but had no effect on LTP induced by high-frequency stimulation or long-term depression induced by low-frequency stimulation. The
The functional output of principal neurons depends critically on
synaptic inhibition by interneurons that release GABA. Drugs
that perturb GABAergic synaptic transmission affect cognitive
functions of human subjects (Barbee, 1993; Ka ¨lvia ¨inen, 1999)
and experimental animals (Sankar and Holmes, 2004). Some
neurological diseases and mental disorders are also associated
with changes in the GABAergic system (Wong et al., 2003; Lewis et
al., 2005). At the physiological level, activity of GABAergic inter-
neurons is known to regulate hippocampal rhythmic activities
(Klausberger et al., 2003; Klausberger and Somogyi, 2008), which
may be important for memory formation (Axmacher et al., 2006).
Blockade of GABAAreceptors (GABAARs) during picrotoxin-
induced epilepsy (Mackenzie et al., 2002) or potentiation of
Brazhnik and Vinogradova, 1986) markedly alters the pattern of
rhythmic activities. Furthermore, GABAergic inhibition exerts a
of local depolarization (Wigstrom and Gustafsson, 1983), and
plasticity (Kleschevnikov et al., 2004; Liu et al., 2005).
Na?- and Cl?-dependent GABA transporters (GATs), among
which GAT1 is predominantly expressed in GABAergic neurons
and tonic GABAergic inhibition (Dalby, 2000; Nusser and Mody,
2002; Semyanov et al., 2003; Keros and Hablitz, 2005). Blocking
GABA uptake with the GAT1 inhibitor tiagabine impaired spatial
learning of rats in Morris water maze (Schmitt and Hiemke, 2002),
whereas elevating GABA uptake by overexpressing GAT1 also re-
In this study, we examined the effect of disrupting GABA
uptake, using the GAT1 gene knock-out (KO) mice or specific
GAT1 inhibitor, on activity-dependent synaptic plasticity, hip-
pairs a specific form of hippocampal long-term potentiation (LTP)
15836 • TheJournalofNeuroscience,December16,2009 • 29(50):15836–15845
induced by theta burst stimulation (TBS), i.e., multiple bursts of
high-frequency (100 Hz) stimuli delivered at the theta frequency
memory. Thus, GABA uptake may serve an important function in
construct, homologous recombination, and genotyping were described
previously (Cai et al., 2006). Briefly, a 1.57 kb DNA fragment that con-
tains the exon 2 and exon 3 of the mouse GAT1 gene was replaced by a
1.37 kb neomycin-resistant gene cassette (neo) to eliminate the GAT1
gene activity. Mouse embryonic stem (ES) cell (CJ7) was electroporated
erated by injecting the recombinant ES cells into C57BL/6J blastocysts
and implanted into ICR females. GAT1 KO mice were backcrossed for
to generate homozygous, heterozygous, and wild-type (WT) littermate
were analyzed by preparing tail DNAs and PCR assay (Cai et al., 2006).
were always done during the light phase of the cycle. Mice had access to
in these experiments followed the guidelines of, and the protocols were
approved by, the Institutional Animals Care and Use Committee of the
Institute of Neuroscience, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences. In all experiments, the investigators were
blind to the genotype of mice. The experiments were performed on the
mice in a randomized order.
In vitro electrophysiology
Transverse hippocampal slices (350 ?m thick) were prepared from 6- to
10-week-old male WT or GAT1 KO littermate mice. After decapitation,
the brain was removed and placed in oxygenated (95% O2/5% CO2)
artificial CSF (ACSF) at 4°C. Slices were cut with a Leica VT1000S vi-
bratome (Leica Instruments) and maintained at room temperature (23–
25°C) in a holding chamber filled with oxygenated ACSF for at least 2 h,
whereas slices for whole-cell recordings were initially incubated in
warmed (32°C) ACSF for 30 min and then maintained at room temper-
it was held between two nylon nets and continuously perfused with ox-
ygenated ACSF (23–25°C) at a flow rate of 2–3 ml/min. The same ACSF
ing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1 NaH2PO4, 1.3 MgSO4, 26.2
osmolarity of the ACSF was 310–320 mOsm/L.
