Neuroscience Research Laboratory, Department of Pharmacology and Therapeutics, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Shew, T.,
S. Yip, and
B. R. Sastry.
Mechanisms Involved in Tetanus-Induced Potentiation of Fast IPSCs
in Rat Hippocampal CA1 Neurons.
J. Neurophysiol. 83: 3388-3401, 2000.
In the present study, possible
mechanisms involved in the tetanus-induced potentiation of
-aminobutyric acid-A (GABA-A) receptor-mediated inhibitory
postsynaptic currents (IPSCs) were investigated using the whole cell
voltage-clamp technique on CA1 neurons in rat hippocampal slices.
Stimulations (100 Hz) of the stratum radiatum, while voltage-clamping the membrane potential of neurons, induces a long-term potentiation (LTP) of evoked fast IPSCs while increasing the number but not the
amplitude of spontaneous IPSCs (sIPSCs). The potentiation of fast IPSCs
was input specific. During the period of IPSC potentiation, postsynaptic responses produced by
4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride and
baclofen, GABA-A and GABA-B agonists respectively, were not
significantly different from control. CGP 36742, a GABA-B antagonist,
blocked the induction of tetanus-induced potentiation of evoked and
spontaneous IPSCs, while GTP
S, an activator of G proteins,
substitution for GTP in the postsynaptic recording electrode did not
occlude potentiation. Since GABA-B receptors work through G proteins,
our results suggest that pre- but not postsynaptic GABA-B receptors are
involved in the potentiation of fast IPSCs. A tetanus delivered when
GABA-A responses were completely blocked by bicuculline suggests that
GABA-A receptor activation during tetanus is not essential for the
induction of potentiation. Rp-cAMPs, an antagonist of protein kinase A
(PKA) activation, blocks the induction of potentiation of fast IPSCs. Forskolin, an activator of PKA, increases baseline evoked IPSCs as well
as the number of sIPSCs, and a tetanic stimulation during this
enhancement uncovers a long-term depression of the evoked IPSC.
Sulfhydryl alkylating agents, N-ethylmaleimide and
p-chloromercuribenzoic acid, which have been found to
presynaptically increase GABA release and have been suggested to have
effects on proteins involved in transmitter release processes occurring
in nerve terminals, occlude tetanus-induced potentiation of evoked and
spontaneous IPSCs. Taken together our results suggest that LTP of IPSCs
originates from a presynaptic site and that GABA-B receptor activation,
cyclic AMP/PKA activation and sulfhydryl-alkylation are involved.
Plasticity of IPSCs as observed in this study would have significant
implications for network behavior in the hippocampus.
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INTRODUCTION |
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Even though normal function of the hippocampus is
critically dependent on inhibition by GABAergic interneurons,
plasticity of inhibitory synapses has received little attention when
compared with studies on the plasticity of excitatory synapses. Only in recent years has attention been paid to the plasticity of GABAergic synaptic transmission. Tetanus-induced enhancements of GABAergic synaptic transmission were first described in the hippocampus (Morishita and Sastry 1991; Xie and Sastry
1991
; Xie et al. 1995
). Subsequently, plasticity
of GABAergic inhibition was shown in deep cerebellar nuclei
(Ouardouz and Sastry 1999
), visual cortex (Komatsu 1994
, 1996
; Komatsu and Iwakiri
1993
) and medulla (Glaum and Brooks 1996
).
In the mammalian CNS, GABA activates both GABA-A and GABA-B receptors,
activation of the former produces an increase in
Cl conductance that generates the fast
inhibitory postsynaptic current (Eccles et al.
1977
) and the latter a G-protein-mediated increase in
K+ conductance that results in the slow IPSC
(Bowery et al. 1980
).
There is evidence that GABA-B receptors exist both pre- and
postsynaptically (Ault and Nadler 1982; Bowery et
al. 1980
; Olpe et al. 1982
; Peet and
McLennan 1986
). In the hippocampus, the activation of
presynaptic GABA-B receptors has been shown to decrease transmitter
release (Ault and Nadler 1982
; Bowery et al.
1980
; Diesz and Prince 1989
; Thompson and
Gahwiler 1989
) and activation of postsynaptic receptors causes
a hyperpolarization of neurons (Alger 1984
). These
actions have been shown to modulate the induction of activity-dependent
plasticity at excitatory synapses (Davies and Collingridge
1996
; Davies et al. 1991
). Although from
previous studies we have shown that activation of GABA-B receptors is
not associated with the maintenance of tetanus-induced potentiation of
CA1 neuronal fast inhibitory postsynaptic potentials (IPSPs) (Xie et al. 1995
), it is unclear if they participate in
the induction of the potentiation.
Protein kinase A (PKA) can phosphorylate a variety of cellular
substrates such as ion channels, receptors, and proteins on synaptic
vesicles and presynaptic membranes of nerve terminals to modulate their
function (Raymond et al. 1993). Forskolin binds to the
catalytic subunit of adenylyl cyclase, greatly increasing intracellular
cAMP concentration, which can stimulate protein kinase A. The
hippocampus contains forskolin binding sites (Gehlert et al.
1985
), and in the CA1 area, adenylyl cyclase is more densely located in dendritic spines as well as in presynaptic terminals, than
in somata (Mons et al. 1995
). Modulation of excitatory
synapses and long-term potentiation (LTP) of excitatory synaptic
transmission by protein kinase activity has been intensely investigated
(Malenka et al. 1986
, 1987
). Recently stimulation of PKA
has been shown to potentiate hippocampal inhibitory synaptic
transmission by a presynaptic mechanism of action (Capogna et
al. 1995
). It is, however, unclear whether the induction of
tetanus-induced plasticity of IPSCs requires activation of PKA.
It has been shown that activation of PKA inhibits GABA-B
receptor-mediated effects, and GABA-B receptor activation inhibits forskolin-induced cAMP accumulation (Malcangio and Bowery
1993; Xi et al. 1997
; Yoshimura et al.
1995
). However, it has also been shown that GABA-B agonists
increase intracellular cAMP and enhance neurotransmitter(noradrenaline, isoproterenol,
-adrenergic
agonists) stimulated intracellular cAMP accumulation (Knight and
Bowery 1996
; Oset-Gasque et al. 1993
;
Scherer et al. 1989
). In addition, GABA-B receptors have
been shown to regulate GABA-A receptor function through G proteins
linked to the adenylyl cyclase pathway (Barthel et al.
1996
). Whether GABA-B and PKA pathways interact to modulate tetanus-induced potentiation of the GABA-A mediated IPSC is currently unknown.
