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GABA Transporter-1 (GAT1)-Deficient Mice: Differential Tonic Activation
of GABA
A
Versus GABA
B
Receptors in the Hippocampus
Kimmo Jensen,
1,
* Chi-Sung Chiu,
2,
* Irina Sokolova,
2
Henry A. Lester,
2
and Istvan Mody
1
1
Departments of Neurology and Physiology, University of California Los Angeles School of Medicine, Los Angeles, California 90095;
2
Division of Biology, California Institute of Technology, Pasadena, California 91125
Submitted 13 March 2003; accepted in final form 9 June 2003
Jensen, Kimmo, Chi-Sung Chiu, Irina Sokolova, Henry A. Lester,
and Istvan Mody. GABA transporter-1 (GAT1)-deficient mice: dif-
ferential tonic activation of GABA
A
versus GABA
B
receptors in the
hippocampus. J Neurophysiol 90: 2690–2701, 2003. First published
June 18, 2003; 10.1152/jn.00240.2003. After its release from inter-
neurons in the CNS, the major inhibitory neurotransmitter GABA is
taken up by GABA transporters (GATs). The predominant neuronal
GABA transporter GAT1 is localized in GABAergic axons and nerve
terminals, where it is thought to influence GABAergic synaptic trans-
mission, but the details of this regulation are unclear. To address this
issue, we have generated a strain of GAT1-deficient mice. We ob-
served a large increase in a tonic postsynaptic hippocampal GABA
A
receptor-mediated conductance. There was little or no change in the
waveform or amplitude of spontaneous inhibitory postsynaptic cur-
rents (IPSCs) or miniature IPSCs. In contrast, the frequency of quantal
GABA release was one-third of wild type (WT), although the densi-
ties of GABA
A
receptors, GABA
B
receptors, glutamic acid decarbox-
ylase 65 kDa, and vesicular GAT were unaltered. The GAT1-deficient
mice lacked a presynaptic GABA
B
receptor tone, present in WT mice,
which reduces the frequency of spontaneous IPSCs. We conclude that
GAT1 deficiency leads to enhanced extracellular GABA levels result-
ing in an overactivation of GABA
A
receptors responsible for a
postsynaptic tonic conductance. Chronically elevated GABA levels
also downregulate phasic GABA release and reduce presynaptic sig-
naling via GABA
B
receptors thus causing an enhanced tonic and a
diminished phasic inhibition.
INTRODUCTION
After its release, GABA is taken up both to terminate inhib-
itory transmission and for reuse by neurons and glia. Because
inhibition of GABA uptake has antiepileptic, cognition en-
hancing and neuroprotective effects (O’Connell et al. 2001),
there is an extensive interest in physiological and clinical
aspects of GABA uptake. Four distinct GABA transporters
(GATs 1–3 and a betaine/GABA transporter) have been iden-
tified in mammalian tissues. The GATs have unique anatomi-
cal distributions in the rodent CNS, and the major subtype,
GAT1 (Guastella et al 1990), is particularly abundant in areas
rich in GABAergic neurons, such as the hippocampus, neocor-
tex, cerebellum, and retina (Borden 1996). The GABA uptake
process by GAT1 is electrogenic and is driven by Na
⫹
influx
in the ionic ratios of 1 GABA:2 Na
⫹
:1 Cl
⫺
(Cammack et al.
1994; Lester et al. 1994). Recently, GAT1 has been visualized
in knock-in mice that express a mGAT1-green fluorescent
protein fusion (mGAT1-GFP) (Chiu et al. 2002). The fluores-
cence is observed in axons and nerve terminals of GABAergic
interneurons. At the ultrastructural level, GAT1 is found in
inhibitory axons and nerve terminals (Conti et al. 1998; Minelli
et al. 1995), and this organization is well suited for functions
associated with GABA uptake. Interestingly, GAT1 protein has
also been observed at sites away from GABAergic synapses
such as bare astrocytic processes (Conti et al. 1998), and a
GAT1 mRNA signal has also been detected in the pyramidal
cell layer of the hippocampus (Frahm et al. 2000). These data
suggest that GAT1 is present both at synaptic and extrasynaptic
sites, where it lowers the ambient [GABA]
o
near GABA
A
receptors and GABA
B
auto- and heteroreceptors (Mitchell and
Silver 2000). According to present concepts, inhibition of the
GABA uptake system leads to micromolar levels of ambient
GABA in the extracellular space (Dalby 2000).
We have examined the functional role of GAT1 in GABA
signaling by performing electrophysiological measurements in
GAT1-deficient mice. In the course of generating the mGAT1-
GFP strain, we constructed a strain that contains a 2.2-kb neo
cassette in intron 14 of the GAT1-GFP fusion gene (Chiu et al.
2002). Although this gene has a theoretically intact set of
exons, such large insertions often produce splicing errors and
lead to decreased protein expression. We report here that
homozygotes from this strain are so hypomorphic for GAT1 in
hippocampal axons and terminals, that they are functional
knockouts (KOs), and for convenience the strain will be termed
GAT1 KO.
We examined GABA
A
receptor conductances and the regu-
latory functions of GABA
B
receptors in the GAT1 KO mice.
We found several distinct alterations, which generally support
the essential role of GAT1 in regulating transmission by both
GABA
A
and GABA
B
receptors.
