1Department of Physiology and 2Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
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Jensen, Kimmo, John D. C. Lambert, and Morten Skovgaard Jensen. Activity-Dependent Depression of GABAergic IPSCs in Cultured Hippocampal Neurons. J. Neurophysiol. 82: 42-49, 1999. Short-term depression of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) evoked between pairs of cultured rat hippocampal neurons was investigated using dual whole cell patch-clamp recordings. Paired stimuli applied to the GABAergic neuron resulted in paired-pulse depression (PPD) of the second IPSC (IPSC2) at interpulse intervals from 25 to 2,000 ms. CGP 55845A, but not CGP 35348, reduced PPD marginally. Brief paired-pulse applications of exogenous GABA indicated that postsynaptic factors made only minimal contribution to PPD of IPSCs. IPSC1 and PPD was reduced on lowering [Ca2+]o and enhanced on increasing [Ca2+]o. The potassium-channel blocker 4-aminopyridine (4-AP), which increases presynaptic Ca2+ influx, enhanced IPSC1 and PPD. Chelation of residual Ca2+ in the GABAergic boutons with EGTA-AM enhanced PPD. Stimulation of the presynaptic neuron at frequencies (f) ranging from 2.5 to 80 Hz resulted in tetanic depression of IPSCs, which declined rapidly and reached a plateau depending on f and [Ca2+]o. CGP 55845A decreased tetanic depression in the first part of the train, but this could be overcome with continued stimulation. We show that GABAergic IPSCs are robustly depressed by paired-pulse stimulation in cultured hippocampal neurons. The depression of IPSCs is mainly independent of presynaptic GABAB receptors and could be caused by depletion of releasable vesicles. Depleted synapses recover with a slow time course, depending on factors that regulate [Ca2+]i in the GABAergic boutons.
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INTRODUCTION |
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GABA is the major inhibitory neurotransmitter in
the CNS, where it acts on GABAA and
GABAB receptors. GABA is released in response to
an increase in Ca2+ influx through N- and
P/Q-type Ca2+ channels located near the secretory
apparatus (Ohno-Shosaku et al. 1994a). The release of
neurotransmitter at single synapses depends on their history of
activation, because different patterns of stimulation can evoke
frequency-dependent facilitation or depression (McCarren and
Alger 1985
). In experiments with hippocampal slices, inhibitory
postsynaptic potentials (IPSPs) evoked at fast rates are usually
depressed for several seconds due to feedback of GABA onto presynaptic
GABAB receptors (Nathan and Lambert
1991
; Roepstorff and Lambert 1994
), which
depresses GABA release by a G protein coupled mechanism
(Thompson et al. 1993
). However, a fraction of
inhibitory postsynaptic current (IPSC) paired-pulse depression (PPD) is
insensitive to GABAB receptor antagonists
(Lambert and Wilson 1994
). Glutamatergic synapses are
also subject to PPD, which seems to depend on mechanisms associated
with the basal secretory apparatus at the active zones (Dobrunz
et al. 1997
). It is therefore likely that excitatory and
inhibitory synapses share basal secretory mechanisms, as suggested by
Lambert and Wilson (1994)
. These mechanisms can be
studied in detail in cultured hippocampal neurons because GABAergic
IPSCs show a substantial PPD, which is insensitive to
GABAB receptor antagonists (Wilcox and
Dichter 1994
; Yoon and Rothman 1991
), although
functional GABAB receptors are present on the
terminals (Harrison 1990
).
We have made paired whole cell patch-clamp recordings from cultured hippocampal neurons and stimulated the presynaptic GABAergic neuron to evoke monosynaptic IPSCs, which were used to study the frequency-dependent depression of GABAergic IPSCs. Our results indicate that depletion of synaptic vesicles is responsible for depression of repetitively evoked IPSCs, although "spill-over" of GABA onto presynaptic GABAB autoreceptors does occur to a small extent. We also examined whether an increased level of internal Ca2+ contributes to refilling of depleted active zones. The latter process would be important for sustained release of transmitter during prolonged high-frequency activity at inhibitory GABAergic synapses.