All electrophysiological recordings were performed at room tempera-
analyzed using a Digidata 1322A interface and Clampfit 9.0 software
(Molecular Devices). For extracellular recordings in the CA1 region of
the hippocampus, a bipolar platinum-iridium stimulating electrode was
The field EPSPs (fEPSPs) were recorded via a glass micropipette filled
with ACSF (1–3 M?) placed in the stratum radiatum. GABAAR antago-
nists were not present during the LTP/long-term depression (LTD) ex-
periments. Stimuli (0.1 ms duration) were delivered every 30 s. Test
pulses were recorded for 10–20 min before data collection to ensure
stability of the response. To induce LTP, the stimulation intensity under
control conditions was adjusted to evoke ?30–50% of the maximum
response. TBS and high-frequency stimulation (HFS) (100 Hz for 1 s)
were used to induce LTP. Unless otherwise stated, TBS consisted of 5
repeated once at 20 s. In some experiments, TBS was modified to multi-
ple burst stimulations (MBS) with distinctive interburst intervals (100–
1000 ms). To induce LTD, low-frequency stimulation (LFS) (1 Hz for
900 s) was used and the stimulation intensity was adjusted to evoke
?40–60% of the maximum response. The slope of fEPSPs was deter-
mined by Clampfit 9.0 software.
Whole-cell recordings were also made from the CA1 region of hip-
pocampal slices. The neurons were visually identified using an upright
contrast optics and an infrared camera. Patch pipettes were made from
borosilicate glass (1.5 mm OD) with a micropipette puller (PC-830, Na-
rishige). The internal pipette solution for voltage-clamp recording con-
EGTA, 10 HEPES. The pH was adjusted to 7.2, and the osmolarity was
300–310 mOsm/L. To block action potentials, 2 mM QX-314 was added
above internal solution was 3–5 M?. Under voltage-clamp conditions,
all the cells were held at ?70 mV. Series resistances were usually 10–20
M?. To record IPSCs, 10 ?M 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX) and 20 ?M D-2-amino-5-phosphonopentanoic acid (D-APV)
were added to the ACSF to block glutamatergic responses. A bipolar
platinum–iridium stimulating electrode was placed at the Schaffer col-
lateral axons to evoke IPSCs. To mimic the burst stimulation in TBS, a
which had a markedly longer decay time than the IPSC induced by a
single pulse (see Fig. 2B). The stability of recording was monitored by
GABAAR-mediated currents were examined by applying the selective
GABAAR antagonist picrotoxin into the slice chamber in a final concen-
shift in the holding current.
In vivo electrophysiology
In vivo electrophysiology was performed as described previously (Lin et
al., 2006). In brief, the 96-channel recording array (in stereotrode for-
GAT1 KO mice. The electrodes were advanced slowly until reaching the
CA1 area. The position of the electrode in the CA1 pyramidal layer was
determined by the presence of fast oscillations (“ripples”) in association
with synchronous discharge of neurons (Buzsa ´ki et al., 2003). Data were
obtained from the electrode with the maximum ripple amplitude. Once
in the layer, maximum ripple amplitudes were used as an online refer-
ence for consistent electrode placement between animals. Electrical ac-
sleep and novelty exploration, two parameters closely associated with
Plexon system. Histological staining, with (1% cresyl violet) was used to
confirm the electrode positions.
used throughout all behavioral tests.
20 cm. The water surface was covered with floating black resin beads.
Yellow curtains were drawn around the pool (50 cm from the pool pe-
Before training, a 60 s free swim trial without the platform was run.
For training, a submerged (1.5 cm below the surface of the water,
invisible to the animal) platform was fixed in the center of a quadrant
so that the animal had to learn the location of the platform which was
the only getaway from the water. The training session consisted of 7 d (4
trials per day). A trial was terminated when the mouse had climbed onto
60 s. Swimming paths for training session and probe test was monitored
using an automatic tracking system. This system was used to record the
swimming trace and calculate the latency to the platform and the time
spent in each quadrant.
Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTPJ.Neurosci.,December16,2009 • 29(50):15836–15845 • 15837
Passive avoidance. Mice were individually
habituated to the lighted compartment before
test. During training session, each mouse was
placed into lighted compartment and the la-
tency to enter the dark compartment was re-
corded. When the mouse entered the dark
compartment with all four paws, a foot shock
was delivered. During retention session 24 h
later, each mouse was placed into lighted com-
partment again and the latency to enter the
dark compartment was recorded.
Contextual fear conditioning. The contextual
Infrared (NIR) Video Fear Conditioning Sys-
for 3 min and the freezing response was re-
corded as baseline freezing. After contextual
learning, two 2 s foot shocks (0.75 mA) were
terward, mice remained in the chamber for 25 s
before being returned to the home cage. During
the retention test 24 h later, each mouse was
All drugs and chemicals in these experi-
ments were purchased from Sigma. In the
experiments with hippocampal slices, drugs
were applied to the bathing medium. All the
data were shown as the mean ? SEM, with
statistical significance assessed by Kolmog-
orov–Smirnov test or Student’s t test. All sta-
tistical analysis was performed using Origin
To study the function of GAT1-mediated
GABA uptake in hippocampal synaptic
plasticity, we used the GAT1 KO mice,
which do not express GAT1 and exhibit
markedly impaired GABA uptake activity
in hippocampal synaptosomes (Cai et al.,
tions, we found no detectable differences
pus between GAT1 KO and WT mice
(supplemental Fig. S1, available at www.
jneurosci.org as supplemental material).
Furthermore, immunostaining of several
markers of GABAergic and glutamatergic
synapses (NMDA receptor subunits: NR1, NR2A, NR2B; AMPA
receptor subunits: GluR1 and GluR2/3; GABAAR subunit ?2/3;
glutamic acid decarboxylases GAD65 and GAD67) did not reveal
at www.jneurosci.org as supplemental material). Consistently,
basal excitatory synaptic functions were similar between the two
groups of mice, as shown by the same input–output relation of
stimulus intensity versus the slope of field EPSPs (fEPSPs), and
ent interpulse intervals (50, 100, 150 ms) (supplemental Fig. S3,
other hand, consistent with impaired GABA uptake, the GAT1
KO mice showed a significantly larger tonic GABA-induced cur-
6, p ? 0.01) (supplemental Fig. S4A,C,D, available at www.
jneurosci.org as supplemental material) [see also Jensen et al.
(2003) and Chiu et al. (2005)]. Together, these results suggest
that genetic disruption of GAT1 in mice impaired GABA uptake
without significant effect on excitatory synaptic structure and
function in the hippocampus.
Further experiments were performed to examine the effect of
GAT1 gene deletion on hippocampal synaptic plasticity in mice.
Field EPSPs were recorded from the CA1 area of hippocampal
collateral stimulation, including theta burst stimulation [TBS,
five bursts (four pulses at 100 Hz) delivered at 5 Hz and repeated
group). Typical fEPSP recordings were shown before TBS (1) or 40 min after LTP induction (2). B, Cumulative probability of
15838 • J.Neurosci.,December16,2009 • 29(50):15836–15845Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTP
once at 20 s], HFS (100 Hz for 1 s), and LFS (1 Hz for 900 s). We
found that in hippocampal slices from WT mice, TBS induced a
4% of the baseline level, n ? 12, p ? 0.01) (Fig. 1A), indicating
of the baseline level, n ? 12, p ? 0.01) (Fig. 1A), which was
mogorov–Smirnov test) (Fig. 1B). Interestingly, for LTP in-
duced by HFS (Fig. 1C,D) and LTD induced by LFS (Fig.
or depression between the WT and GAT1 KO mice, indicating that
due to acute absence of GABA uptake through GAT1 or indirect
chronic effect of GAT1 gene deletion, we examined the effect of
acute blockade of GAT1 function with the specific GAT1 inhibi-
tor NO711 (Suzdak et al., 1992) in hippocampal slices from WT
mice. The presence of NO711 (20 ?M) had no effect on the
basal excitatory synaptic transmission, as
shown by the input–output function and
PPF of fEPSPs (supplemental Fig. S5,
available at www.jneurosci.org as supple-
mental material), but significantly in-
creased the tonic GABA current (from
5.7 ? 1.1 to 14.2 ? 2.0 pA, n ? 6, p ?