METHODS
Generation of GAT1-deficient mice
GAT1-deficient mice were generated as an intermediate in the
construction of the mGAT1-GFP strain (Chiu et al. 2002). By homol-
ogous recombination, a 2.2-kb neo cassette was inserted into intron 14
of the GAT1-GFP fusion gene in ES cells (Fig. 1A). Blastocysts were
*K. Jensen and C.-S. Chiu contributed equally to this work.
Address for reprint requests and other correspondence: I. Mody, Dept. of
Neurology, The David Geffen School of Medicine at UCLA, 710 Westwood
Plaza, RNRC 3-155, Los Angeles, CA 90095-1769 (E-mail: mody@ucla.edu).
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.
J Neurophysiol 90: 2690–2701, 2003.
First published June 18, 2003; 10.1152/jn.00240.2003.
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injected with recombinant ES cells and implanted into C57Bl6 fe-
males (Chiu et al. 2002). The strain is maintained on a C57Bl6
background and originally named intron 14-neo-mGAT1 (Fig. 1, B
and C). These mice are hypomorphic for GAT1 expression in the
brain such that they are functional KOs. Accordingly, this strain will
be termed GAT1 KOs in this paper. Individual mice were genotyped
as described earlier (Chiu et al. 2002).
Synaptosomal preparation and GABA uptake assay
Mice were anesthetized with halothane, and brains were dissected
and collected on ice. Tissue (⬃25 mg) was homogenized in 20⫻
(wt/vol) homogenization buffer (0.32 M sucrose, 0.1 mM EDTA, and
5 mM HEPES, pH 7.5; 1 ml) (Nagy and Delgado-Escueta 1984). The
mixture was centrifuged at 1,000⫻gfor 10 min. The supernatant was
further centrifuged at 10,000⫻gfor 20 min to produce the crude
mitochondria pellet. The mitochondria pellet was washed once with 1
ml homogenization buffer. The particulate (synaptosome) fraction
from the 10,000⫻gwas suspended with 1 ml homogenization buffer.
For GABA uptake assays, 20
l of the suspension was mixed with
280
l of uptake buffer, which contained (in mM) 128 NaCl, 2.4 KCl,
3.2 CaCl
2
, 1.2 MgSO
4
, 1.2 KH
2
PO
4
, 10 glucose, and 25 HEPES, pH
7.5 (Lu et al. 1998) and incubated at 37°C for 10 min. Subsequently,
100
l GABA and [
3
H]GABA (5 and 0.05
Mfinal, respectively)
was added and incubated for additional 10 min. Uptake was termi-
nated by placing the samples on ice, followed by two washes with
uptake buffer containing same concentration of cold GABA at
10,000⫻g. The GAT1-specific inhibitor NO-711 (30
Mfinal) was
included to measure the nonGAT1 uptake activity; the NO-711-
sensitive fraction accounted for 75–85% of WT activity.
Brain slice preparation and electrophysiology
Wildtype littermates (WT) and GAT KO mice (P15–P25) were
anesthetized with halothane before decapitation, and the brains were
removed and placed into an ice-cold artificial cerebrospinal fluid
(ACSF), in accordance with a protocol approved by the UCLA Chan-
cellor’s Animal Research Committee. The ACSF contained (in mM)
126 NaCl, 26 NaHCO
3
, 1.25 NaH
2
PO
4
, 2.5 KCl, 2 CaCl
2
, 2 MgCl
2
,
10 D-glucose, 0.2 L-ascorbic acid, 1 pyruvic acid, and 3 kynurenic
acid, pH 7.3, bubbled with 95% O
2
-5% CO
2
. The brain was glued to
a platform, and 350-
m-thick slices were cut in the coronal plane with
a Leica VT1000S vibratome. The slices were stored in bubbled ACSF
at room temperature for ⱖ1 h until transferred individually to the
recording chamber.
During experiments, the slices were perfused with ACSF at 1.5
FIG. 1. Generation and screening of the GABA transporter-1 (GAT1) knockout (KO) mouse strain. A: modification of mGAT1
genomic DNA to generate a targeting plasmid that contains a floxed neomycin selection cassette in intron 14 and an mGAT1-GFP
fusion sequence at the C-terminus of exon 15. B: portion of the mGAT1 gene in the neo-intact mGAT1-green fluorescent protein(or
GAT1 KO) mouse. C: example of PCR genotyping results. Lanes 1–3 show PCR screening for mice that are respectively
homozygous, heterozygous, and wild type (WT) for the neo cassette. D: hippocampal synaptosomal GABA uptake. Ratios of
NO-711-sensitive and NO-711-insensitive synaptosomal [
3
H]GABA uptake activities among the 3 genotypes (means ⫾SE;
triplicate assays from each of 4 experiments with all 3 genotypes).
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ml/min at 32–33°C. CA1 pyramidal cells in hippocampus were visu-
ally identified [Zeiss Axioscope infrared differential interference con-
trast (IR-DIC) videomicroscopy, ⫻40 water-immersion objective].
