 8 April 1999; accepted in final form 7 July 1999.
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REFERENCES |
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