0.01) (supplemental Fig. S4A,B,D, avail-
able at www.jneurosci.org as supplemen-
tal material). As expected, we found that
NO711 treatment selectively impaired
12, p ? 0.01) as that found in GAT1 KO
mice ( p ? 0.1, Kolmogorov–Smirnov
test) (Fig. 1B), but had no effect on either
HFS-induced LTP (Fig. 1C,D) or LFS-
Thus, acute disruption of GAT1 activity
gene knock-out. Furthermore, NO711
LTP in GAT1 KO mice (supplemental Fig.
S6, available at www.jneurosci.org as
supplemental material), indicating that
GAT1 KO had occluded the NO711 ef-
fect, consistent with common underly-
ing mechanism for the impairment
of TBS-induced LTP in these two
To understand how GABA uptake affects
the induction of LTP by TBS, we exam-
ined the effect of GAT1 disruption on the
pyramidal cells to monitor IPSCs evoked
by Schaffer collateral stimulation, in the
Materials and Methods). Consistent with
previous reports (Dingledine and Korn,
ity by NO711 (20 ?M) increased the decay time of IPSCs (WT,
49 ? 6 ms; NO711, 132 ? 25 ms; n ? 7, p ? 0.01) (Fig. 2A,C)
without affecting the average IPSC amplitude (Fig. 2A,D). Sim-
ilarly, for slices obtained from GAT1 KO mice, IPSCs decay time
was longer (88 ? 5 ms, n ? 9; p ? 0.01) than that found for WT
mice (Fig. 2A,C).
To examine GABAergic inhibition during TBS, we applied a
striking (?4–5-fold) prolongation of burst-induced compound
IPSCs (Fig. 2B,C). The prolonged IPSCs were mediated by
GABAARs, because they were largely abolished by GABAAR an-
tagonist picrotoxin (100 ?M, PTX) (Fig. 2B). Similar to that
found for IPSCs evoked by a single stimulus, NO711 treatment
did not affect the amplitude of burst-induced compound IPSCs
(Fig. 2B,D). Together, these results indicate that disruption of
GABA uptake greatly enhances GABAergic inhibition for a few
Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTPJ.Neurosci.,December16,2009 • 29(50):15836–15845 • 15839
longing the time course of GABA action.
For TBS at 5 Hz, the subsequent burst stimulation arrives at 200
ms following the preceding one (Fig. 2B, dashed line) when the
difference in the amplitude of compound IPSCs is the largest
between WT and NO711-treated or GAT1 KO slices. It is ex-
pected that the excitatory action of subsequent bursts will be
induced fEPSP, we analyzed the total area of burst-fEPSP, which
mice or NO711-treated slices from WT mice, the decline of the
burst-fEPSP area during TBS was much faster than that in WT-
untreated slices (Fig. 3B,C). Thus, GAT1 disruption resulted in
significant suppression of burst-induced fEPSPs, indicating a
Given the time course of prolonged inhibition after burst
stimulation, we expect that the excitatory action of subsequent
bursts during TBS depends on the precise frequency of TBS. As
shown in Figure 4A, the difference of compound IPSCs between
WT and GAT1 KO, highlighted in the shaded area, reaches the
maximum at ?200 ms, but it was not evident at shorter (?100
ral window, we redesigned the induction protocol of LTP by
changing the interburst interval during TBS from 200 ms to 100,
143, 333, or 1000 ms (corresponding to 10, 7, 3, or 1 Hz). In WT
hippocampal slices, this multiple burst stimulation (MBS) in-
was the most effective, whereas MBS at 1 and 10 Hz was least
effective, leading to a bell-shaped frequency–response relation-
ship (Fig. 4B–F). Consistent with the prediction based on the
MBS-induced LTP in WT and GAT1 KO mice was only signifi-
cant when the MBS was given at 3, 5 (TBS), and 7 Hz, but not at
1 and 10 Hz (Fig. 4B–F). Interestingly, the MBS-induced LTP in
of stimulation, with the maximum LTP at 5 Hz (Fig. 4F). How-
ever, GAT1 deletion abolished this bell-shaped dependence (Fig.
different frequencies (Fig. 4B–E, inset) showed a similar fre-
quency dependence in the differences between WT and GAT1
KO mice (Fig. 4G), with the maximal difference at 5 Hz. Impor-
the physiological theta frequency. Thus, GABAergic inhibitory
dynamics attained by GAT1 activity plays a specific role in regu-
lating TBS-induced LTP.