Secretory machinery, cytoskeletal proteins and proteins involved in the
interaction of the synaptic vesicle with the presynaptic membrane, have
many putative sites for phosphorylation. Recently it has been suggested
that activation of PKA directly facilitates the probability of
exocytosis of individual vesicles in response to a constant
Ca2+ signal (Trudeau et al. 1997).
The same study concluded that PKA directly facilitates secretory
machinery at a step downstream of Ca2+ influx and
vesicle docking. In separate studies, cAMP has been shown to enhance
the phosphorylation of microtubule associated protein (MAP-2),
synapsins and RAB 3A, proteins suggested to be involved in
neurotransmitter release (Koszaka et al. 1991
;
Lonart and Sudhof 1998
).
Many proteins have recently been cloned and suggested to be involved in
neurotransmitter release. N-ethylmaleimide-sensitive factor
(NSF), an ATPase whose hydrolysis of ATP, has been shown to be
essential for cellular vesicular fusion and transmitter release
(Whiteheart et al. 1994). N-ethylmaleimide
(NEM), a sulfhydryl alkylating agent, has been shown to inhibit
vesicle-membrane fusion in the Golgi apparatus, to decouple G proteins
from their associated receptors (Kitamura and Nomura
1987
; Shinoda et al. 1990
) and more recently to
enhance GABA release from presynaptic nerve terminals and block
depolarization-induced suppression (DSI) of sIPSCs and of evoked IPSCs
(Morishita et al. 1997
). The effects of NEM on physiologically measured plasticity of inhibitory synaptic transmission is not well understood.
Previous studies from our laboratory have shown that in the presence of
DL2-amino-5-phosphonovaleric acid (APV) and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a tetanic stimulation
delivered to the stratum radiatum induced a posttetanic potentiation of
hippocampal CA1 neuronal, GABA-A-mediated fast inhibitory postsynaptic
potentials (IPSPs) (Xie et al. 1995
). In this study, it
was also concluded that the potentiation was at least partly localized
to GABAergic synapses on CA1 neurons, although the contributions made
by pre- and postsynaptic mechanisms were unknown. In the present study,
we further characterize and investigate possible mechanisms involved in
the tetanus-induced potentiation of fast IPSCs in rat hippocampal CA1
neurons with the use of whole cell patch-clamp recording techniques.
Our results suggest that the potentiation arises from a presynaptic
site of origin and that GABA-B receptor activation, cyclic AMP/PKA
activation, and sulfhydryl-alkylation are involved in the
tetanus-induced potentiation of GABA-A receptor-mediated IPSCs.
Preliminary results from this study were presented at a scientific
meeting (Shew and Sastry 1998
).
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METHODS |
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Slice preparation and recording solutions
Halothane was used to deeply anesthetize rats before
decapitation. Transversely sectioned hippocampal slices (400 µM) were then obtained from 2-wk-old male Wistar rats as previously described (Xie et al. 1995). The CA3 region of the slices was
dissected free to minimize the influence of spontaneous activity from
CA3 neurons. The slices were then placed in an incubating chamber and
allowed to recover for
1 h before being transferred and submerged in
a constant-flow (2-2.5 ml/min) recording chamber (Pandanaboina and Sastry 1984
). A single hippocampal slice was held between two nylon nets and then transferred into a recording chamber, which was
superfused with oxygenated (95%
O2-5%CO2) physiological medium (pH = 7.4). The physiological medium contained (in mM) 120 NaCl, 3 KCl, 1.8 NaH2PO4,
26 NaHCO3, 2 MgCl2, 2 CaCl2, and 10 D-glucose. All
experiments were performed in the presence of 40 µM
DL
2-amino-5-phosphonovaleric acid [APV, an
N-methyl-D-aspartate (NMDA) antagonist] and 20 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) to pharmacologically
isolate inhibitory responses from excitatory responses and to minimize
the possibility of polysynaptic influences. All experiments were
performed at room temperature (24-26°C). Since these studies were
conducted at 24-26°C, it would be important to determine if similar
changes occur at physiologically normal conditions.
For some studies on evoked IPSCs, patch electrodes with resistances of
4-6 M were filled with patch solution containing (in mM) 135 K-gluconate, 10 HEPES, 10 KCl, 1 K4-bis-(o-aminophenoxy)-N, N,N',N'-tetraacetic acid (K4-BAPTA), 5 Mg-ATP, 0.1 CaCl2, 10 Na2-phosphocreatine, and 0.4 Na3-GTP. Creatine phosphokinase was added to
produce a final concentration of 50 U/ml (pH adjusted to 7.20-7.25
with KOH). For studies of evoked IPSCs and spontaneous IPSCs, the patch solution contained (in mM) 85 CH2O2SCs, 50 CsCl, 10 HEPES, 10 KCl, 1 BAPTA, 5 Mg-ATP, 0.1 CaCl2, 10 Na2-phosphocreatine, and 0.4 Na3-GTP and creatine phosphokinase 50 U/ml (pH
adjusted to 7.20-7.25 with CsOH). ATP-regenerating patch solutions
were used to maintain cytosolic levels of ATP and ensure that there was no rundown of GABA-A receptor-mediated responses.
All drugs were bath applied except for bicuculline methiodide. Bicuculline (100 µM) was applied locally through a glass micropipette with a tip diameter of ~40-50 µm. This micropipette was connected to a perfusing barrel and the drug was forced through by using overhead pressure. The flow of drug was calculated to be 0.3-0.5 ml/min. DNQX and NEM were made by dissolving the drug in dimethylsulfoxide. The final concentration of dimethylsulfoxide in the recording solution was 0.05%. Bicuculline methiodide, BAPTA, baclofen, and NEM were obtained from Sigma. APV, DNQX, and forskolin were purchased from Precision Biochemicals and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP) and Rp-cAMPs were from Research Biochemicals International. CGP 36742 was a gift from Ciba-Geigy.
Recording and analysis
CA1 pyramidal cell recordings were obtained using the blind,
whole cell patch-clamp recording technique. Neurons were
voltage-clamped at 70 mV for studies on sIPSCs and at cell resting
membrane potential for studies only looking at evoked IPSCs. Acceptable
neurons used for experiments had resting potentials between
58 and
65 mV immediately after break-in and input resistances >120 M
.
Series resistances of neurons were between 10 and 30 M
and were
compensated by
70%, except in experiments on sIPSCs. If series
resistance changed >15%, the experiments were discarded.
Intracellular responses in hippocampal CA1 neurons were recorded using
an Axoclamp 2A (Axon Instruments, Foster City, CA) in voltage-clamp
mode and the pClamp 6.0 software.