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METHODS |
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Hippocampal culture preparation
Pregnant Sprague-Dawley rats were anesthetized by pentobarbital
sodium (50 mg/kg ip) at gestational day 17-18. Fetuses were removed and decapitated, and the hippocampi were dissected free. The
tissue was triturated mechanically in a HEPES-buffered dissection medium and plated on poly-D-lysine-coated coverslips in
35-mm Petri dishes. Plating medium consisted of minimal essential
medium with Earle's salts and Glutamax-1 (glutamine) supplemented with horse serum (HS, 10%), fetal calf serum (FCS, 10%), penicillin (50 IU
ml1), and streptomycin (50 µg/ml). Cultures
were grown in 5% CO2 and 10%
O2 at 37°C. Plating medium was fully replaced
by 2 ml feeding medium after 1 day in vitro, and thereafter 1 ml was
exchanged twice weekly. Feeding medium had the same composition as
plating medium except that FCS was omitted and HS was reduced to 5%.
The mitosis inhibitors 5'-Fluoro-2'-Deoxyuridine (FUDR, 15 µg/ml) and
uridine (35 µg/ml) were added after 3-4 days to inhibit glial overgrowth.
Electrophysiology
Coverslips with the cultured cells were placed in a chamber
mounted on an inverted Nikon Diaphot 200 microscope and perfused (1 ml/min) with an extracellular (control) medium containing (in mM) 140 NaCl, 3.5 KCl, 2.5 CaCl2, 2.5 MgCl2, 10 glucose, and 10 HEPES; pH 7.35 with
NaOH (22°C), osmolality 305 mosm/kg (Wescor 5500 osmometer).
Patch-clamp electrodes (3-6 M) were fabricated from borosilicate
glass (1.2 mm OD) on a Flaming/Brown P-97 puller (Sutter Instruments).
The presynaptic electrode contained (in mM) 140 KOH, 11 EGTA, 1 CaCl2, 2 MgCl2, 15 NaCl, 10 HEPES, 0.10 leupeptin, 2 MgATP; pH adjusted to 7.3 with methanesulfonic
acid, 290 mosm/kg. To increase the driving-force for
Cl
and to block Na+ and
K+ currents, the postsynaptic electrode contained
(in mM) 120 CsCl, 10 TEACl, 5 QX-314, 11 EGTA, 1 CaCl2, 1 MgSO4, 0.10 leupeptin, 4 MgATP, pH adjusted to 7.3 with CsOH, 290 mosm/kg. Whole
cell recordings were made at a holding potential
(Vhold) of
70 mV using Axopatch 200 and 200A amplifiers in voltage clamp. Excitatory synaptic responses
were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM)
and DL-2-amino-5-phosphonopentanoic acid
(DL-AP5; 50 µM). GABAergic neurons were identified by the
presence of autaptic IPSCs following a 3-ms depolarizing pulse to 0 mV.
Stimulation pulses (3 ms at 0 mV) were delivered by a pulse-generator (Master 8, AMPI) to the presynaptic GABAergic neuron. Paired-pulse stimulation was delivered at interpulse intervals (IPI) ranging from 25 ms to 1 s at a rate of 0.1 Hz and at IPIs of 2-4 s at 0.067 Hz. Stimulus trains consisting of 80 pulses were given at 2.5-80 Hz. Whole cell currents were low-pass filtered at 10 kHz, monitored on a pen recorder (Servogor 220), digitized using an AD converter (Instrutech VR-100 B), and stored simultaneously on a VTR and a Pentium PC equipped with Clampex (pClamp v. 6.0. software, Axon Instruments).
Drug application
Active substances were dissolved as stock solutions at 1,000 times the final concentration, diluted in extracellular medium just before use and perfused through the bath (exchange time 2-3 min). All changes in [Ca2+]o were compensated by changes in [Mg2+]o to keep the total extracellular divalent cation concentration constant at 5 mM. For experiments in which a rapid change of the extracellular medium was required, the neurons were continuously superfused from a three-barrel gravity-feed pipette (tip opening ~100 µm). Brief (10 ms duration) applications of GABA (1 mM) were made by applying pressure to a patch electrode with an opening of 4-5 µm placed close to the neuron. Response kinetics were improved by rapid removal of the applied GABA through a larger (100-µm opening) suction pipette placed nearby. In these experiments, TTX (0.2 µM) was added to the bathing solution to inhibit spontaneous synaptic currents.