To further investigate the subtype of GABA receptors mediating
the LTP impairment induced by GAT1 disruption, we first used
PTX to block GABAARs in both WT and GAT1 KO mice. In the
much stronger in GAT1 KO mice (from 126 ? 4 to 160 ? 8%,
2B), indicate a key role of GABAARs in regulating GABAergic
inhibitory dynamics after GAT1 disruption.
tic GABABreceptors (GABABRs) in TBS-induced LTP (Davies et
al., 1991; Mott and Lewis, 1991). However, the impairment of
TBS-induced LTP in GAT1 KO mice persisted following the
treatment of GABABR antagonist CGP 54626 (10 ?M) (n ? 9 for
more effective in reducing LTP in the WT mice (from 149 ? 4 to
136 ? 4%, p ? 0.05) (Fig. 5B,C), but it had little effect on TBS-
0.1) (Fig. 5B,C). Presynaptic blockade of inhibitory GABAB
mice (Sta ¨ubli et al., 1999). Our result is consistent with the idea
that GABABR blockade could not further increase the effective
GABA concentration, which already reached saturation due to
GAT1 disruption (Fig. 5D).
15840 • J.Neurosci.,December16,2009 • 29(50):15836–15845 Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTP
oscillation in vivo
The commonly used TBS for LTP induction mimics the endoge-
nous hippocampal theta rhythm (Otto et al., 1991), which plays
an important role in learning and memory (Axmacher et al.,
2006). We have recorded delta, theta, gamma, and fast ripple
oscillatory field potentials from the CA1 pyramidal layer in vivo.
During REM sleep and novelty explora-
tion, spectral analysis of hippocampal lo-
cal field activities revealed a significant
change in theta band (4–10 Hz) activities
(Fig. 6), which are involved in encoding
and storing hippocampus-dependent in-
theta band was not significantly altered in
GAT1 KO mice during either REM sleep
(WT, 7.16 ? 0.57% of total power spectral
density; KO, 6.28 ? 0.48%; n ? 6 record-
each group, p ? 0.05) (Fig. 6B). However,
the frequency at which theta power
reached maximum was significantly
shifted from 8.7 ? 0.2 Hz to 5.8 ? 0.2
Hz during REM sleep (n ? 6 recordings
of 3 mice for each group, p ? 0.01) (Fig.
6A,C), and from 9.0 ? 0.2 Hz to 6.8 ?
6B,C). Thus, GAT1 activity does not affect
tion activity, but modulates the precise fre-
ing tremor and gait abnormality, as re-
ported by a previous study (Chiu et al.,
2005). The reduction of theta oscillation
frequency in the hippocampus during
ysis of the locomotor behaviors of the mice
mice exhibited hyperlocomotion (distance
mental Fig. S7, available at www.jneurosci.
org as supplemental material), consistent
showed reduced anxiety and depression-
reduction of theta oscillatory frequency in
GAT1 KO mice during novelty exploration
Synaptic plasticity is generally viewed as a
cellular mechanism for learning and
memory (Bliss and Collingridge, 1993;
Whitlock et al., 2006), but the exact functions of different forms
hippocampal LTP described above suggest that GAT1 KO mice
al., 2005), their swimming speed were comparable to that of WT
0.05; **p ? 0.01; N.S., no significant difference, compared with WT group; Student’s t test. G, The differences of the fifth
Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTP J.Neurosci.,December16,2009 • 29(50):15836–15845 • 15841
KO, 0.15 ? 0.01 m/s, n ? 12; p ? 0.1).
eighth day, GAT1 KO mice showed a sig-
nificantly longer latency in finding the
platform during the learning session (n ?
