Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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Overstreet, Linda S. and Gary L. Westbrook. Paradoxical Reduction of Synaptic Inhibition by Vigabatrin. J. Neurophysiol. 86: 596-603, 2001. GABAergic inhibition, a primary target for pharmacological modulation of excitability in the CNS, can be altered by multiple mechanisms including alteration of GABA metabolism. Gamma-vinyl GABA (vigabatrin, GVG) is an irreversible inhibitor of the GABA catabolic enzyme GABA transaminase, thus its anticonvulsant properties are thought to result from an elevation of brain GABA levels. We examined the effects of GVG on GABAergic synaptic transmission in hippocampal slices. GVG unexpectedly reduced miniature and evoked inhibitory postsynaptic currents (IPSCs) in dentate granule cells. The reduction in synaptic events was accompanied by an increase in tonic GABAA receptor-mediated current. These effects developed slowly and persisted following wash out of GVG. The GVG pretreatment reduced sucrose-evoked GABA release as well as postsynaptic sensitivity to exogenous GABA, indicating that both pre- and postsynaptic mechanisms contributed to the reduction in synaptic currents. These results suggest that tonic rather than phasic increases in GABA underlie the anticonvulsant properties of GVG, and that mechanisms that elevate brain neurotransmitter levels do not necessarily correlate with enhanced synaptic release.
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INTRODUCTION |
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The GABA synthetic enzyme
glutamate decarboxylase (GAD) and the catabolic enzyme GABA
transaminase affect CNS excitability by regulating transmitter levels
in GABAergic neurons. Experimental manipulation of GAD activity
suggests that the cytoplasmic GABA concentration in presynaptic
terminals may influence vesicular GABA release. For example, genetic
deletion of GAD65, the GAD isoform located in nerve terminals, reduces
K+-induced GABA release (Hensch et al.
1998) as well as GABA that is released during sustained
stimulation (Tian et al. 1999
). The increased seizure
susceptibility observed in GAD65 knockout mice may be a consequence of
reduced GABA release (Asada et al. 1996
; Kash et
al. 1997
). Pharmacological inhibition of GAD activity also
reduces GABA release (Golan and Grossman 1996
), and it
has been proposed that estradiol suppresses GABAergic inhibition via inhibition of GAD (Murphy et al. 1998
). Surprisingly,
the reduction in synaptic GABA release in GAD65-deficient animals is
due to fewer vesicles released rather than a decrease in the number of molecules per vesicle (Tian et al. 1999
). Together these
results suggest that the level of GABA in the presynaptic terminal
influences the release of synaptic vesicles.
Presynaptic GABA levels may be elevated by interfering with its
degradation. For example, gamma-vinyl GABA (vigabatrin, GVG), an
irreversible inhibitor of GABA transaminase, increases GABA levels in
synaptosomes (Löscher et al. 1989). In addition to elevating the intracellular concentration of GABA (Jung et al. 1977
), GVG increases the basal level of extracellular GABA and K+-stimulated GABA release (Abdul-Ghani et
al. 1981
; Neal and Shah 1989
; Qume and
Fowler 1997
; Qume et al. 1995
). The
anticonvulsant properties of GVG in animal models of temporal lobe
epilepsy and human partial epilepsy are thought to arise from an
elevation of brain GABA levels. But it is not clear whether the
anticonvulsant properties result from the elevation in basal
extracellular GABA or from an increase in activity-dependent GABA release.
We investigated the action of GVG on GABAergic synaptic transmission in dentate granule cells of rat hippocampal slices. Contrary to the notion that elevated intracellular GABA enhances synaptic GABA release, our results indicate that GVG dramatically reduces evoked GABA release. This reduction was accompanied by an increase in ambient GABA as measured by tonic GABAA receptor-mediated currents. Our results suggest that the anticonvulsant properties of GVG result from an elevation of basal extracellular GABA rather than enhanced synaptic release.