Spontaneous IPSCs were quantified manually, and all traces were
visually inspected, using pClamp 6.0 and a minianalysis program (version 3.0.1.) developed by Jaejin Software. Spontaneous IPSCs were
filtered at 1 kHz and digitized at 5 kHz. Rigorous visual inspection of
all traces was performed to ensure that the analysis was not corrupted
by the presence of events with abberent waveforms or periods of
unstable holding potentials. IPSCs were evoked continuously (at 0.05 Hz, filtered at 3 kHz, digitized at 10 kHz) with a bipolar concentric
stimulating electrode (SNEX-100, Rhodes Medical Instruments, Tujunga,
CA) placed in the stratum radiatum of the CA1 region and was only
interrupted when a tetanic stimulation (2 trains of 100 Hz for 1 s, 20-s interval) was delivered. Stimulation strengths (50-150 µA,
0.2 ms) were adjusted so that control IPSCs were ~50% of the maximum
response, leaving adequate room for potentiation. Data for IPSCs were
obtained from the average of two to five consecutive traces. Data for
number of spontaneous IPSCs were collected in 3- to 5-min bins. Control
responses had to be stable for 10-20 min before drugs or tetanic
stimulations were applied. Posttetanic responses were followed for
30
min. Both DNQX (20 µM) and APV (40 µM) were continually present in
the recording medium.
In experiments in which input specificity of tetanus-induced
potentiation of IPSPs was tested, two stimulating electrodes were
positioned in the stratum radiatum, one on each side of the recorded
CA1 neuron. APV and DNQX were present in the superfusing media
throughout the experiment. IPSPs were evoked by stimulation with one
(A) or the other (B) electrode. Twin pulse stimulation by either A or B
(40-ms delay between pulses) resulted in a suppression of the second of
the two IPSPs, in each case. When stimulations were given by A and B,
with an interstimulation delay of 40 ms, there was no suppression of
the second response whether A or B was stimulated first, indicating
that the two inputs do not overlap. After ascertaining this, control
recordings of IPSPs (A and B) were taken for 15 min before
tetanically stimulating (2 trains of 100 Hz for 1 s, 20-s
interval) one of the inputs. A or B was stimulated at 0.025 Hz,
alternating the stimulations, so that the delay between successive
stimulation was same throughout, therefore the IPSPs were recorded at
0.05 Hz. The posttetanic IPSPs were then followed for
30 min.
IPSCs illustrating the tetanus-induced potentiation were calculated by measuring the peak amplitude of IPSC after the tetanic stimulation and expressing them as a percentage of the averaged control IPSCs. Data are reported as the means ± SE. Statistical analysis of the data were performed with two-tailed Student's t-tests, an ANOVA followed by Duncan's multiple comparisons test and Kolmogorov-Smirnov (K-S) tests for cumulative frequency distributions of spontaneous IPSCs. The level of significance was set at P < 0.05.
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RESULTS |
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Tetanus-induced potentiation of evoked IPSCs
Using electrodes filled with either K-gluconate-based or CsCl-based patch solutions, bicuculline-sensitive GABA-A receptor-mediated fast IPSCs were recorded from hippocampal CA1 neurons with low-frequency stimulation (0.05 Hz) of the stratum radiatum. On tetanic stimulation (2 trains at 100 Hz for 1 s, 20-s interval) significant potentiations of evoked IPSCs and spontaneous IPSCs were observed in 60-70% of cells.
In neurons patched with gluconate-based patch solution, tetanic
stimulation of the stratum radiatum induced a significant potentiation
of the GABA-A receptor-mediated fast IPSC that developed gradually,
reached its peak after ~7 min and maintained for 30 min
posttetanus (Fig. 1A). This
potentiation of the fast IPSC was reliably induced when the tetanus was
delivered in both current- and voltage-clamp conditions. The magnitude,
time of onset, and duration of potentiation were not significantly
different in these cells so their data were pooled together. The
average potentiation of IPSC amplitude was 125.0 ± 3.0% of
control (n = 8, P < 0.05, ANOVA, Fig.
1A). The reversal potential of the IPSC was not
significantly changed during the potentiation (see also Xie et
al. 1995
). When the peak amplitude of the control IPSC was
scaled up to the potentiated IPSC, there was no difference in the shape
of the IPSC, suggesting that the kinetics of the IPSC were not changed
during the potentiation (Fig. 1A).
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In recordings of IPSCs from neurons filled with
Cl-based patch solution, a tetanic stimulation
of the stratum radiatum also produced a significant posttetanic
potentiation. As in gluconate-filled cells, the potentiation occurred
in neurons that were either current- or voltage-clamped during the
tetanic stimulation. The potentiation of the IPSC, however, occurred
immediately with the first or second evoked response after the tetanus.
The average potentiation of the IPSC was 160.7 ± 2.7% of control
and maintained for 30 min posttetanus (n = 7, Fig.
1B, P < 0.05, ANOVA). The kinetics of the
IPSC did not appear to have changed, as there was no difference in the
shape of the pre- and posttetanus IPSC.
Tetanus-induced potentiation of evoked IPSCs does not appear to be dependent on postsynaptic neuron membrane potential changes induced during the tetanus since a potentiation could be obtained whether the postsynaptic membrane potential was allowed to fluctuate during the tetanus or not. Potentiation of evoked IPSCs could be induced in neurons with either K-gluconate or CsCl as the major patch solution constituent (others components of the patch solution were identical and were of the similar concentrations).
It is interesting that CA1 neurons filled with CsCl-based patch solution displayed a larger and immediate potentiation of the evoked IPSC while the potentiation of gluconate-filled neurons was smaller and developed gradually peaking at 7-10 min posttetanus (Fig. 1). While these differences were not due to changes in reversal potential, the exact reasons are unclear at present.
Tetanus-induced potentiation of sIPSCs
When recording from CA1 neurons filled with
Cl-based patch solution, sIPSCs can be observed
in the presence of APV and DNQX. sIPSCs were recorded continuously
along with evoked IPSCs for
10-15 min before a tetanus was
delivered. A tetanic stimulation delivered in either current- or
voltage-clamp conditions produced an significant immediate increase in
the number of sIPSCs (posttetanus 10 min C-clamp: 212.5 ± 15.0%
of control; posttetanus 10 min V-clamp: 206.2 ± 12.5% of
control, n = 8 and 6, respectively, P < 0.05, ANOVA with Duncan's multiple comparisons test) but did not
change the mean amplitude (C-clamp control: 21.7 ± 2.8 pA,
C-clamp posttetanus: 22.5 ± 2.1 pA; V-clamp control: 25.6 ± 1.9 pA, V-clamp posttetanus: 27.1 ± 2.3 pA, P > 0.05, ANOVA with Duncan's multiple comparisons test) or amplitude
distribution of spontaneous IPSCs (Kolmogorov-Smirnov). The increase in
sIPSC frequency sustained for
20 min. In Fig. 2 are typical examples of neurons that
exhibited tetanus-induced potentiation of sIPSCs when the tetanus was
delivered under and current- and voltage-clamp conditions. These
results suggest that it is worthwhile to examine whether presynaptic
mechanisms are involved in the potentiation.