Whole cell recordings were made using an Axopatch 200B amplifier
(Axon Instruments, Union City, CA). Patch electrodes were pulled
(Narishige PP-83, Tokyo, Japan) from borosilicate glass (1.5 mm OD,
1.10 mm ID; Garner, Claremont, CA) and filled with a solution
containing (in mM) 140 CsCl, 2 MgCl
2
, and 10 HEPES, titrated to a
pH of 7.2 with CsOH (osmolarity: 275–290 mosmol/l). In a few
experiments, MgATP was used instead of MgCl
2
, which did not
influence the GABA
A
currents. When excitatory and inhibitory
postsynaptic currents (EPSCs and IPSCs) were recorded simulta-
neously, the patch pipette contained (in mM) 135 Cs-gluconate, 10
CsCl, 5 TEA, 0.1 EGTA, and 15 HEPES, titrated to pH 7.2 with
CsOH. Postsynaptic GABA
B
receptor-mediated currents were re-
corded with patch pipettes containing (in mM) 130 K-methylsulfate,
0.3 NaGTP, 2 MgATP, 10 KCl, and 10 HEPES, pH 7.2 with KOH.
The resistances of the electrodes were between 3 and 6 M⍀when
filled with solution. The pyramidal cells were recorded in voltage-
clamp mode at a holding potential (V
hold
)of⫺70 mV, unless other-
wise indicated. The series resistance and whole cell capacitance were
monitored repeatedly during the experiments. The series resistance
was compensated by 70–85% using lag values of 7–8
s, and
recordings were discontinued if the series resistance increased by
⬎50%. In a random sample of cells, the average precompensation
series resistance was 13.4 ⫾1.3 (SE) M⍀(n⫽10; range: 7–19 M⍀).
No differences were noted in the series resistances of recordings
between different groups of cells. SR95531, furosemide, and CGP
62349 were dissolved in 50% DMSO, whereas TTX, NO-711, and
GABA were dissolved in water. The final concentration of DMSO
(⬍1%) had no effect per se on the GABA receptor currents. All
chemicals were purchased from Sigma or Tocris (Ellisville, MO).
CGP62349 was a generous gift from W. Froestl (Novartis, Basel).
Analysis of phasic and tonic GABA receptor-mediated
currents
Recordings were low-pass filtered (8-pole Bessel, Brownlee 210A)
at 3 kHz and digitized on-line at 20 kHz using a PCI-MIO 16E-4
data-acquisition board (National Instruments, Austin, TX). Spontane-
ous and miniature synaptic events were detected with amplitude- and
kinetics-based criteria (events were accepted when they exceeded a
threshold of 6–8pAfor⬎0.5 ms) using custom-written LabView-
5.1-based software (National Instruments) running on a Pentium III
computer. Traces were imported into a custom-written analysis pro-
gram, where signals were analyzed and averaged and amplitudes and
kinetics measured. All IPSCs and EPSCs were also inspected visually,
and sweeps were rejected or accepted manually.
Tonic GABA
A
receptor-mediated currents were examined by in-
jecting the selective GABA
A
antagonist SR95531 into the slice cham-
ber in a final concentration of 100–150
M (30–35
lofa6-to
8-mM SR95531 solution; chamber volume: 1.8–2.0 ml) (Brickley et
al. 1996). This SR95531 application will be referred to in text and
figures as “SR95531 ⬎100
M.”When a tonic GABA
A
current was
present, this led to an outward shift in the holding current (E
Cl
⬃0
mV). To analyze the holding current, we measured the mean current
in 5-ms-long epochs at 100-ms intervals. Epochs were rejected if
contaminating IPSCs were present as described earlier (Nusser and
Mody 2002). The mean holding current was calculated in three
5-s-long periods (a,band c). a: 20 s before SR95531 application, b:
immediately before SR95531 application, c: 20 s after the application.
The tonic GABA
A
current was calculated as c–b. The difference
between band awas used to judge the spontaneous baseline changes
without adding drugs and is termed “no treatment”in Table 1. Tonic
GABA
B
receptor-mediated postsynaptic currents were quantified in a
similar manner, using bath injection of 35–40
l of a 0.5-mM CGP
62349 solution, which led to an estimated final concentration of 8–12
M and is referred to as “CGP 62349 ⬎8
M.”Although these drugs
were added as a 30- to 40-
l highly concentrated bolus to the bath, the
concentrations that reached the cells were more than an order of
magnitude higher than that required for full block of the respective
receptors.
Paired and unpaired t-test were performed in Microsoft Excel (v.
2000), whereas for comparing multiple means, Duncan’s multiple
range test was employed using Statistica 6.0 software (StatSoft, Tulsa,
OK). Significance level was set to P⬍0.05, and data are expressed
as means ⫾SE with nindicating the number of cells.
Immunocytochemistry
Mice were anesthetized with halothane and were perfused with 4%
paraformaldehyde in PBS, pH adjusted to 7.6 with Na
2
HPO
4
. Brains
were dissected and kept in 4% paraformaldehyde for1hat4°C, then
incubated with 25% sucrose in phosphate-buffered saline for ⬃20 h.
The brains were embedded in optimal cutting temperature (OCT)
medium (Sakura, Torrance, CA) for sagital sections and sliced with a
cryostat at 35
m. Brain slices were stored in (in mM) 11 NaH
2
PO
4
,
20 Na
2
HPO
4
, 30% ethylene glycol, and 30% glycerol, pH 7.5, at
⫺20°C.