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METHODS |
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Whole cell voltage-clamp recordings of granule
cells in transverse hippocampal slices were made from postnatal
day 14-20 (P14-20) Sprague-Dawley rats. During
recordings, slices were continuously perfused with an extracellular
solution containing (in mM) 125 NaCl, 25 NaHCO3,
2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 25 D-glucose, bubbled with 95% O2-5%
CO2. Patch pipettes were filled with (in mM) 140 KCl, 10 EGTA, 10 HEPES, and 2 Mg2ATP, adjusted to
pH 7.3 and 310 mOsm (1.5-4 M resistance). Visually identified
granule cells were voltage clamped at
70 mV using an Axopatch 200B
amplifier (Axon Instruments) and maintained at room temperature.
Currents were filtered at 2 kHz and sampled at 10 kHz. Miniature
inhibitory postsynaptic current events (mIPSCs) were isolated by adding
TTX (0.5 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 µM)
to the extracellular solution. CGP55845 (1-2 µM) was used to block
GABAB receptors. GVG (100-400 µM) was either included in the incubation chamber (pretreatment), or bath applied in
the recording chamber (acute treatment). mIPSCs were detected with the
template-matching procedure of Axograph 4.0 (Axon Instruments), using
the sum of one rising (
= 400 µs) and one falling (
= 20 ms) exponential as a template. To prevent overlapping events, the
mIPSC decay time course was measured by averaging the subset of events
separated by
100 ms. The decay was fitted with the sum of two
exponential functions, and the weighted decay was calculated from the
equation Ax
1 + Ax
2, where
A is the relative amplitude of each component and
is its
time constant. Tonic GABAA receptor-mediated currents were measured during a 1-min segment in the presence and
absence of SR95531 (5 µM). All points histograms were then fitted
with a Gaussian function. Because the amplitude of tonic GABAergic
currents was small and occasionally obscured by gradual changes in the
holding current, tonic currents were sometimes reported as the standard
deviation of the current. IPSCs were evoked with a bipolar stimulating
electrode (FHC, Bowdoinham, ME) placed in the granule cell layer (0.1 ms duration, 2-20 mV). For measurements of
ECl
, CsCl replaced KCl, and QX314 (10 mM) was included in the intracellular solution to block voltage-gated
K+ and Na+ currents,
respectively. Junction potentials were not compensated. GABA and high
osmolar solutions (extracellular solution increased to 650 mOsmol with
sucrose) were applied via pressure ejection (2-4 psi, Picospritzer,
General Valve) from a recording pipette placed near the soma of the
recorded cell. Series resistance was monitored throughout each
experiment (4-12 M
before 70-90% compensation); data were
discarded if substantial increases were observed. Data are expressed as
means ± SE. Unless noted, statistical significance was determined
by two-tailed t-tests or Mann-Whitney U tests at the P < 0.05 level.
All drugs were dissolved in water or DMSO (final concentration <0.3%) and added to the bath perfusion solution or incubation chamber. CNQX, 2-amino-5-phosphonopentanoic acid (AP5), and CGP55845 were purchased from Tocris Cookson (Ballwin, MO). 1-(2-(((Diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NO-711) and SR95531 were purchased from Research Biochemicals (Natick, MA). GABA, gamma-vinyl GABA, strychnine, and TTX were purchased from Sigma (St. Louis, MO).
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RESULTS |
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Vigabatrin pretreatment reduces GABAergic IPSCs
To examine the effect of elevated GABA on inhibitory synaptic transmission, hippocampal slices were incubated for 2-5 h in GVG (100-400 µM). This protocol resulted in an accumulation of extracellular GABA (see below) following irreversible inhibition of GABA transaminase. Recordings were made from pretreated slices following wash out of GVG (>20 min). GVG pretreatment resulted in a concentration-dependent reduction in mIPSCs recorded in 5 µM CNQX, 0.5 µM TTX, and the GABAB receptor antagonist CGP55845 (2 µM, Fig. 1). GVG (400 µM) reduced the median mIPSC amplitude by 57% (10.9 ± 1.0 pA, mean ± SE, n = 11, vs. 25.2 ± 2.5 pA in untreated controls, n = 8) and greatly reduced the mIPSC frequency (interevent interval 10 ± 4 s vs. 1.0 ± 0.2 s in controls). The decay of mIPSCs was unaffected by GVG (Fig. 1C, inset). Thus GVG pretreatment did not affect the gating of the underlying channels, but fewer channels were activated.