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As in evoked IPSCs, fluctuations in CA1 neuron membrane potential during the tetanus do not appear to significantly influence the potentiation of sIPSCs since in both cases the increase in the number of IPSCs was of similar magnitude. To minimize postsynaptic membrane potential fluctuations during the tetanus and possible postsynaptic modifications that may occur, in subsequent experiments, all neurons were voltage-clamped during the delivery of the tetanus.
Attempts were made to examine the effects of tetanus on miniature (mIPSC) amplitude and frequency; however, we were unable to effectively wash out TTX or STX in a reasonable amount of time so that a tetanus could be delivered. We tried to mimic the tetanus with high levels of K+ as well as block action-potential-driven spontaneous events by various methods that would allow faster recovery so that a tetanus could be delivered. These attempts, however, were also unsuccessful not effectively mimicking tetanus.
Input specificity of tetanus-induced potentiation of IPSPs
When IPSPs were evoked in CA1 neurons by two electrodes positioned in stratum radiatum such that the two inputs did not overlap, a tetanic stimulation of one input produced a significant potentiation of IPSPs induced by that, but not by the untetanized input, indicating input specificity of the potentiation (n = 5, Fig. 3, P < 0.05, ANOVA).
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Postsynaptic responses to applied GABA-A and GABA-B agonists are not changed during tetanus-induced potentiation of IPSCs
To determine if the tetanus altered the postsynaptic sensitivity of GABA receptors to applied agonists, in neurons filled with gluconate-based patch solution, CA 1 neuron responses to the GABA-A agonist THIP (20 µM, for 1 min) were compared before and during the potentiation of the IPSC. During potentiation of the IPSC (posttetanus 7 min 126.0 ± 5.6% of control, n = 6, Fig. 4A1), the amplitude and shape of THIP-induced currents were not increased (96.2 ± 6.6% of control, n = 6, Fig. 4A2, P > 0.05, Student's t-test). Note that the potentiated IPSC was not altered by THIP (posttetanus 25 min 124.3 ± 4.0% of control, n = 6, Fig. 4A1). In other experiments, THIP was applied at twice the concentration and duration (40 µM, 2 min), as previously applied so that the drug-effect reached a plateau. In six of six neurons, neither the shape nor amplitude of the THIP-induced current (posttetanus THIP: 95.6 ± 6.2% of control, n = 6, P > 0.05, Student's t-test, not shown) was significantly altered after the tetanic stimulation. These results suggest that the sensitivity of postsynaptic GABA-A receptors to applied agonists does not change during the tetanus-induced potentiation of the IPSC and that there was no change in the desensitization of THIP responses.
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To test for changes in CA1 postsynaptic sensitivity to baclofen, the agent (50 µM, 1 min)-induced responses were compared before and during the potentiation of the IPSC (Fig. 4B1, n = 5). The baclofen-induced current was also not significantly altered (86.2 ± 8.2% of control, n = 5, Fig. 4B2, P > 0.05, Student's t-test) during the potentiation of the IPSC. In addition, baclofen did not alter the potentiated IPSCs. It, therefore appears that potentiation of the fast IPSC is not secondary to tetanus-induced modifications of GABA-B receptors.
Tetanus-induced potentiation of evoked IPSCs in neurons containing BAPTA
Changes in postsynaptic Ca2+ have been
shown to be important for the induction of LTP of excitatory synapses
in CA1 neurons (Lynch et al. 1983; Malenka et al.
1988
; Malinow et al. 1989
). To determine if the
potentiation of the IPSC was dependent on increases in postsynaptic
Ca2+ concentration that may occur during and/or
after the tetanus, experiments were performed in which the
Ca2+ chelator, BAPTA (10 mM in the patch
solution) was included in the Cl
-based patch
solution. A tetanic stimulation delivered to the stratum radiatum,
while neurons were current-clamped, produced a potentiation of the
evoked IPSC. The onset of potentiation was immediate after the first or
second response after tetanus and the potentiation in the IPSC was
significantly larger than in neurons that did not contain BAPTA
[average potentiation: 202.2 ± 19.7% of control
(n = 6, not shown) as compared with 160.7 ± 2.7%
of control for non-BAPTA containing neurons, see Fig. 1B, P < 0.05, ANOVA] while the kinetics of responses were
not altered. Spontaneous IPSCs number increased immediately
(posttetanus 10 min: 234.0 ± 41.0%, n = 6, P < 0.05, ANOVA with Duncan's multiple comparisons
test) and maintained for 20 min while mean sIPSC amplitude remained
unchanged (pretetanus: 27.8 ± 2. 2 pA, posttetanus: 30.0 ± 2.0 pA, n = 6, P > 0.05, ANOVA with
Duncan's multiple comparisons test, not shown). These results suggest
that postsynaptic Ca2+ is not essential for the
potentiation and as suggested in earlier reports from this laboratory
(Morishita and Sastry 1991
; Xie at al.
1995
) may even dampen the tetanus-induced potentiation of the IPSC.
It has been shown that elevations in postsynaptic
Ca2+ concentrations suppress GABA-A-mediated
responses through destabilizing the phosphorylation of the GABA-A
receptor complex by the activation of several phosphatases
(Chen et al. 1990; Morishita and Sastry 1996
; Pitler and Alger 1992
, 1994
). Although
further investigation into effects of postsynaptic
Ca2+ levels on tetanus-induced potentiation was
not the theme of this study and therefore not further pursued, our
results are consistent with reports in literature.
Reports in literature show that increases in intracellular
Ca2+ can lead to long-lasting suppression of
GABA-A receptor function in central neurons (Chen and Wong
1995; Morishita and Sastry 1996
; Stelzer
and Shi 1994
). Since APV was present throughout our
experiments, Ca2+ entry into postsynaptic neurons
during the tetanus is unlikely to be via NMDA channels. Tetanic
stimulation of afferent fibers could, however, conceivably result in
intracellular Ca2+ accumulation through other
routes such as release from intracellular stores, activation of
voltage-gated Ca2+ currents, etc.