Sections were incubated for2hatroom temperature in a blocking
solution [10% normal goat serum (NGS), 0.3% triton in PBS, pH 7.6],
followed by incubation with the primary antibody for 36–48hat4°C
with rotational mixing. Primary antibodies and their dilutions were
rabbit anti-GABA
A
receptor
␣
1 (Upstate; 1:100), guinea pig anti-
GABA
B
-R1a/b (Chemicon; 1:1,000), rabbit anti-glutamate decarbox-
ylase (GAD) 65 (Chemicon; 1:1,000), rabbit anti-calbindin D28k
(Swant; 1:2,500), rabbit anti-calretinin (Chemicon; 1:1,000), goat
anti-parvalbumin (Swant; 1:2,500), and rabbit anti-vesicular GABA
transporter (vGAT; Synaptic Systems; 1:100). After treating with
primary antibody, the brain slices were washed with PBS containing
0.5% triton, followed by two washes with PBS. The slices were then
incubated in solutions containing the appropriate rhodamine red-x-
conjugated secondary antibodies. These secondary antibodies are goat
anti-rabbit, or goat anti-guinea pig, or donkey anti-goat secondary
antibodies (Jackson Laboratories; 1:200). After three washes with
PBS, slices were rinsed with PBS and mounted with Vectashield
(refractive index: 1.4577).
Image analysis and bouton quantification
Brain slices were imaged using a Leica TCS SP1 confocal micro-
scope system. Images were taken with a 100⫻plan apochromatic
objective, NA 1.4 (Leica, No. 506038). The pinhole was set at 152
m
as recommended by the manufacturer. The scan speed was 200 lines/s
(slow mode) and the image size was 1,024 ⫻1,024 pixels. Each
image was scanned with four repeats. The laser power was adjusted
using the microscope’s acousto-optical tunable filter so that the fluo-
rescence of a sample fell within the linear range of the detection
system.
The ImageJ package was used for analysis, and immunopositive
structures were counted using the “analyze particle”function. The
criterion for detectable fluorescence intensity was set to ⬎2.5 times
average intensity where little or no background fluorescence was
included. Only structures ⬎20 or 25 pixels (⬃0.2–0.24
m
2
), de-
pending on bouton size, were included. Counted structures were
inspected visually at the end of each analysis. When several boutons
were closely interspaced, fluorescence became confluent and the
structures were counted as one. Such large particles were observed
with similar density in WT and KO.
2692 JENSEN, CHIU, SOKOLOVA, LESTER, AND MODY
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RESULTS
GAT1 KO mice have little or no NO-711-sensitive GABA
transport activity
Both heterozygous and homozygous GAT1 KO mice are
viable and fertile. The coding region of mGAT1 in these mice
contains a C-terminal GFP fusion, and the neo-deleted
mGAT1-GFP strain described previously has nearly normal
GAT1 activity (Chiu et al. 2002). In the strain described in this
paper, measurements of [
3
H]GABA uptake confirmed the lack
of GAT1 activity. Crude synaptosome preparations from hip-
pocampus of the GAT1 KO homozygotes showed ⬍2% NO-
711 sensitive [
3
H]GABA uptake activity (0.17 ⫾0.08 pmol/10
min/mg) compared with WT littermates (17.1 ⫾1.4 pmol/10
min/mg), and heterozygotes displayed intermediate activity
(10.9 ⫾0.4 pmol/10 min/mg; Fig. 1D). The NO-711-insensi-
tive [
3
H]GABA uptake measured in hippocampal synapto-
somes was unaffected in GAT1 KO animals or in heterozy-
gotes (Fig. 1D), indicating that GABA uptake mediated by
other transporters was not upregulated to compensate for the
absence of GAT1 function.
GAT1 KO mice show increased tonic GABA
A
currents
in CA1 pyramidal cells
To assess the functional changes in GAT1 KO mice, we
made whole cell recordings from hippocampal CA1 pyramidal
cells in GAT1 KO and WT slices. First, we examined the
influence of GAT1 deletion on phasic and tonic GABA
A
re-
ceptor-mediated currents. Neurons loaded with CsCl were
clamped at a V
hold
of ⫺70 mV in the presence of the broad-
spectrum ionotropic glutamate receptor antagonist kynurenic
acid (3 mM). Phasic currents appeared as spontaneous IPSCs
(sIPSCs), and in both WT and GAT1 KO mice, sIPSCs were
blocked by the GABA
A
receptor antagonist SR95531 (⬎100
M) (Fig. 2, Aand B). In GAT1 KO slices, SR95531 also
revealed a tonic, steady GABA
A
receptor-mediated current of
45.8 ⫾11 (n⫽5) versus 4.9 ⫾2.3 pA in WT (P⬍0.05, n⫽
4). These tonic currents were measured by assessing the hold-
ing current every 100 ms in 5-ms-long epochs (see METHODS).
GABA-uptake inhibitors produce tonic GABA
A
currents
in WT slices
In WT slices, a tonic GABA
A
current (mean: 25.2 ⫾6.0 pA,
n⫽5, Fig. 2C, middle) could be recorded in the presence of
the specific GAT1 inhibitor NO-711 (10
M). Further increas-
ing the NO-711 concentration to 50
M in WT slices, reduced
the tonic current by 50% to 12.6 ⫾3.5 pA (n⫽4, Fig. 2C,
right) consistent with a possible GABA
A
receptor blocking
effect of the uptake blocker (Overstreet et al. 2000). The
potentiating effect of NO-711 (10
M) on the tonic current was
absent in GAT1 KO slices demonstrating that NO-711-sensi-
TABLE 1. Tonic GABA
A
receptor-mediated currents in CA1
pyramidal cells in wild-type (WT) and in GABA transporter 1
(GAT1) knockout (KO) mice under various treatments
Treatment SR95531-Sensitive Tonic
Current, pA No. of
Cells
No treatment 5.7 ⫾1.0 20
WT 4.9 ⫾2.3 4
WT ⫹10
M NO-711 25.2 ⫾6.0 5
WT ⫹50
M NO-711 12.6 ⫾3.5 4
WT ⫹10
M NO-711 ⫹0.8
M GABA 45.4 ⫾21 4
GAT1 KO 45.8 ⫾11 5
GAT1 KO ⫹10
M NO-711 38.1 ⫾14 7
GAT1 KO ⫹1
M TTX 17.0 ⫾4.9 5
GAT1 KO ⫹0.6 mM furosemide 18.9 ⫾3.9 4
GAT1 KO ⫹1 mM furosemide 17.9 ⫾6.3 4
Values are means ⫾SE. No treatment: spontaneous holding current fluc-
tuations during 20 s with no applied SR95531.