Drugs and chemicals were purchased from Sigma except CNQX and DL-AP5 (Tocris Cookson) and EGTA-AM (Molecular Probes). CGP 35348 was a gift from Ciba-Geigy, and CGP 55845A was provided by Novartis. Culturing media were purchased from GIBCO, except for FUDR, uridine, and poly-D-lysine (Sigma).
Analysis
The traces shown in the figures are averages of eight consecutive responses, unless otherwise indicated. Analysis of IPSCs and responses to GABA was performed in pClamp (Axon Instruments). IPSCs were inspected visually and rejected if spontaneous activity disturbed the measurements. PPD is expressed as the percentage of the net amplitude of the second IPSC (IPSC2) compared with the first (IPSC1). In the paired-pulse experiments, decays of IPSCs and responses to exogenous GABA were fitted by a monoexponential function using the Clampfit routine and inspected visually. Data were imported into a spreadsheet (Excel v. 7.0a), where statistical calculations were performed. All data are presented as means ± SE with n indicating the number of pairs of neurons tested. Changes were considered to be significant at P values < 0.05.
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RESULTS |
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Monosynaptic GABAAergic IPSCs
Monosynaptic GABAAergic IPSCs were
investigated in pairs of rat hippocampal neurons, which were
continuously perfused with CNQX (10 µM) and DL-AP5 (50 µM) to block glutamatergic excitation. The presynaptic GABAergic
neuron was stimulated by stepping from 70 to 0 mV for 3 ms, which
evoked a short-latency (1-3 ms) IPSC in most nearby neurons. The IPSC
was blocked by bicuculline (10 µM) and had a reversal potential near
ECl (n = 5), indicating that it
is mediated by GABAA receptors. In spite of the
inclusion of MgATP in the pipette solutions, minor rundown of IPSC
amplitudes was usually seen. Rundown has previously been calculated to
be ~10% after the first 20 min of recording (Jensen et al. 1999b
).
PPD of IPSCs
To study use-dependent depression of the IPSCs, pairs of stimuli at an IPI of 150 ms were delivered at 0.1 Hz to the presynaptic GABAergic neuron. Single traces showed that the amplitude of IPSC2 varied considerably more than the amplitude of IPSC1 (Fig. 1A), and on rare occasions it was larger than IPSC2 (paired-pulse facilitation, not shown). Normally, eight consecutive pairs of IPSCs were averaged and used as the basis for further analysis. Averaged traces disclosed that the amplitude of IPSC2 was always depressed with respect to the first, commonly termed PPD. The time course of PPD was investigated by varying IPI between 25 and 4,000 ms (Fig. 1B). At short IPIs, IPSCs showed temporal summation with IPSC2 riding on the tail of IPSC1. IPSC2 was then measured as the net response after subtraction of the tail of IPSC1. When results from such experiments were pooled from seven pairs of neurons, significant PPD was seen at IPIs from 25 to 2,000 ms (Fig. 1C). Maximal depression occurred at the shortest IPI of 25 ms and was by 55.2 ± 7.4% (mean ± SE, P < 0.01).
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The decay of the IPSCs could be fitted by a monoexponential function
with a time constant, decay, of 41.9 ± 5.3 ms for IPSC1.
decay
for IPSC2 at an IPI of 250 ms (where the 2 IPSCs
were completely separated) was 39.3 ± 4.5 ms, which was not
significantly different from
decay of
IPSC1 (n = 7; P > 0.05). This is in contrast to IPSCs in intact neuronal preparations
in which
decay of IPSC2 is modulated by paired-pulse stimulation (Roepstorff and Lambert 1994
).
Effects of changing [Ca2+]o on IPSCs and PPD
The probability of transmitter release at cultured hippocampal
GABAergic synapses is steeply dependent on
[Ca2+]o
(Ohno-Shosaku et al. 1994b). In 30 experiments performed
on 14 pairs of neurons, we examined the effect of changing
[Ca2+]o in the range of
0.8-4.0 mM on pairs of IPSCs elicited at an IPI of 150 ms (Fig.
2A). We measured
IPSC1 relative to IPSC1 in 2.5 mM Ca2+ and plotted this against PPD (Fig.
2C;
). Linear regression showed that PPD was correlated
to the size of IPSC1 (P < 0.05), and for every 100% increase in IPSC1, PPD
increased by 33%.