12 for each group, p ? 0.01 for each day)
(Fig. 7A). During the probe test, GAT1
preference for the target quadrant (WT,
48.2 ? 4.2% of total time, n ? 12; KO,
cation (WT, 4.6 ? 0.9, n ? 12; KO, 0.9 ?
0.2, n ? 12; p ? 0.01) (Fig. 7C). These
results indicate that GAT1 KO mice ex-
hibited an impaired spatial learning and
memory in the Morris water maze test.
Passive avoidance test was also per-
formed to examine the hippocampus-
dependent memory in GAT1 KO mice.
KO mice were placed into lighted com-
partment and showed a similar latency in
entering the dark compartment (WT,
a foot shock. In the test session 24 h after
the training, mice were placed into the
lighted compartment again. The GAT1
KO mice showed a much shorter latency
in entering the dark compartment than
the WT mice (WT, 209 ? 25 s, n ? 12;
Finally, we examined the hippocampus-
dependent contextual fear conditioning.
During the training session, each mouse
was placed into the shock chamber for 3
min and the freezing response was re-
corded as baseline freezing. Consistent
with the hyperlocomotion, GAT1 KO
mice showed lower baseline freezing than
WT mice (WT, 12.8 ? 2.0%, n ? 10; KO,
6.4 ? 1.3%, n ? 10; p ? 0.05) (Fig. 8B).
During the retention test 24 h later, each
mouse was placed back into the same
shock chamber and the contextual freez-
ing response was recorded for 5 min. WT
mice showed much stronger freezing re-
sponses above the baseline (32.4 ? 2.0%;
n ? 10; p ? 0.01) (Fig. 8B), whereas the
freezing responses of GAT1 KO mice were
not significantly different from the baseline
In this study, we demonstrated that GAT1 disruption specifi-
cally impaired TBS-induced LTP, without affecting HFS-induced
LTP and LFS-induced LTD. In vivo electrophysiological recordings
showed that GAT1 disruption specifically altered the temporal
pattern of hippocampal theta oscillations by reducing the oscil-
lation frequency. Furthermore, these specific changes in theta
network activity and synaptic plasticity were accompanied by
severe impairment of hippocampus-dependent learning and
memory. Our data highlight a specific role of GABA uptake in
modulating rhythmic theta activities in the hippocampus, and
suggest a potential explanation of the optimal TBS frequency for
inducing hippocampal LTP.
from A and B showing that PTX rescued the impairment of LTP induction in GAT1 KO mice. *p ? 0.05; **p ? 0.01; N.S., no
ripple oscillatory field potentials were recorded in vivo from hippocampal CA1 pyramidal layer in free-moving mice (see
A and B, showing the decreased frequency of theta oscillation in GAT1 KO mice. **p ? 0.01, compared with WT group;
15842 • J.Neurosci.,December16,2009 • 29(50):15836–15845 Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTP
The GAT1 is primarily responsible for the removal of GABA
transmission. Consistently, pharmacological blockade and genetic
deletion of GAT1 significantly enhanced the tonic GABAAR-
transmission. Previous studies showed that the effect of gluta-
mate transporter blockade strongly depended on the input stim-
higher frequencies, indicating an important physiological function
Wadiche, 2007). In the present study, we found that GAT1 dis-
ruption resulted in only modest prolongation of IPSCs induced
by a single pulse, but much more robust prolongation of burst
stimulation (four pulses at 100 Hz)-induced IPSCs. Thus, the
effect of GAT1 on GABAergic transmission depends strongly on
the pattern of synaptic stimulation.
Furthermore, we found that GAT1 disruption significantly
impaired TBS-induced LTP, but had no effect on either HFS-
induced LTP or LFS-induced LTD. Blockade of GAT1 had no
significant effect on either basal fEPSPs or paired-pulse facilita-
tion but significantly impaired the burst stimulation-induced
sequential bursts, presumably due to the robust prolongation of
GABAergic inhibition that suppressed the subsequent burst-
induced responses. Consistent with the temporal window deter-
found that GAT1 disruption only affected LTP induced by stim-
ulation with multiple bursts given at the theta frequency (3–7
Hz), indicating the specific role of GAT1 in modulating TBS-
induced LTP. However, if GAT1 disruption exerts its effect on
LTP by increasing GABAergic inhibition, why was HFS-induced
LTP not affected? There are several possible explanations. First,
the elevated GABA inhibition after the GAT1 disruption is too
small to affect HFS-induced LTP induction. Second, HFS-
induced LTP is less sensitive to GABAergic inhibition, due to,
perhaps, intracellular accumulation of Cl?caused by repetitive
high-frequency stimulation within a short time (Thompson and
Gahwiler, 1989; Isomura et al., 2003) or activity-dependent
2006), or due to desensitization of the inhibitory function of
GABAergic modulation of LTP.