FIG. 2. GAT1 KO mice display a large tonic
GABA
A
receptor-mediated current. A: in WT slices,
spontaneous IPSCs (sIPSCs) were recorded in Cl
⫺
-
loaded CA1 pyramidal cells (V
hold
⫽⫺70 mV).
Injection of the GABA
A
antagonist SR95531 into the
bath (⬎100
M) blocked the sIPSCs. B: in GAT1
KO slices, SR95531 blocked the sIPSCs but also
abolished a steady inward current (⬃85 pA in this
example). This current reflects a tonic GABA
A
re-
ceptor-mediated conductance. C: to illustrate the
tonic GABA
A
currents, the holding current of 3
representative WT pyramidal cells is plotted vs.
time. In the WT control cell (left), the SR95531-
sensitive tonic current was ⬃5 pA. Addition of 10
M NO-711 produced a tonic GABA
A
current of 15
pA in this example (middle), while 50
M NO-711
caused no further increase. D: in a cell in an un-
treated GAT1 KO slice, a tonic current of ⬃80 was
observed (left). The tonic current was strongly re-
duced by TTX (1
M; middle). Furosemide (0.6
mM) reduced the tonic current by ⬃60% (right)
compared with the untreated GAT1 KO slice.
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tive GABA uptake was not functional in these animals. Perfu-
sion of NO-711 (10
M) slightly reduced the tonic current in
GAT1 KO slices (mean: 38.1 ⫾13.7 pA, n⫽7, not shown),
consistent with a blocking action of NO-711 on the GABA
A
receptors responsible for the tonic current. This confounding
effect of NO-711 could be overcome by adding a small con-
centration of agonist. The tonic current observed in GAT1 KOs
could be reproduced in WT slices perfused with the combina-
tion of 10
M NO-711 and 0.8
M GABA: this treatment
induced a tonic current of 45.4 ⫾21 pA (n⫽4, not shown)
comparable to that observed in GAT1 KOs.
Modulation of tonic GABA
A
currents in GAT1 KOs
by furosemide and TTX
Next, we asked whether the tonic GABA
A
conductance in
GAT1 KOs could be modulated. We tested whether the in-
creased GABA levels in the GAT1 KO mice originated from
action potential-dependent release. TTX (1
M) reduced the
tonic GABA
A
current by 63% to 17.0 ⫾4.9 pA compared with
45.8 pA without TTX (P⬍0.05, n⫽4; Fig. 2D, middle). This
result indicates that presynaptic action potentials are partially
responsible for the GABA release that elevates [GABA]
o
.In
GAT1 KO slices, furosemide (0.6 –1 mM), which blocks a
subset of postsynaptic GABA
A
receptors with distinct subunit
compositions (Jackel et al. 1998), reduced the tonic GABA
A
currents by 62% to 18.4 ⫾3.5 pA (n⫽8, P⬍0.05; Fig. 2D,
right). The effects of 0.6 or 1 mM furosemide on tonic currents
were indistinguishable, and furosemide did not affect s- or
mIPSCs (following text). A summary of the tonic currents
under various treatments is shown in Table 1.
sIPSCs are not altered in GAT1 KO mice
As summarized in Table 2, sIPSCs were not significantly
different in GAT1 KO mice when compared with those re-
corded in WT mice (Fig. 3, Aand B). Their amplitudes were
36.6 ⫾2.6 pA in WT (n⫽6) and 54.3 ⫾13 pA in GAT1 KO
(n⫽6, P⬎0.05). Application of NO-711 (10
M; n⫽5) or
NO-711 (10
M) ⫹GABA (0.8
M; n⫽4) in WT slices did
not significantly affect sIPSC, amplitudes, frequencies, or
waveforms. Furosemide also failed to affect sIPSC amplitudes
TABLE 2. Properties of spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal cells
sIPSCs WT WT ⫹NO-711 WT ⫹NO-711 ⫹0.8
M GABA GAT1 KO
Amplitude, pA 36.6 ⫾2.6 48.0 ⫾6.7 35.7 ⫾4.8 54.3 ⫾13
Frequency, Hz 24.1 ⫾4.7 15.9 ⫾4.5 22.2 ⫾4.9 16.8 ⫾5.8
Rise time (10–90%),
s 331 ⫾55 330 ⫾38 303 ⫾32 369 ⫾47
Decay constant, ms (weighted) 4.0 ⫾0.2 4.0 ⫾0.3 3.6 ⫾0.2 4.1 ⫾0.2
No. of cells 6 5 4 6
Properties of sIPSCs in CA1 pyramidal cells in WT slices, in WT slices perfused with NO-711 (10
M), and in GAT1 KO slices. GABA
A
receptor-mediated
sIPSCs were recorded in Cl
⫺
-loaded cells at a V
hold
of ⫺70 mV. No significant changes were observed.