GABA-A receptor activation during the tetanus is not required for potentiation of IPSC
To test whether activation of the fast IPSC during the tetanus was required for the potentiation of the IPSC to occur in gluconate-containing neurons, 100-Hz stimulations were delivered when the IPSC was blocked (0.13 ± 0.12% of control, n = 6, Fig. 5) by the GABA-A antagonist, bicuculline methiodide (100 µM, 2 min, locally applied). Bicuculline completely abolished the fast IPSC within 1 min of application, revealing in two of six neurons a much smaller slow GABA-B-mediated IPSC. In control experiments, recovery of the fast IPSC occurred after a washout period of >25 min (n = 6). In test experiments, a tetanus was delivered when the fast IPSC had been completely abolished for 30 s, that is, at 1.5 min after the initiation of the bicuculline application. In tetanized neurons, the IPSC recovered more quickly from bicuculline antagonism than in control experiments (posttetanus 15 min: 104.5 ± 11.6% of control, control wash 15 min: 65.1 ± 10.7% of control; posttetanus 25 min: 126.8 ± 3.0% of control, control wash 25 min: 83.9 ± 8.9% of control, Fig. 5, P < 0.05, ANOVA). These results suggest that activation of GABA-A receptors, during tetanus, is not necessary for potentiation of the IPSC.
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GABA-B receptor activation during the tetanus is important for tetanus-induced potentiation of fast IPSCs
Many reports in literature suggest the presence of presynaptic
GABA-B receptors (autoreceptors) on GABAergic nerve terminals and their
role in reducing GABA release (for review, see Misgeld et al.
1995). However, its functional role is still unclear. More recently, presynaptic GABA-B receptors have been suggested to be
involved in the enhancement of transmitter release (Brenowitz et
al. 1998
; Glaum and Brooks 1996
).
Previous studies from this laboratory (Xie et al. 1995)
have shown that inhibition of GABA-B receptors does not effect already existing tetanus-induced potentiation of the fast IPSP, suggesting that
GABA-B receptors are not involved in the maintenance of the potentiation of GABA-A-mediated synaptic transmission and that the
increase in the observed fast IPSP was not secondary to changes in
GABA-B-mediated responses.
In the present study, experiments were performed to determine whether GABA-B receptor activation is required for the induction of potentiation of the IPSC and sIPSCs. A tetanic stimulation was delivered to the stratum radiatum in the presence of the GABA-B antagonist CGP 36742 (500 µM, for 10 min before, during the tetanic stimulation and then for the remainder of the experiment, n = 6). While CGP 36742 did not affect the control response, only an initial transient increase in the evoked IPSC amplitude, which lasted for 1-3 min following the tetanus, could be induced in its presence (Fig. 6A, P > 0.05, ANOVA). Spontaneous IPSC number also increased transiently, but at 5 min after the delivery after the tetanus, they had returned to control levels (Fig. 6B, P > 0.05, ANOVA with Duncan's multiple comparison's test). Mean amplitude of sIPSCs did not change in the presence of CGP 36742 or posttetanus (pretetanus: 15.5 ± 1.6 pA, CGP 36742: 15.2 ± 1.3 pA, posttetanus: 16.0 ± 1.6 pA, P > 0.05, ANOVA with Duncan's multiple comparison's test) It was observed that in two of six neurons, CGP 36742 reduced the frequency of sIPSCs substantially but when results were pooled together this decrease was not significant. The kinetics for the IPSC and sIPSCs remained similar during the period of enhanced amplitude and after return to control levels. These observations suggest that activation of GABA-B receptors is an essential step in tetanus-induced potentiation of the fast IPSC.
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Actions of GABA-B receptors are mediated via G proteins (Andrade
et al. 1986; Dutar and Nicoll 1988
;
Thalmann 1988
). It has been shown that when GTP
S, a
nonhydrolyzable analogue of GTP that activates G proteins, was applied
intracellularly, postsynaptic GABA-B-mediated responses could not be
seen (Thalmann 1988
). In the present study, in some
neurons an equimolar substitution of GTP
S for GTP in gluconate patch
solution was made. As expected these neurons had much more negative
resting membrane potentials and lower input resistances, and
bath-applied baclofen (50 µM, 1 min) elicited negligible responses
(not shown). In these neurons, a tetanic stimulation induced a
potentiation of evoked IPSCs that was seen with the first response
after the 100-Hz stimulations (average potentiation:136.8 ± 3.0%
of control, n = 6, Fig. 6C, P < 0.05, ANOVA) and lasted for 30 min. The kinetics
of control and posttetanus IPSCs were not significantly different.
Postsynaptic GABA-B receptor-coupled K+ channels
are known to be blocked by Cs+ (Gahwiler
and Brown 1985; Jarolimek et al. 1995
); in
addition high concentrations of intracellular
Cl
(such as concentrations used in this study)
have been shown to greatly decrease postsynaptic GABA-B responses
(Lenz et al. 1997
). With high concentrations of
Cs+ and Cl
together in
the postsynaptic electrode, one would expect postsynaptic GABA-B
receptors to be nearly completely antagonized. GTP
S,
intracellularly, by activating G proteins occludes subsequent
activation of postsynaptic GABA-B receptors. Our observation of
tetanus-induced potentiation of the fast IPSC under both of the
above-mentioned conditions strongly suggest that postsynaptic GABA-B
receptors are not involved in the induction of potentiation of fast
IPSCs. However, in experiments with CsCl in the recording electrode,
tetanic stimulation delivered during the application of the GABA-B
antagonist CGP 36742 blocked the potentiation of the fast IPSC. Taken
together, these results suggest that presynaptic GABA-B receptors are
involved in the induction of GABA-A-mediated potentiation.
Dependence of the potentiation on PKA activity
Several subunits of GABA-A receptors have putative phosphorylation
sites for PKA. The involvement of these protein kinases in inhibitory
synaptic transmission has only recently been studied (Capogna et
al. 1995; Trudeau et al. 1997
). The
involvement of these enzymes in plasticity of GABAergic synapses is not
well understood.