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Consistent with the dramatic reduction in mIPSCs, GVG pretreatment also
reduced the amplitude of evoked IPSCs. GVG pretreatment (200 µM)
reduced the amplitude of IPSCs evoked by focal stimulation of the
granule cell layer in CNQX (591 ± 167 pA following GVG pretreatment, n = 11, vs. 1,099 ± 156 pA in
untreated controls, n = 6, not shown). Evoked IPSCs
were reduced in GVG-pretreated slices even though greater stimulus
intensities (10 ± 2 mV vs. 3.6 ± 0.5 mV in untreated
controls) were required to evoke responses. However, GVG did not appear
to effect the release probability or the GABA transient. Paired-pulse
depression (200-ms interval) was not affected by GVG pretreatment
(0.82 ± 0.03, n = 7 vs. 0.88 ± 0.06 in
untreated controls, n = 6), suggesting that there was not a sizeable change in the probability of release. Displacement of a
low-affinity antagonist by synaptically released transmitter can be
used to estimate the concentration profile of the transmitter in the
cleft (Clements et al. 1992). The low-affinity
GABAA receptor antagonist SR95301 (5 µM)
(Jones et al. 1998
) reduced the IPSC by a similar degree
in GVG-pretreated and control cells (by 62 ± 4% vs. 61 ± 5% in untreated controls, n = 3), suggesting that GVG
did not alter the GABA transient in the synaptic cleft.
Vigabatrin pretreatment elevates tonic GABA
GVG pretreatment increased baseline current noise by 177% (n = 13 control, n = 17 GVG). A saturating concentration of the high-affinity GABAA receptor competitive antagonist SR95531 reduced the baseline noise by 60 ± 5% in GVG-treated slices (n = 8), compared with 30 ± 12% in control slices (n = 7, Fig. 2). This indicates that the reduction in mIPSC amplitude and frequency was accompanied by an elevation of ambient extracellular GABA. Tonic GABAA receptor-mediated currents were quantified by making Gaussian fits to all-points histograms from stable 1-min current segments in the presence and absence of the antagonist. The contribution of synaptic events to the Gaussian fits was negligible. When GABAA receptors were blocked with SR95531, there was no difference in the mean or standard deviation of the membrane noise between control and GVG-pretreated cells (13.0 ± 1.2 pA, n = 8, vs. 15.6 ± 1.8 pA in control, n = 7).
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We next compared the effects of acute applications of GVG or exogenous
GABA to untreated slices in the presence of TTX, CNQX, and CGP55845. In
each cell GVG increased tonic current fluctuations after several
minutes of application (Fig.
3A, asterisk). After 20 min,
GVG increased the current to 221 ± 11% (n = 5),
compared with 105 ± 7% in untreated control cells
(n = 5). Acute treatment with GVG reduced the mIPSC
amplitude (to 69 ± 4% control), while the interevent interval
was unaffected (105 ± 11%). In control slices the mIPSC
amplitude and interevent interval were stable over the same time period
(n = 5). The tonic current and mIPSC amplitude
reduction produced by GVG were similar to that produced by a low
concentration of exogenous GABA (Fig. 3C), although the effects of GVG developed more slowly. GABA (5 µM, n = 4) increased tonic current fluctuations (230 ± 100%) and
decreased the mIPSC amplitude (to 78 ± 5%), without changing the
mIPSC interevent interval (99 ± 18%). The increased tonic
current and reduced synaptic current were not due to a direct action of
GVG on GABAA receptors because GVG (400 µM) did
not significantly affect currents activated by GABA (10 mM, 400 ms) in
nucleated patches from untreated granule cells (93 ± 3% of
control, n = 3, not shown). GVG also did not activate a
current in nucleated patches (n = 3). Thus the acute effects of GVG are compatible with an accumulation of extracellular GABA in the micromolar range that desensitizes postsynaptic
GABAA receptors (Overstreet et al.