FIG. 3. sIPSCs are similar in WT and GAT1 KO
mice. A: sIPSCs recorded in Cl
⫺
-loaded pyramidal
cells in the presence of kynurenic acid (3 mM). B:
the average of 50 sIPSCs in WT and GAT1 KO
(right). In the GAT1 KO pyramidal cell, the sIPSC
waveform was not different from that in WT, al-
though the sIPSCs occurred on a noisier baseline.
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in GAT1 KOs (38.6 ⫾4.2 pA, n⫽11), frequencies (21.4 ⫾
5.2 Hz, n⫽11), 10–90% rise times (285 ⫾38
s, n⫽5), or
the weighted decay time constants (4.4 ⫾0.36 ms, n⫽5).
These values are not significantly different from the GAT1 KO
control data in Table 2 (P⬎0.05).
mIPSCs have reduced frequencies in GAT1 KO mice
mIPSCs recorded in the presence of TTX (1
M) had similar
10–90% rise times, decay time constants, and amplitudes in
WT (n⫽9) and GAT1 KO pyramidal cells (n⫽9, P⬎0.05;
Table 3 and Fig. 4). Yet, the frequency of mIPSCs in GAT1
KO animals was reduced to about one-third of control frequen-
cies (6.4 ⫾1.6 Hz in GAT1 KO compared with 17.8 ⫾2.1 Hz
in WT; P⬍0.01). One possible explanation for the finding of
reduced mIPSC frequency, but no reduction in amplitudes, is
that the GAT1 KO slices have a reduced number of functioning
GABAergic synapses onto pyramidal cells. Another reason for
the change in frequency could have been a differential detec-
tion of mIPSCs by our analysis software, resulting from a
noisier baseline (more tonic current) in GAT1 KO cells. This
second possibility is highly unlikely as the average peak-to-
peak noise as well as the SD of the baseline current were not
statistically different between the recordings from WT and
GAT1 KO slices (Table 3). This contrasts with the tonic
inhibition-dependent increase in baseline variance recorded in
small compact neurons such as dentate gyrus granule cells
(Stell and Mody 2002). However, in the larger and leakier
pyramidal cells the tonic current is only a small fraction of the
total holding current (13.8% in GAT KO and 9.7% in WT ⫹10
M NO-711), and therefore it does not significantly contribute
to the baseline variance. Furthermore, the lowest amplitude
sIPSCs detected by the analysis software were also comparable
between WT and GAT1 KO (12.8 ⫾0.6 vs. 13.8 ⫾0.7 pA,
P⬎0.05), and the fraction of the events found in the low-
amplitude bins were not different between the two preparations
(e.g., see lowest 25% of the events in the cumulative proba-
bility plots of amplitudes depicted in Fig. 4C). Nevertheless, it
might have been possible for two nonsignificant effects (i.e., a
slight reduction in amplitude and a small increase in detection
threshold) to combine and artificially yield a significant reduc-
tion in mIPSC frequency. To examine this possibility, we have
modeled such a combined effect by generating log-normal
distributions that fit the amplitude distributions of mIPSCs in
WT neurons. Next, we shifted the mean to lower amplitudes by
10%, and the detection threshold was shifted toward larger
amplitudes by 8%, corresponding to the experimental values
obtained in GAT1 KO slices. In such simulations, the number
of detected events was reduced by ⬍10%. Therefore we con-
clude that combined small shifts in amplitude and detection
threshold are not responsible for the dramatic 64% reduction in
mIPSC frequency in the GAT1 KO animals.
Lack of tonic postsynaptic GABA
B
receptor activation
The results so far indicate that slices from GAT1 KO mice
have elevated ambient [GABA]
o
levels, which persistently
activate postsynaptic GABA
A
receptors. Perfusion of NO-
711 and GABA (0.8
M) onto WT slices mimicked this
condition. In the presence of such elevated [GABA]o, we
also expected postsynaptic GABA
B
receptors to be tonically
activated in CA1 pyramidal cells. We tested this hypothesis
using K-methylsulfate-filled pipettes to record from neurons
voltage-clamped at –50 mV while the GABA
B
receptor
antagonist CGP 62349 was injected into the bath to yield a
final concentration of ⬎8
M (Fig. 5A). Surprisingly, nei-
ther NO-711 (10
M, n⫽2) nor NO-711 plus 0.8
M
GABA (n⫽3) induced any detectable postsynaptic GABA
B
TABLE 3. Properties of GABA
A
-receptor-mediated miniature
IPSCs (mIPSCs) in CA1 pyramidal cells
mIPSCs WT GAT1 KO
Amplitude, pA 37.5 ⫾3.1 34.1 ⫾2.9
Frequency, Hz 17.8 ⫾2.1 6.4 ⫾1.6**
Baseline peak-to-peak, pA 10.80 ⫾0.78 9.73 ⫾0.58
Baseline SD, pA 4.61 ⫾0.2 4.06 ⫾0.23
10–90% rise time,
s 298 ⫾11 280 ⫾9
Weighted decay time
constant, ms 4.6 ⫾0.2 4.6 ⫾0.2
No. of cells 9 9
Properties of mIPSCs in CA1 pyramidal cells of WT and GAT1 KO slices.
mIPSCs were recorded in the presence of 1
M TTX at a V
hold
of ⫺70 mV.