To determine if PKA activation is necessary for induction of tetanus-induced potentiation, a tetanus was delivered in the presence of the competitive PKA antagonist, Rp-cAMPs (20 µM, 10 min prior to the delivery of, during the tetanic stimulation and then for the remainder of the experiment). In the presence of Rp-cAMPs, no significant potentiation of the evoked IPSC was observed (posttetanus 5 min 102.0 ± 9.5% of control, posttetanus 20 min: 107.0 ± 13.3% of control, n = 6, Fig. 7A, P > 0.05, ANOVA). Tetanic stimulation failed to produce an increase in the number of sIPSCs (posttetanus 5 min: 92.5 ± 13.2% of control, n = 6, Fig. 7B, P > 0.05, ANOVA with Duncan's multiple comparison's test) and did not change the mean sIPSC amplitude or distribution (pretetanus: 17.3 ± 1.1pA, Rp-cAMPs: 17.5 ± 1.1 pA, posttetanus: 17.2 ± 1.0 pA, P > 0.05, ANOVA with Duncan's multiple comparison's test). Rp-cAMPs itself does not effect the control IPSC amplitude (Rp-cAMPs: 99.6 ± 2.7% of control, P > 0.05, ANOVA with Duncan's multiple comparison's test), the basal sIPSC frequency (96.1 ± 2.2% of control, P > 0.05, ANOVA with Duncan's multiple comparison's test) or the mean amplitude of sIPSCs.
|
In separate experiments, forskolin was used to stimulate PKA. In
control experiments, bath applied forskolin (20 µM, 25 min) increased
the amplitude of evoked IPSCs and the number but not the amplitude of
sIPSCs, similar results are described by Capogna et al.
(1995) (Fig. 8, A and
B). These effects persisted even after 20 min of washout
(evoked IPSC 20-min washout: 150.4 ± 13.6% of control,
P < 0.05, ANOVA, sIPSC frequency 20-min washout:
164.0 ± 8.6% of control, n = 7, Fig. 8,
A and B, P < 0.05, ANOVA with Duncan's multiple comparison's test). To determine if a tetanic stimulation could induce a potentiation of the IPSC in addition to that
produced by forskolin in different neurons, a tetanus was delivered
during the period of increased GABAergic synaptic transmission 15 min
after the beginning of the application of forskolin. In these
experiments, no further increase in the evoked IPSC was observed,
instead by 10 min posttetanus, while forskolin was still present, the
evoked IPSC returned to control levels and maintained there for the
rest of the experiment (pretetanus in forskolin: 155.3 ± 8.4% of
control, posttetanus 10 min in forskolin: 108.1 ± 6.3% of
control, n = 9). At posttetanus, 10 min evoked fast
IPSCs were significantly smaller than control neurons in which
forskolin was applied without tetanus (P < 0.05, ANOVA). Interestingly, the tetanic stimulation did not significantly
decrease sIPSC number (pretetanus in forskolin: 163.6 ± 8.1% of
control, posttetanus 10 min in forskolin: 181.2 ± 32.8% of
control, n = 9, P > 0.05, ANOVA with
Duncan's multiple comparison's test) or sIPSC mean amplitude as might
be expected in light of the evoked IPSC results. In fact, posttetanus
sIPSC frequency was not statistically different from control frequency
until posttetanus 25 min. The tetanic stimulation did not alter the
kinetics of evoked or spontaneous IPSCs nor did it change sIPSC
amplitude.
|
Results from the cAMP antagonist suggest that PKA activation is needed for tetanus-induced potentiation of evoked and sIPSCs. If this is the case, PKA activation by forskolin would be expected to at least partially occlude potentiation induced tetanus. This was observed for sIPSCs but not observed for the evoked IPSC, instead it appeared that the tetanus caused a depotentiation of the evoked IPSC or induced a long-term depression of the IPSC.
Although our results suggest PKA activation is important in tetanus-induced potentiation of evoked IPSCs and sIPSCs, it is clear that further investigation is required to elucidate the exact mechanisms.
Sulfhydryl-alkylation is important for potentiation of IPSCs
NEM is a sulfhydryl alkylating agent that has been shown to block
pertussis-toxin-sensitive GABA-B actions and increase the release of
GABA from presynaptic terminals in the rat hippocampus by an unknown
presynaptic mechanism (Morishita et al. 1997). To determine if the alkylation of sulfhydryl groups is important for
tetanus-induced potentiation of fast IPSCs, the effects of NEM on
tetanus-induced potentiation was investigated. Control studies showed
that the NEM (250 µM, 11 min) caused significant increases in
amplitude of the fast IPSC (P < 0.05, ANOVA) and the
number, but not the mean amplitude (control: 25.2 ± 3.2 pA, NEM:
26.7 ± 2.6 pA, P > 0.05, ANOVA with Duncan's
multiple comparison's test) of spontaneous IPSCs that returned to
control levels after ~20 min of washout (Fig.
9, A and B). In
separate experiments, a tetanus was delivered to the stratum radiatum
during the 11th min of NEM application, during the peak of NEM-induced
increase in inhibitory synaptic transmission. No further potentiation
of the IPSC was observed, NEM occluded further potentiation of both evoked (Fig. 9A, P > 0.05, ANOVA) and
sIPSCs without changing mean sIPSC amplitude (NEM: 26.7 ± 2.6 pA,
posttetanus 27.1 ± 2.9 pA, Fig. 9B, P > 0.05, ANOVA with Duncan's multiple comparison's test).
|
To further test if the observed NEM effects were due to its effects on sulfhydryl groups, a different sulfhydryl alkylating agent p-chloromercuribenzoic acid (PCMB, 250 µM, 11 min) was tested. In control experiments, PCMB was also found to increase the amplitude of evoked IPSCs and the number but not the mean amplitude of sIPSCs, and these effects maintained for 30 min after washout (Fig. 9, C and D). PCMB then was applied for 11 min and a tetanus was delivered during the 11th min of drug application. PCMB was also found to occlude the potentiation of evoked and sIPSCs, with no significant increases in IPSC amplitude (Fig. 9C, P > 0.05, ANOVA) or sIPSC frequency (Fig. 9D, P > 0.05, ANOVA with Duncan's multiple comparison's test).
These results suggest that NEM modulates a component(s) of the pathway that leads to tetanus-induced potentiation of inhibitory synaptic transmission and that the modification involves the alkylation of sulfhydryl groups.
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DISCUSSION |
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Plasticity of excitatory synapses has been extensively studied in
laboratories around the world. However, plasticity of inhibitory synapses has not received such attention. In this paper we report that
GABAergic synapses in the CA1 area of the hippocampus undergo plasticity. Previous studies from our laboratory have shown that a
tetanic stimulation in the stratum radiatum induces a sustained potentiation of GABA-A receptor-mediated fast IPSPs recorded from rat
hippocampal CA1 neurons (Morishita and Sastry 1991;
Xie and Sastry 1991
; Xie et al. 1995
).