2000). However, the dramatic reduction of mIPSC frequency
observed in the pretreatment protocol was not observed during acute
applications of either GVG or GABA.
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Presynaptic mechanisms
The dramatic reduction in mIPSC frequency following GVG pretreatment potentially could be explained either by a presynaptic reduction in GABA release or a marked postsynaptic reduction in GABA sensitivity such that mIPSCs were below detection thresholds. Activation of presynaptic GABAB receptors by elevated tonic GABA was an obvious possibility that was ruled out because mIPSCs were recorded in the presence of the GABAB receptor antagonist CGP55845. Inclusion of CGP55845 (10 µM) in the incubation chamber also failed to block the reduction in mIPSC frequency caused by GVG pretreatment (median interevent interval 15.6 ± 5.7 s in GVG + CGP, n = 5; 10.6 ± 4.1 s in GVG, n = 11, Fig. 4, B and D). In the absence of CGP55845, acute treatment with GVG reduced the mIPSC frequency in three of five cells (median interevent interval increased by 223%, n = 3; Mann-Whitney U test, Fig. 4, A and C), whereas no frequency reduction was observed in the presence of CGP55845 (n = 5, Fig. 4C). Thus elevated ambient GABA can reduce the frequency of mIPSCs via presynaptic GABAB receptors, but it does not account for the dramatic reduction of mIPSCs that occurs following prolonged pretreatment with GVG.
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A second explanation for the dramatic reduction in mIPSCs could be a
decrease in the availability or filling of synaptic vesicles. This
would be manifested as a decrease in the readily releasable vesicle
pool evoked by hyperosmotic sucrose (Rosenmund and Stevens 1996; Stevens and Tsujimoto 1995
). Indeed, the
response elicited by hypertonic extracellular solution (650 mOsmol) was
greatly attenuated in GVG-pretreated slices. In control cells, sucrose elicited a barrage of mIPSCs (11 ± 0.7 Hz measured for 5 s
following application, n = 5) compared with 2.3 ± 0.4 Hz in GVG-pretreated slices (n = 6, Fig.
5A). Thus GVG appears to
reduce the readily releasable pool of GABAergic vesicles.
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Postsynaptic mechanisms
Because GVG reduced mIPSC amplitudes as well as frequency, we also tested the possibility that there was a reduction in postsynaptic receptor sensitivity. Responses evoked by a brief local application of GABA (10 mM) were reduced in GVG-pretreated slices. In control, the GABA-evoked current was 8.6 ± 0.5 nA (n = 8), whereas following GVG pretreatment the GABA-evoked current was 4.5 ± 0.8 nA (n = 10, Fig. 5C). However, a concentration of SR95531 expected to produce at least a 90% reduction in postsynaptic responsiveness did not significantly alter the measured increase in sucrose-evoked mIPSC frequency in untreated slices (7.8 ± 1.6 Hz, n = 4, Fig. 5B). This suggests that the reduction in postsynaptic sensitivity following GVG pretreatment did not reduce sucrose-evoked mIPSCs below detectable amplitudes, thereby causing an artifactual reduction in mIPSC frequency. Thus both pre- and postsynaptic mechanisms contribute to the reduction in synaptic currents.