** P⬍0.01.
FIG. 4. Miniature IPSCs (mIPSCs) in GAT1 KO mice have reduced fre-
quencies compared with WT but similar kinetics. A: mIPSCs were recorded in
TTX (1
M) in WT and GAT1 KO CA1 pyramidal cells. In GAT1 KOs, the
mIPSCs had longer inter-event intervals. B: the amplitudes and kinetics of
mIPSCs were similar in WT and GAT1 KO. The traces are averages of 50
mIPSCs. In the displayed cells, the mean peak amplitudes were 46 pA for WT,
and 45 pA for GAT1 KO. C: cumulative probability plots show the distribution
of mIPSC inter-event intervals (left) and amplitudes (right) from representative
WT and GAT1 KO cells. Longer inter-event intervals were observed for GAT1
KO mIPSCs.
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currents in WT slices (Fig. 5A, 1.3 ⫾1.4 pA, the 5 cells
pooled). Similarly, a tonic postsynaptic GABA
B
receptor
current could not be detected in GAT1 KO slices (1.3 ⫾0.9
pA, n⫽3). To ascertain the presence of functional postsyn-
aptic GABA
B
receptors (Fig. 5B), we perfused the GABA
B
agonist baclofen (10
M). This induced a robust CGP
62349-sensitive current of 50.4 ⫾13.9 pA in WT (n⫽9)
and 41.2 ⫾5.9 pA in GAT1 KO (n⫽7). Although the
GAT1 KO baclofen response was somewhat smaller, this
difference did not reach statistical significance (i. e., P⬎
0.05, when compared with WT, unpaired t-test assuming
unequal variances, or Mann-Whitney nonparametric test).
The results indicate that postsynaptic GABA
B
receptors are
not activated by the levels of GABA that activate GABA
A
receptors to produce the tonic current.
Presynaptic GABA
B
receptor tone is lost in GAT1 KO mice
In light of the abundance of presynaptic GABA
B
receptors
(Schuler et al. 2001), we next examined a possible tonic
activation of such receptors by ambient GABA in WT and
GAT1 KO slices. CA1 pyramidal cells were recorded with
Cs-gluconate filled electrodes at V
hold
between –20 and –30
mV, where inward sEPSCs and outward sIPSCs of roughly
similar amplitudes could be observed (Fig. 6A). Under the
conditions of such reduced driving forces, the detectable events
had frequencies of about 1/10 of those recorded under control
conditions at two to three times the driving force (Stell and
Mody 2002). In WT slices in the absence of NO-711, CGP
62349 (⬎8
M) caused a large increase in sIPSC frequency
from 1.5 ⫾0.3 to 4.9 ⫾2.3 and 5.7 ⫾0.3 Hz, at 20 and 30 s,
respectively, after CGP application (n⫽4, P⬍0.01). The
mean sIPSC amplitudes increased also from 26.1 ⫾2.7 to
31.0 ⫾2.5 pA in CGP 62349 (P⬍0.05, n⫽4). The frequency
of sEPSCs changed less dramatically from a control frequency
of 1.7 ⫾0.4 to 3.1 ⫾1.3 Hz at 20 s and to 3.7 ⫾1.9 Hz at 30 s
after CGP 62349 (n⫽4, P⬎0.05), without a change in mean
FIG. 6. A presynaptic GABA
B
tone is lost in GAT1 KO mice. A: simulta-
neous recordings of sEPSCs (inward currents) and sIPSCs (outward currents)
using Cs-gluconate pipettes. V
hold
was about –30 mV to obtain similarly sized
inward and outward currents. Inset: the events on a faster time scale (I: sIPSC,
E: sEPSC). After injection of CGP 62349 (⬎8
M), sIPSCs increased in
frequency and amplitude, whereas sEPSCs did not change. B: the increase in
sIPSC frequency after the CGP 62349 injection was abolished by 6,7-dinitro-
quinoxaline-2,3-dione (DNQX; 10
M) and APV (50
M), which block
glutamatergic excitation. This indicates that tonically active GABA
B
receptors
control the presynaptic network in WT. C: in GAT1 KO slices, no increases in
the frequencies of sIPSCs or sEPSCs were observed after CGP 62349 appli-
cation.
FIG. 5. Tonic GABA
B
receptor-mediated postsynaptic currents are not
present in WT or GAT1 KO CA1 pyramidal cells. A: no postsynaptic outward
currents were present in WT as tested by the GABA
B
receptor antagonist CGP
62349 (⬎8
M). This experiment was performed in the presence of NO-711
(10
M). In an untreated GAT1 KO slice (right), a tonic postsynaptic GABA
B
current was also absent. The pyramidal cells were recorded with a K-based
intracellular solution-filled electrode and held at –50 mV. Slices were perfused
with kynurenic acid (3 mM) and picrotoxin (50
M) to block ionotropic
glutamate and GABA
A
receptors, respectively. B: baclofen (10
M) induced a
robust GABA
B
receptor-mediated current, which was revealed during antag-
onism by CGP 62349 (⬎8
M). The GABA
B
current reached 55 pA in the
exemplar WT cell and 60 pA in the GAT1 KO cell. Scale bars apply to all
traces in Aand B. C: summary of postsynaptic GABA
B
currents in CA1
pyramidal cells. The WT data consist of the pooled results from 5 cells where
NO-711 (n⫽2) or NO-711 ⫹0.8
M GABA (n⫽3) was present. No tonic
GABA
B
currents were observed (1.3 ⫾1.4 pA, n⫽5). When NO-711 and
GABA were replaced by 10
M baclofen, the GABA
B
currents reached
50.4 ⫾13.9 pA (n⫽9). For GAT1 KO slices, the GABA
B
current reached
1.3 ⫾1.4 pA (n⫽3) in the absence and 41.2 ⫾5.9 pA (n⫽7) in the presence
of baclofen (10
M). The baclofen-induced currents were not different be-
tween WT vs. GAT1 KO (P⬎0.05). Error bars indicate SE.