This potentiation is likely localized to GABAergic synapses rather than
other areas (see Xie et al. 1995
), and our current
results suggest that it is input specific. Postsynaptic neuronal
protein kinase C (PKC) activation and Ca2+
accumulation are not necessary for, and may even dampen, the tetanus-induced potentiation of fast IPSPs (Xie and Sastry
1991
; Xie et al. 1995
). Major findings from the
present study strongly suggest that the potentiation of GABA-A
receptor-mediated synaptic transmission is due to modifications that
occur presynaptically and involve GABA-B receptors and PKA.
GABA receptor involvement in tetanus-induced potentiation of IPSCs
Activation of GABA-A receptors during the tetanic stimulation
appears to be unnecessary for the induction of a potentiation of the
fast IPSCs. Moreover the potentiation could be induced if the tetanus
was given while the CA1 neurons were voltage-clamped, indicating that
alterations in postsynaptic neuronal membrane potentials were not
needed to induce the potentiation. While blocking postsynaptic GABA-B
actions by including Cs+ and
Cl together (Lenz and Alger
1997
) or occluding these effects by GTP
S, in the
postsynaptic neurons, a potentiation of fast IPSCs could still be
induced by high-frequency stimulation, suggesting that activation of
postsynaptic GABA-B receptors is not needed for the induction of the
potentiation. Interestingly, in neurons in which postsynaptic GABA-B
actions were blocked, CGP 36742 still antagonized the induction of
tetanus-induced potentiation, suggesting the involvement of presynaptic
GABA-B receptors. The maintenance of tetanus-induced potentiation of
fast IPSPs was not affected by blocking GABA-B receptors (Xie et
al. 1995
), suggesting that changes in GABA-B responses could
not account for the observation of enhanced GABA-A IPSCs. Neither
postsynaptic responsiveness to GABA-A receptor agonist, THIP, nor
desensitization of GABA-A receptors to a prolonged application of this
agent was altered during the posttetanic sustained potentiation of the IPSC.
Many studies indicate the role of presynaptic GABA-B receptors in
reducing GABA release from terminals (Baumann et al.
1990; Calabresi et al. 1991
; Davies and
Collingridge 1993
; Davies et al. 1990
;
Diesz and Prince 1989
; Olpe et al. 1994
;
Pittaluga et al. 1987
). Presynaptic GABA-B receptors
were also implicated in an enhancement of GABAergic synaptic
transmission in different areas of the CNS (Brenowitz et al.
1998
; Glaum and Brooks 1996
). In our studies, it
is unclear how activation of presynaptic GABA-B receptors during a
tetanic stimulation can set up a long-lasting enhancement of GABAergic
synaptic transmission. The fact that baclofen by itself does not induce
a long-term change in fast IPSCs, however, suggests that the activation
of presynaptic GABA-B receptors, if involved, must work in conjunction
with other factors influenced by high-frequency stimulation. It is also
possible that bath-applied baclofen simply does not accurately mimic
the activation of presynaptic GABA-B receptors by endogenously released GABA during tetanic stimulation.
It is unclear why the potentiation of the IPSC was immediate and not
gradual when GTPS was included in the gluconate-patch solution.
GTP
S is not specific to GABA-B related G proteins and as a result
can upregulate other G-protein-linked receptor systems. Staley
et al. (1995)
showed that mechanisms modulating
activity-dependent collapse of transmembrane Cl
currents are inhibited by PKC activation; it is conceivable that GTP
S would cause an activation of PKC. If this was the case, GTP
S
would indirectly block the collapse of the transmembrane Cl
gradient during tetanus allowing for an
immediate or early potentiation of IPSCs; this would be consistent with
other findings in this study. However, the sustained potentiation of
the IPSCs observed in our studies was not associated with a change in
the reversal potential.
Involvement of PKA
There are several consensus sites for phosphorylation within major
intracellular domains of GABA-A receptor subunits for cAMP-dependent protein kinase (Gehlert et al. 1985). There are
conflicting reports on how PKA functionally affect GABA-A receptor
actions. Studies have suggested that activation of presynaptic PKA
enhances basal GABA release from GABAergic interneurons by independent
mechanisms of action (Capogna et al. 1995
). Our
finding that Rp-cAMPs, a competitive antagonist of cAMP, blocks
long-lasting potentiation of the evoked IPSC and sIPSCs without
affecting sIPSC amplitude is consistent with findings by Capogna
et al. (1995)
and Trudeau et al. (1997)
, who
suggested that PKA activation enhances evoked and spontaneous GABA-A
mediated synaptic transmission. In addition, adenylyl cyclase
activator, forskolin, which is known to activate PKA, enhanced GABA-A
receptor-mediated evoked IPSCs as well as basal sIPSC frequency but not amplitude.
If PKA activation is involved in the tetanus-induced potentiation of
IPSCs, it was felt that forskolin would occlude a tetanus-induced increases in IPSCs. In fact, a slowly developing depotentiation or a
long-term depression (LTD) of evoked IPSCs was observed when tetanized
in the presence of forskolin. Activity-mediated LTP and LTD at
excitatory synapses have been shown to occur simultaneously (Bear and Malenka 1994), but factors that determine
which phenomenon predominates under certain circumstances still need to
be determined. It is possible that LTP and LTD at inhibitory synapses
can also co-exist in a similar manner. In our laboratory, it has
recently been shown that IPSCs in deep cerebellar nuclear cells undergo LTD (Morishita and Sastry 1996
) or LTP depending on the
induction parameters (Ouardouz and Sastry 1999
). If a
similar case exists in the hippocampus; a tetanus delivered when
synapses were maximally potentiated could reveal a previously unseen depression.
It is unclear as to how tetanus can induce an enhancement of GABA
release. Activation of presynaptic PKA can theoretically affect any
part of the stimulation secretion-coupling pathway, from
Ca2+ entry into the presynaptic terminal to
transmitter release processes. Investigations to understand how GABA-B
and PKA pathways interact in the mammalian CNS are currently being
pursued by various groups (Barthel et al. 1996;
Malcangio and Bowery 1993
; Xi et al.
1997
; Yoshimura et al. 1995
). It has been
suggested that GABA-B receptors are linked to the adenylyl cyclase
pathway via G proteins (Wojcik et al. 1989
; Xi et
al. 1997
). By activating GABA-B receptors, one can modulate
cAMP production and subsequently PKA activity (Bowery
1993
; Oset-Gasque et al. 1993
; Scherer et
al. 1989
; Wojcik et al. 1989
; Xi et al.
1997
). It has been suggested (Barthel et al.
1996
; Xi et al. 1997
) that through this pathway
that GABA-B receptors can acutely suppress GABA-A currents. However,
results in our study as well as those of others (Capogna et al.