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Effect of 4-aminopyridine (4-AP) on IPSCs and PPD
4-AP inhibits A-type K+ channels and delays
repolarisation after APs. This prolongs presynaptic
Ca2+ influx and enhances transmitter release
(Buckle and Haas 1982). During paired stimulation, 4-AP
(20-100 µM) enhanced IPSC1 and PPD (Fig.
2B). From the plot of PPD as a function of
IPSC1 (Fig. 2C;
), it is seen that
PPD did not increase as steeply with
IPSC1 as was the case in the experiments
in which [Ca2+]o was
increased. We also wanted to examine recovery from PPD after depletion
of the entire readily releasable pool of vesicles. We found that this
could be accomplished by perfusing 100 µM 4-AP, which enhanced PPD to
nearly 100% at the shortest IPIs. Results from a single pair of
neurons are shown in Fig. 2D. The time course of PPD in the
presence of 4-AP was similar to the control
(t1/2 ~500 ms, and full recovery
following PPD ~3 s in both cases).
PPD is reduced by a GABAB receptor antagonist
It has been reported that PPD of IPSCs in cultured hippocampal
neurons is completely insensitive to the GABAB
receptor antagonists 2-OH saclofen (Yoon and Rothman
1991) and CGP 35348 (Wilcox and Dichter 1994
).
Synaptically released GABA is, therefore, not thought to activate
presynaptic GABAB receptors in this preparation.
Accordingly, we found that bath perfusion of CGP 35348 (100 µM) had
no effect on either IPSC1 or PPD, which was
36.0 ± 6.3% in control compared with 35.1 ± 5.1% in CGP
35348 (P > 0.05, n = 6, paired
t-test). The GABAB agonist, baclofen
(10 µM) depressed IPSCs by 41.8 ± 6.9% (n = 7, not shown), and this effect could be blocked by CGP 35348 (n = 3), demonstrating that this antagonist was indeed
able to block actions at presynaptic GABAB receptors.
In contrast to CGP 35348, the newer potent blocker CGP 55845A (1-5
µM) (Davies et al. 1993) caused a significant
reduction of PPD (Fig. 3B)
from 39.4 ± 7.9% to 31.8 ± 10.4% (P < 0.05, n = 6, paired t-test). Full reversal
of the effect on PPD was obtained after wash out of the drug. Because
rundown was usually seen, the effect of CGP 55845A on a single IPSC was
difficult to evaluate. However, when CGP 55845A was applied through a
perfusion pipette that allowed medium exchange within a few seconds
(see METHODS), single IPSCs were slightly enhanced (by 7%
superimposed on a rundown of 2%, n = 2). This could
indicate that presynaptic GABAB receptors are
tonically activated in control conditions.
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IPSCs can be mimicked by exogenous applications of GABA
Investigations of the reversal potentials for pairs of IPSCs
showed that EIPSC did not change
between IPSC1 and IPSC2
(n = 2, not shown). To examine whether desensitization
of postsynaptic GABAA receptors plays a role in
PPD, these were activated directly by applying brief (10 ms) pulses of
GABA (1 mM) to 25 single neurons using a focal perfusion system (see
METHODS). The responses to GABA qualitatively mimicked
GABAergic IPSCs, although the response to GABA (Fig.
4Ab) had slower kinetics
(decay 82.7 ± 7.3 ms, n = 25) than IPSCs (41.9 ± 5.3 ms). Paired application of GABA was
made in 71 trials at different IPIs. The absolute amplitudes of the two
responses to GABA were similar, whereas the net amplitude of the second
response (GABA2) depended on whether a tail of
the first response was still present (Fig. 4B). PPD of the
net amplitude of GABA2 was therefore mainly a
result of temporal summation. This is as illustrated in Fig.
4Ca, where the decay of GABA1 (dotted line) is shown in relation to PPD of the GABA responses. The curve for
PPD deviates from the decay of GABA1 in the
interval 200-1,000 ms, indicating that there is a slight decrease in
postsynaptic responsiveness during GABA2. The
GABAergic IPSCs showed profound PPD at intervals up to 3 s,
irrespective of the decay of IPSC1 (Fig.
4Cb), indicating that PPD of IPSCs was not affected by
temporal summation. Taken together, these data suggest that PPD mainly involves a presynaptic mechanism.