protocol to induce LTP, and a number of in vitro and in vivo
studies have shown that LTP induction by burst stimulation is
optimal when the time interval between the bursts is ?200 ms
(Larson and Lynch, 1986; Greenstein et al., 1988). Why burst
stimulation at the theta frequency is particularly effective in LTP
induction is largely unknown. A possible mechanism is that each
burst may modulate several ion channels, e.g., inactivating
A-type K?current (Hoffman et al., 1997) or activating Ihcon-
ductance (Cobb et al., 1995), thus priming the effect of the sub-
Student’s t test. During the retention test, WT but not GAT1 KO mice showed much stronger
Gongetal.•GAT1ModulatesThetaOscillationandTBS-LTPJ.Neurosci.,December16,2009 • 29(50):15836–15845 • 15843
sequent burst. Brain-derived neurotrophic factor (BDNF) and
TrkB signaling is known to selectively affect TBS-induced LTP
remains to be demonstrated. Our results further showed that
GABA uptake also selectively affects TBS-induced LTP. Interest-
ingly, another component in GABAergic system, presynaptic
GABABautoreceptor, is also involved in TBS-induced LTP
(Davies et al., 1991; Mott and Lewis, 1991). In TBS- but not
HFS-induced LTP, a priming effect occurs among multiple
bursts. During the interburst interval, GABA released from in-
hibitory interneurons feeds back onto presynaptic GABABRs to
depress further GABA release. Thus, a common pathway for
GAT1 and presynaptic GABABR to be involved in TBS-induced
LTP is that they both cause the reduction of GABA level in the
effect of GABABR blockade on TBS-induced LTP appeared to be
occluded by GAT1 disruption, consistent with common under-
Can the blockade of GAT1 account for the modification of
theta oscillation frequency? During learning, the typical firing
mode of hippocampal pyramidal cells consists of several sequen-
tial high-frequency bursts (of 3–5 spikes per burst at 100–400
Hz) occurring at the theta frequency, which is mimicked by the
1993; Skaggs et al., 1996). Each high-frequency burst may cause
intense activation of recurrent GABAergic inhibition and abun-
dant release of GABA, and GAT1 disruption effectively prolongs
the IPSP during each theta cycle, thus broadening the refractory
period of high-frequency burst activities and decreasing the os-
cillation frequency of theta activities. We suggest that GABA up-
take is an effective mechanism for modulating the rhythm of
theta activity, which may originate from the recurrent circuit in
a complex phenomenon involving several brain structures and
different mechanisms. Our finding does not rule out the contri-
other mechanisms besides GABA uptake. For example, septal
GABAergic neurons rhythmically hyperpolarize the hippocam-
pal basket interneurons, and may be critically involved in the
rhythm generation of theta oscillation (Buzsa ´ki, 2002). Whether
there exists endogenous regulation of GAT1 activity as a means
for physiological modulation of hippocampal theta oscillation
awaits further investigation.
Synaptic plasticity is known to be a cellular mechanism for
learning and memory (Bliss and Collingridge, 1993; Whitlock et
al., 2006), but the exact roles of different forms of synaptic plas-
described above suggest that GAT1 KO mice represent an excel-
and memory. Indeed, we found that GAT1 KO mice showed
severe impairment of learning and memory in parallel to that of
haviors are critical for learning (Buzsa ´ki, 2002). Given that theta
oscillations were altered and similar processes to TBS-induced
LTP might take place during learning, these could explain the
cause of the impaired hippocampus-dependent learning and
memory functions in GAT1 KO mice.
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