1995
; Trudeau et al. 1997
) suggest that
activation of PKA can lead to a long-lasting enhancement of GABA
release and IPSCs. It is tempting to speculate that the long-term
effect of presynaptic GABA-B receptor-mediated and tetanic
stimulation-induced increase in PKA activity is to increase GABA release.
Several proteins recently identified as being associated with neuron
cytoskeletal elements such as synapsin, GAP-43, dynamin, MAP-2, and RAB
3A, and proteins involved in the fusion of presynaptic vesicles with
the presynaptic membrane such as -SNAP, SNAP-25, and NSF, all having
putative sites for protein kinase phosphorylation (for review, see
Whatley and Harris 1996
). Phosphorylation of any of
these proteins could potentially lead to enhanced GABA release and
IPSCs. Trudeau et al. (1997)
suggested that PKA
activation causes synaptic facilitation by directly elevating
probability of exocytosis of individual vesicles at a step downstream
from Ca2+ influx. Forskolin has been shown to enhance
phosphorylation of synapsin and rabphilin, the effector protein of
Rab3A, both proteins are essential for LTP at excitatory synapses
(Lonart and Sudhof 1998
).
Sulfhydryl-alkylating agents and tetanus-induced potentiation
The sulfhydryl-alkylating agent, NEM has been reported to enhance
GABA release, potentiating evoked IPSCs and increasing the frequency of
sIPSCs, from presynaptic terminals in the rat hippocampus via a
presynaptic mechanism (Morishita et al. 1997). During
NEM- and PCMB-induced enhancement of GABAergic IPSCs, a tetanus-induced potentiation of these synaptic transients was occluded. The simplest explanation for this observation is that tetanus- and
sulfhydryl-alkylating agent-induced enhancement of GABA-A
receptor-mediated responses share common pathways. No changes in sIPSC
kinetics, amplitudes, or amplitude distribution, which might indicate
tetanus-induced postsynaptic modifications, were observed.
NEM has been shown to de-couple G protein receptors from their
substrates in central neurons (Kitamura and Nomura 1987;
Shinoda et al. 1990
). If NEM and PCMB block presynaptic
GABA-B actions in this manner and activation of these GABA-B receptors
is some how important for induction of potentiation, as we have shown, then NEM and PCMB could block potentiation of IPSCs. In this case, the
mechanism of how sulfhydryl-alkylating agents occlude GABAergic plasticity may not be related to how they affect GABA release from
presynaptic terminals.
NEM directly modulates NSF, an ATPase whose function is essential for
vesicular fusion to presynaptic membranes and thus transmitter release
(Whiteheart et al. 1994). If tetanus and NEM share
similar pathways that affect GABAergic plasticity, it is possible
tetanus modulates factors that directly affect proteins important in
secretory machinery. It is also possible that tetanus can affect NSF
indirectly through presynaptic GABA-B receptors and PKA.
It would be interesting to investigate if and how presynaptic GABA-B receptors and PKA interact and if they together regulate GABA-A-mediated synaptic plasticity. Does PKA modulate proteins important in presynaptic vesicular release and if so how do these proteins modulate potentiation of fast IPSCs?
In conclusion, based on the following observations, we suggest that the
tetanus-induced potentiation of GABAergic IPSCs is presynaptic in
origin. 1) With stimulation of two separate inputs that
elicited fast IPSPs in the same neuron, only the pathway subjected to
high-frequency stimulation showed a long-lasting potentiation;
therefore the potentiation appears to be input specific. 2)
Changes in postsynaptic membrane potential, during the tetanic stimulation, are not required to induce the potentiation. 3)
Tetanic stimulation produced an increase in the frequency of
spontaneous synaptic events without an increase in the mean sIPSC
amplitude or a change in amplitude distribution of sIPSCs.
4) Presynaptic rather than postsynaptic GABA-B receptors
appear to be needed for the induction of the tetanus-induced
potentiation of evoked and sIPSCs. 5) NEM, which has been
suggested to increase GABA release by a presynaptic mechanism (see
Morishita et al. 1997), occludes tetanus-induced
potentiation of evoked as well as sIPSCs.
While the potentiation of GABAergic IPSCs in CA1 neurons may involve an increase in GABA release at synapses, it is, however, possible that tetanic stimulation recruits and activates previously inactive presynaptic terminals and/or release sites as well as silent GABA-A receptor clusters. Another possibility is that the observed increase in evoked IPSCs might be due to an increase in the safety factor for action potential propagation at axon branching into collaterals, leading to an increase in the number of detected sIPSCs in our samples and, therefore in the size of evoked IPSCs.
Significance of plasticity of GABAergic synaptic transmission
A great deal of information about activity-mediated plasticity at
hippocampal excitatory synapses has accumulated in literature. It
should, however, be realized that the hippocampus functions in complex
network patterns and that inhibitory interneurons form numerous
connections with, and regulate many functional aspects of, principal
neurons (for review, see Freund and Buzsaki 1996). Moreover, interneurons have been shown to communicate with each other
and in doing so, can strongly influence hippocampal function (Csicsvari et al. 1999
; Freund and Buzsaki
1996
; Hajos and Mody 1997
). GABA is the major
inhibitory transmitter and network behavior in the CNS is dependent on
the balance between excitation and inhibition. Therefore potentiation
of inhibition, as observed in our studies, could significantly modulate
this balance and affect network function. The hippocampus has long been
implicated as a site that is important in learning processes and the
storage of memory (Bliss and Lomo 1973
; Lynch et
al. 1979
). Neurons in hippocampus can exhibit seizure activity
(Stelzer et al. 1987
) and can be greatly
affected by neurodegenerative (Rapport et al. 1985
;
Wiley et al. 1998
) and aging processes (Shefer
1977
). The sustained potentiation of GABA-A receptor-mediated
fast IPSCs described in this study, thus would have important
implications in developmental neurobiology, hippocampal physiology as
it relates to learning and memory, and in neurological diseases and
disorders. Our studies were conducted on 2-wk-old rats. Whether the
observed plasticity exists in other age groups is unknown and should be determined.
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ACKNOWLEDGMENTS |
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We thank CIBA-Geigy for the generous gift of CGP 36742.
This work was supported by Medical Research Council studentships to T. R. Shew and S. Yip and National Institute of Neurological Disorders and Stroke Grant NS-30959 to B. R. Sastry.
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FOOTNOTES |
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Address reprint requests to B. R. Sastry.
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 4 November 1999; accepted in final form 29 February 2000.
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REFERENCES |
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