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EGTA-AM increases PPD
It has recently been reported that the rate of recovery from PPD
at excitatory glutamatergic synapses depends on residual Ca2+ in the boutons, which is thought to
stimulate refilling of depleted vesicles at the active zones
(Dittman and Regehr 1998; Stevens and Wesseling
1998
; Wang and Kaczmarek 1998
). It has not been established whether a similar mechanism is present at inhibitory GABAergic synapses. To investigate this, we made recordings from presynaptic neurons in the perforated-patch configuration to preserve the internal environment. We evoked pairs of IPSCs at an IPI of 500 ms
(each 15 s) and monitored PPD (Fig.
5). After stable control recordings had
been obtained, perfusion of the membrane-permeable Ca2+ chelator, EGTA-AM (100 µM), was started.
PPD increased over the course the next minute and stabilized at a
higher level (Fig. 5B), whereas no effect was seen on
IPSC1. In four pairs of neurons, PPD increased
from 26.6 ± 4.1% in control to 42.9 ± 1.7% in EGTA-AM (P < 0.05, Fig. 5C). These results indicate
that chelation of Ca2+ in the boutons by
exogenously applied Ca2+ buffers depresses
IPSC2.
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Tetanic depression of IPSCs depends on frequency and [Ca2+]o
Because a substantial depression of IPSC2
was observed with paired stimulation, it was expected that IPSCs would
be further reduced in response to prolonged train stimulation. We
applied train stimuli consisting of 80 pulses at frequencies
(f) ranging from 2.5 to 80 Hz (37 trains in 20 pairs of neurons). At all frequencies, a gradual decrement of net IPSC
amplitudes (tetanic depression) was observed, which reached a plateau
after about five pulses (Fig.
6B). The constants for the
initial decrease in IPSC (depr) did not change
in any consistent pattern with f (Table
1, middle column). On the
other hand, when the first IPSC was compared with the net amplitudes of
the last 10 IPSCs in the train, the plateau phase during tetanic
depression depended strongly on f (Table 1, right
column).
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Tetanic depression also depended on extracellular
Ca2+ as shown when this was decreased from 2.5 to
1.2 mM or increased to 4.0 mM (Fig. 6C).
depr was 1.55 stimuli in 1.2 mM
Ca2+, 0.90 stimuli in 2.5 mM
Ca2+, and 0.41 stimuli in 4.0 mM
Ca2+. In 1.2 mM
[Ca2+]o the last 10 IPSCs
were depressed by 76.3 ± 3.1%, in 2.5 mM Ca2+ by 82.1 ± 1.4% and in 4.0 mM
Ca2+ by 89.9 ± 1.3%.
Effects of CGP 55845A on tetanic depression
To see whether increasing the level of extracellular GABA could cause a greater activation of GABAB autoreceptors, we stimulated the presynaptic neuron at 80 Hz for 1 s and tested the effect of CGP 55845A on the response (Fig. 7, n = 8). CGP 55845A reduced tetanic depression during the early part of the train but, surprisingly, not later in the train (Fig. 7). To quantify this differential effect, IPSC areas were measured with respect to the baseline during an early (at 150-300 ms) and a later (at 850-1,000 ms) period of the train (stippled thin lines in Fig. 7B). Areas were normalized to the area of a single pretetanic IPSC in each solution. Accordingly, a value of 1.0 means that the IPSC area was the same as the area of a single pretetanic IPSC. In CGP 55845A, the early area was 2.28 ± 0.3, which was 16% larger than in control solution (1.96 ± 0.2, P < 0.05, Fig. 7C). There was no significant difference between the late areas, which were 2.45 ± 0.3 in CGP 55845A compared with 2.28 ± 0.3 (P > 0.05). This indicates that the effect of GABAB autoreceptor activation is overcome by continued tetanic stimulation.