It is unlikely that the reduction in sensitivity was due to a direct
action of GVG on postsynaptic GABAA receptors
because GVG was washed from the slice before experiments began, and GVG had no effect on GABA-activated currents in nucleated patches. The
elevation in tonic ambient GABA could reduce postsynaptic GABAergic
responses by several mechanisms, including agonist-dependent receptor
internalization (Tehrani and Barnes 1991). To block
agonist-dependent internalization, we pretreated slices with GVG and an
antagonist cocktail of bicuculline (10 µM), CNQX (5 µM), and
CGP55845 (2 µM). This treatment did not block the effects of GVG
(n = 6, not shown), as the median mIPSC interevent
interval (12.5 ± 3 s after GVG vs. 1.4 ± 0.3 s in
control) and tonic GABAA receptor-mediated current (13 ± 2 pA, mean ± SD, after GVG vs. 4 ± 2 pA
in control) were increased compared with slices pretreated only with
the antagonist cocktail (n = 5). Furthermore, if
agonist-induced internalization accounted for the reduction in mIPSCs,
we would expect that prolonged GABA pretreatment would mimic the
effects of GVG pretreatment. However, slice pretreatment with 10 µM
GABA (>2 h) did not alter the mIPSC amplitude (24 ± 2 pA,
n = 6) or frequency (interstimulus interval 2.5 ± 0.7 s, P = 0.06).
Two other possible explanations for the GVG-induced reduction in
postsynaptic sensitivity are indirect changes in membrane properties
(Frerking et al. 1999), and a shift in the IPSC reversal potential (Thompson and Gahwiler 1989
). A tonic
current could reduce the membrane resistance, causing a reduction in
IPSC amplitude as a result of space-clamp error (Frerking et al.
1999
; Spruston et al. 1993
). A space-clamp error
would be expected to reduce the amplitude of EPSCs as well as IPSCs.
However, in GVG-pretreated slices, mEPSCs were not larger when
GABAergic currents were blocked by SR95531 (2 µM, 15.8 ± 1.0 pA
in the absence of SR95531 vs. 13.1 ± 1.5 pA in SR95531,
n = 3). Finally, we examined the possibility that GVG
reduced GABAergic inhibition by a shift in the
Cl
reversal potential. Using an intracellular
solution containing CsCl and QX314, we found that the reversal
potential for the evoked IPSC was unaffected by GVG pretreatment (10 vs. 7 mV), but the synaptic conductance was reduced approximately
fivefold (n = 4 control and GVG-pretreated cells, not
shown). Thus GABAA receptor internalization, a
change in membrane resistance, and a shift in the
Cl
reversal potential do not appear to account
for the reduction in postsynaptic sensitivity caused by GVG pretreatment.
Role of GABA transport
In GVG-pretreated slices, application of the GABA uptake blocker NO711 (100 µM) produced a robust inward current and increase in membrane noise, whereas only a small current was detected in control slices (average current 56 ± 15 pA following GVG vs. 12 ± 5 pA in control; n = 6). This current was blocked by SR95531, consistent with activation of GABAA receptors by endogenous GABA (Fig. 6). A lower concentration of NO711 (20 µM) also increased the tonic GABAA receptor-mediated current fluctuation in GVG-pretreated slices by 430 ± 212% (n = 3) compared with control slices (160%, n = 2). These results suggest that the role of GABA transporters in maintaining low extracellular levels of GABA was enhanced following inhibition of GABA transaminase.
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Interestingly, NO711 (20 µM) applied before and during the acute GVG protocol blocked the reduction in mIPSC amplitude (98% of control) and attenuated the increase in tonic GABA current (122% of control, n = 2, not shown). To further investigate this phenomenon, mIPSCs were recorded in slices after dual pretreatment with NO711 (20 µM) and GVG (400 µM, >3 h). In this protocol NO711 blocked the effect of GVG (Fig. 7, n = 6). mIPSC amplitudes were 27 ± 4 pA versus 25 ± 2 pA in untreated controls, compared with 11 ± 1 pA after GVG alone. The interevent interval was only slightly reduced, 2.1 ± 0.2 s versus 1.0 ± 0.2 s in control, compared with 10 ± 4 s after GVG alone. Tonic current fluctuations measured after dual pretreatment (following wash out of NO711 and GVG, 61 ± 17 pA, mean ± SD, n = 3) were not different from pretreatment with NO711 alone (69 ± 32 pA, n = 3). Following dual pretreatment the tonic current fluctuations in NO711 (101 ± 19 pA, n = 6) were reduced compared with fluctuations in NO711 applied after the GVG pretreatment (386 ± 60 pA, n = 3). Thus NO711 pretreatment blocked both the reduction of mIPSCs and the increase in tonic GABAergic current fluctuations produced by GVG.