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DISCUSSION |
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Presynaptic GABAB receptors in cultured neurons
Activity-dependent depression of IPSPs is a prominent feature of
GABAergic synaptic transmission in the mammalian CNS (Thompson et al. 1993). Experiments with hippocampal and cortical brain slices have shown that PPD of IPSPs is partly caused by activation of
presynaptic GABAB autoreceptors, leading to a
reduction in GABA release (Davies et al. 1990
;
Deisz and Prince 1989
; Mott et al. 1993
;
Nathan and Lambert 1991
). In contrast to structurally intact preparations, investigations of PPD in cultured hippocampal neurons have shown that PPD of IPSCs is independent of presynaptic GABAB receptor activation (Wilcox and
Dichter 1994
), despite the fact that functional
GABAB receptors are present on the presynaptic terminals and can be selectively activated by baclofen (Harrison 1990
). However, we show here that the potent
GABAB antagonist, CGP 55845A, reduces PPD by
~20%, illustrating that presynaptic GABAB
receptors on cultured neurons are activated to a small extent by
synaptically released GABA. A submaximal concentration of baclofen depressed IPSCs by >40%, suggesting a larger depressant potential for
the presynaptic GABAB receptors. That
synaptically released GABA could not activate the
GABAB receptors fully would indicate that these
are located at some distance from the GABA release site. Application of
CGP 55845A also caused a small enhancement of single IPSCs. If this
reflects tonic activation of the high affinity presynaptic
GABAB receptors, the concentration of ambient extracellular GABA would probably be in the submicromolar range (Dittman and Regehr 1997
).
Postsynaptic factors in PPD
Although PPD is probably expressed at a presynaptic locus
(Wilcox and Dichter 1994), postsynaptic factors such as
a shift in ECl (Thompson and Gähwiler
1989
) or desensitization of GABAA receptors (Frosch et al. 1992
; Oh and Dichter
1992
) could contribute to PPD. We can, however, rule out that a
shift in ECl contributes to the depression of
GABAAergic IPSCs because the reversal potentials for IPSC1 and IPSC2 were
similar. To investigate whether desensitization is involved in the
depression of IPSCs, we made paired applications of exogenous GABA to
voltage-clamped neurons in the whole cell mode. The concentration of
GABA in the synaptic cleft has been estimated to be ~500 µM, which
saturates the postsynaptic receptors (Maconochie et al.
1994
). The brief application of GABA (1 mM) used in our
experiments is therefore likely to mimic simultaneous release at all
GABAergic synapses on the entire neuron. Our results show that the
postsynaptic responsiveness was unchanged following the first
application of GABA at all IPIs, thereby illustrating that the
receptors were not desensitized after the first pulse. This would
indicate that desensitization does not contribute to PPD of the IPSCs.
Presynaptic mechanism for PPD
Because activation of presynaptic GABAB
autoreceptors plays only a minor role in PPD, the most likely mechanism
is depletion of releasable vesicles at the active zones. Changing
[Ca2+]o or applying 4-AP
are established ways of modulating probability of release at individual
release sites by altering presynaptic Ca2+ influx
(Klapstein and Colmers 1992; Ohno-Shosaku et al.
1994b
). The results show that PPD was positively related to the
probability of release (Fig. 2C). Because this relationship
was investigated at an IPI of 150 ms, PPD asymptoted toward 70%, which
is the maximal obtainable PPD (see Fig. 2D). In the presence
of 100 µM 4-AP, PPD at the shortest IPIs was close to 100%, and the
probability of release was maximized. Although the readily releasable
pool was maximally depleted by the first stimulation, the rate of
recovery from PPD was nevertheless the same as the control.
Our results indicate that GABAergic boutons are refractory following
exocytosis, as has previously been suggested for excitatory synapses in
culture (Debanne et al. 1996). Depleted active zones are
replenished at a slow rate as found at glutamatergic synapses, where
refilling takes several seconds (Dobrunz and Stevens
1997
; Stevens and Tsujimoto 1995
). Disregarding
the marginal contribution from GABAB receptors,
the plot of PPD as a function of IPI (Fig. 2D) would reflect
replenishment of depleted vesicles at boutons that had released on
pulse1. Furthermore, there is increasing evidence
against the involvement of other possible sites in PPD such as axonal
branch block (MacKenzie et al. 1996
) or inactivation of
presynaptic voltage-dependent Ca2+ channels (VDCCs)
(Dobrunz and Stevens 1997
; Mintz et al.
1995
). The latter could, however, play a role at IPIs of <20
ms (Dobrunz et al. 1997
).