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DISCUSSION |
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Our results indicate that GVG dramatically reduces synaptic
inhibitory currents. The reduction in synaptic currents was accompanied by an increase in tonic GABAA receptor-mediated
currents that presumably reflect an elevation of ambient extracellular
GABA (Abdul-Ghani et al. 1981; Neal and Shah
1989
; Qume and Fowler 1997
; Qume et al.
1995
). Tonic GABAA receptor-mediated
currents reduce cellular excitability of cerebellar granule cells via
shunting inhibition (Brickley et al. 1996
), and we
expect a similar phenomenon to occur following GVG pretreatment.
However, the net change in cellular excitability resulting from the
elevation of tonic inhibition and simultaneous reduction in synaptic
inhibition will depend on a number of factors. The shift in inhibition
from a mode dominated by brief synaptic events to one mediated by a
small but sustained elevation of ambient GABA could thus have a
significant impact on network activity. This situation is particularly
relevant to chronic use of GVG as an anticonvulsant.
Mechanisms of action of GVG
The dramatic reduction in synaptic currents following GVG
pretreatment is consistent with the reduced Ca2+
dependence of K+-stimulated GABA release
following GVG treatment (Qume and Fowler 1997). Our
finding that GVG attenuated GABA release in response to a sucrose
challenge strongly suggests a reduction in the readily releasable
vesicle pool. This is consistent with our observation that the
paired-pulse ratio, a common measure of release probability, was
unaffected, whereas the mIPSC frequency was severely reduced. Although
there may be a correlation between the size of readily releasable pool
and release probability (Rosenmund and Stevens 1996
),
this relationship can be masked by a subset of unaffected terminals
that have normal release characteristics (Augustin et al.
1999
). The GVG-induced reduction of mIPSC frequency is similar to the actions of bafilomycin, an inhibitor of vesicular transport (Zhou et al. 2000
). High concentrations of GVG can also
inhibit the vesicular GABA transporter (IC50 = 7.5 mM) (McIntire et al. 1997
), suggesting that GVG
may reduce the readily releasable pool by inhibition of vesicle
filling. This mechanism could account for the reduction in mIPSC
frequency if GVG accumulates in the presynaptic terminal during
prolonged incubation, and is not readily cleared following GVG wash out.
GVG pretreatment also had postsynaptic effects that persisted after
prolonged wash out of GVG. Most obvious was the elevation of ambient
GABA that resulted in tonic GABAA
receptor-mediated current fluctuations. The rate of glutamate
transport is reduced following elevation of intracellular transporter
substrates (Takahashi et al. 1996). A similar reduction
in GABA transport may occur when intracellular GABA is elevated
(Attwell et al. 1993
; but see Lu and Hilgemann
1999
), thereby allowing extracellular accumulation of GABA.
Direct inhibition of GABA transport by GVG is unlikely to fully account
for the elevation of ambient GABA because it persisted after prolonged
GVG wash out, and GVG blocks GABA uptake with low efficacy
(Eckstein-Ludwig et al. 1999
; Schousboe et
al. 1986
). In addition, GVG increased tonic GABAergic current
fluctuations more gradually than NO711 (Figs. 3A and
6A), presumably reflecting the gradual accumulation of
intracellular GABA. The increase in extracellular GABA was accompanied
by a reduction in postsynaptic receptor sensitivity that was not due a
shift in the IPSC reversal potential, passive shunting, or
agonist-induced receptor internalization. These results suggest that
the increase in ambient GABA desensitized postsynaptic receptors
(Overstreet et al. 2000
), although the lack of an agent
that blocks GABAA receptor desensitization
prevented us from testing this possibility directly.
Our results are consistent with previous findings that acute GVG
reduced GABAergic inhibition of evoked population spikes in CA1
(Jackson et al. 1994). GVG is also reported to reduce
activity-dependent suppression of IPSCs that occurs during a train of
stimuli by reducing the function of presynaptic
GABAB autoreceptors (Jackson et al.
2000
). The accumulation of ambient GABA may be expected to
tonically activate GABAB receptors, thereby
occluding further activation by synaptically released GABA.
GABAB receptor activation has also been
implicated in GVG's inhibition of cocaine-induced increases in nucleus
accumbens dopamine (Ashby et al. 1999
). Our finding that
acute GVG application reduced the mIPSC frequency only in the absence
of GABAB antagonists confirms the ability of GVG
to promote GABAB receptor-mediated modulation,
even though this mechanism cannot account for the dramatic reduction in
synaptic inhibition following prolonged pretreatment with GVG.
Inhibition of GABA transporters following GVG pretreatment revealed an
increase in transporter activity. This may reflect an increase in tonic
GABA transport to counteract the increase in ambient GABA, although an
increase in the number of functional transporters in response to
elevated extracellular GABA could also contribute (Bernstein and
Quick 1999). This result also suggests that reversal of
transport does not underlie the elevated ambient GABA, in which case
NO711 would be expected to reduce tonic GABAA receptor-mediated currents. The simplest explanation for our finding that concurrent treatment with NO711 blocked the effect of GVG is that
NO711 blocked uptake of GVG into neurons and glia
(Eckstein-Ludwig et al. 1999
; see also Schousboe
et al. 1986
), thereby preventing intracellular accumulation
required for inhibition of GABA transaminase.
Relevance to the use of GVG as an anticonvulsant
The concentration of GVG used here is within the range estimated
to occur in the rat CNS following a single anticonvulsant dose of 1500 mg/kg (Abdul-Ghani et al. 1981). A similar dose has time-dependent effects on seizure susceptibility. At 4 h following injection, GVG has proconvulsant effects, whereas anticonvulsant effects appear 24 h later (Löscher et al.
1989
). The dramatic reduction in synaptic inhibitory currents
observed here may correspond to the proconvulsant period following a
single high-dose injection. However, the role of synaptic GABAergic
inhibition in models of temporal lobe epilepsy is unclear. Depending on
the region and parameter measured, synaptic inhibition following
seizure activity can be enhanced, reduced, or unchanged (reviewed by
Ben-Ari and Cossart 2000
). For example, somatic
inhibition of CA1 pyramidal cells is increased by hyperactivity of
inhibitory interneurons, whereas degeneration of a subpopulation of
interneurons reduces synaptic inhibition in the dendrites
(Cossart et al. 2001
). Experimental epileptogenesis is
also associated with an increase in synaptic inhibition in dentate
granule cells (Otis et al. 1994
) and cortical pyramidal
cells (Prince et al. 1997
). Regardless of whether these increases in synaptic inhibition are compensatory or promote seizure activity by enhanced synchronization (Cobb et al. 1995
),
GVG would be expected to counteract enhanced synaptic inhibition by
reducing synaptic GABA release as well as postsynaptic responsiveness. But the contribution of synaptic inhibition to cellular excitability may be overwhelmed by the elevation of ambient GABA produced by GVG.
Tonic GABAergic inhibition arising from a persistent elevation of
extracellular GABA would be expected to reduce excitability of both
interneurons and principal cells. An elevation of tonic inhibition may
also contribute to the clinical properties of the benzodiazepine
midazolam and the anesthetic propofol (Bai et al. 2001
).
Thus tonic inhibition produced by ambient GABA may represent an
important mechanism for modulating cellular excitability and network behavior.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants MH-12400 (L. S. Overstreet) and NS-26494 (G. L. Westbrook) and a grant from the Human Frontiers Science Program (G. L. Westbrook).
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FOOTNOTES |
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Address for reprint requests: L. S. Overstreet, Vollum Institute, L474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201 (E-mail: overstre{at}ohsu.edu).
Received 6 February 2001; accepted in final form 11 April 2001.
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REFERENCES |
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