Recovery of synaptic depression
In three recent reports, it has been shown that recovery from PPD
at excitatory glutamatergic synapses is accelerated by residual Ca2+ in the boutons (Dittman and Regehr
1998; Stevens and Wesseling 1998
; Wang
and Kaczmarek 1998
). In the rat brain stem, refilling of the
readily releasable pool could be slowed by reducing the spike frequency
(Wang and Kaczmarek 1998
). In the cerebellum, the
membrane-permeable Ca2+ chelator, EGTA-AM,
enhanced PPD and delayed recovery (Dittman and Regehr
1998
). Accordingly, biochemical data show that synaptic vesicle
dynamics are regulated by internal free Ca2+ in
the range of 1 µM (reviewed by Burgoyne and Morgan
1995
). Our results would indicate that a similar mechanism
operates at inhibitory GABAergic synapses (Fig. 5). At a fixed IPI of
500 ms, perfusion of EGTA-AM enhanced PPD from 27 to 43%. This is interesting because GABAergic interneurons are believed to have a
strong internal Ca2+ buffering capacity
(Freund and Buzsáki 1996
), which would not allow
any significant accumulation of free Ca2+ in the
boutons following a single action potential. Nevertheless, the results
with EGTA-AM suggest that internal Ca2+ is indeed
elevated after a single stimulus, and that Ca2+
might stimulate replenishment of the readily releasable pool. However,
the precise mechanism responsible for this presynaptic regulation
remains to be determined.
Tetanic depression of IPSCs
Given that depletion is mainly responsible for frequency-dependent
synaptic depression, IPSCs would decline in response to longer stimulus
trains until depletion was in equilibrium with the restorative
processes (slow refilling), at which point the IPSCs would stabilize at
a plateau level. It is apparent that both the initial decline and the
initial plateau level depended on both the stimulating frequency and
the probability of release (Fig. 6, B and C).
However, although IPSCs were unable to follow the stimulation at the
highest frequencies (80 Hz, Fig. 6, A and B), an
appreciable GABAA current was maintained by
asynchronous GABA release that continued 1-2 s after end of
tetanization. This component of release has been analyzed in detail
elsewhere (Jensen et al. 1999b) and can be blocked by EGTA-AM. It is
therefore likely that asynchronous release is caused by an
increase in [Ca2+]i in
the boutons during the train stimulation (Jensen et al. 1999b
).
Bearing this in mind, it was interesting to find that CGP 55845A
reduced tetanic depression early in the train, but that this effect
declined with continued stimulation (Fig. 7B). GABAB-mediated inhibition of N- and P/Q-type
VDCCs at the secretory apparatus (Ohno-Shosaku et al.
1994b
) was overcome with continued stimulation, probably
because transmitter release is maintained during the train by
Ca2+ entering via L-type VDCCs (Jensen et
al. 1999a
). Because GABAB receptors do
not desensitize (Misgeld et al. 1995
), we have
identified an alternative mechanism whereby presynaptic
GABAB receptor function can be attenuated. This
observation is important in the context of understanding the function
of GABAB autoreceptors during high-frequency activity.
Conclusion
We have shown that GABAergic IPSCs in cultured hippocampal neurons show robust PPD that is mediated by a presynaptic mechanism. We suggest that PPD is caused by depletion of vesicles at the active zones, although spill-over of GABA onto presynaptic GABAB receptors is also present to a minor degree. Depleted vesicles are replenished at a slow rate that, however, depended on [Ca2+]i in the boutons. One could speculate that the loss of such Ca2+-mediated acceleration of vesicular recycling at GABAergic synapses in vivo could lead to an increased susceptibility for epileptic discharges. Finally, the effect of activation of GABAB autoreceptors is overcome with prolonged presynaptic activity. This observation is important in the context of understanding the actions of GABAB modulators as antiepileptic agents, and contributes to the understanding of how the action of high extracellular levels of GABA at presynaptic GABAB receptors is counteracted by accumulation of Ca2+ in the boutons during high-frequency activity.
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ACKNOWLEDGMENTS |
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We thank Ciba-Geigy for a gift of CGP 35348. Novartis supplied CGP 55845A, subject to the terms of their Statement of Investigator. We thank K. Kandborg for preparation of the cultures and S. Kristensen for technical assistance.
We are grateful to the Danish Medical Research Council and Aarhus Universitets Forsknings Fond for financial support.
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FOOTNOTES |
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Address for reprint requests: J.D.C. Lambert, Dept. of Physiology, University of Aarhus, Ole Worms Alle 160, DK-8000 Aarhus C, Denmark.
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 6 January 1999; accepted in final form 15 March 1999.
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REFERENCES |
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