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amplitudes (24.8 ⫾3.1 pA in control vs. 25.9 ⫾2.5 pA in CGP
62349). The increase in sIPSC frequency produced by the
GABA
B
receptor antagonist in WT slices was abolished by
blocking glutamatergic excitation with 6,7-dinitroquinoxaline-
2,3-dione (DNQX; 10
M) and D-APV (50
M). Twenty and
30 s after CGP application, the normalized sIPSC frequencies
were 89.7 ⫾6.0 and 86.7 ⫾11.7% of control, respectively
(n⫽4, P⬎0.05). This finding is consistent with a powerful
GABA
B
receptor-dependent control of interneuron activation
by excitatory synapses in WT slices.
In contrast, in GAT1 KO slices with excitation intact
blockade of GABA
B
receptors failed to increase the fre-
quency of sIPSCs. The sIPSC frequency was 1.9 ⫾0.3 Hz
during the control period and remained steady at 2.0 ⫾0.4
and 1.7 ⫾0.6 Hz, respectively, at 20 and 30 s after CGP
62349 (n⫽5, P⬎0.05). For sEPSCs, the frequency during
the control period was 1.8 ⫾0.5 Hz, and 2.0 ⫾0.5 Hz and
2.2 ⫾0.6 Hz, at 20 and 30 s after CGP 62349 (P⬎0.05).
To circumvent the possibility that marginally detectable
sIPSCs were rendered below detection threshold by the
activation of presynaptic GABA
B
receptors, we repeated the
experiments in Cl
⫺
-loaded cells, with glutamatergic excita-
tion blocked by kynurenic acid (3 mM) and a high driving
force (V
hold
⫽⫺70 mV). Under these conditions, i.e., in the
absence of an excitatory drive onto interneurons, blocking
GABA
B
receptors had no significant effect (P⬎0.05;
paired t-test) on the frequencies of the sIPSCs in either WT
(n⫽8) or GAT-1 KO (n⫽5) slices when the frequency of
the events during a control period was compared with 10-s
periods measured 20 and 30 s after CGP 62349 was added
to the bath. To illustrate these results, the normalized fre-
quencies were plotted in the graphs of Fig. 7, A–D. The
histogram in Fig. 7Edepicts the normalized frequencies in
a window of 15–30 s after the CGP 62349 application.
GAT1 KO mice have normal numbers of GABAergic
interneurons and synapses
The decrease in quantal GABA release and loss of pre-
synaptic GABA
B
receptor function prompted us to perform
histological stainings of the hippocampus. Using fluores-
cence immunocytochemistry, we stained for calbindin, cal-
retinin, and parvalbumin, proteins that label various sub-
types of GABAergic interneurons. The expression of these
proteins was intact in the GAT1 KO mice as was the number
of interneurons labeled by these markers (not shown). Im-
munostainings were also performed for the
␣
1-subunit of
GABA
A
receptors (Fig. 8,Aand B), GABA
B
-R1 receptors
(Cand D), GAD-65 (Eand F), and for the vesicular GABA
transporter vGAT (Gand H). Total levels of fluorescence
were similar within 20%. For GABA
B
receptors, glutamic
acid decarboxylase 65 kDa (GAD65) and vGAT, punctate
staining was observed; and we measured the density of
positive structures that were ⬎20 or 25 pixels (⬃0.2–0.24
m
2
). The immunocytochemistry indicated no significant
differences between the density of GABA synapses and
receptors of WT and GAT1 KO mice (Fig. 8I).
DISCUSSION
Uptake of neurotransmitters is presumed to critically influ-
ence synaptic transmission in the CNS (Genton et al. 2001;
Spinks and Spinks 2002). While many experimental studies
have addressed the acute effects of uptake inhibition, we gen-
erated GAT1 KO mice where the GABA uptake in the brain
has been chronically impaired during the entire life span. In
these animals, some of our findings may have been predicted
from the known acute effects of GAT1 inhibitors. These in-
clude an increased GABA
A
receptor-mediated tonic current in
principal cells (Frahm et al. 2001; Nusser and Mody 2002) and
the lack of change in amplitude and decay of mIPSCs (Isaacson
et al. 1993). The present study confirms this lack of effect
without complications from the additional pharmacological
effects of the GAT1 blockers previously employed.
Other effects were not expected. First, there is a decrease in
the frequency of quantal GABA miniature release. Second, a
presynaptic GABA
B
tone is present in WT slices but not in the
GAT1 KOs. These findings indicate that chronically elevated
FIG. 7. Summary of the presynaptic GABA
B
receptor activation in WT
and GAT1 KO mice. A: in WT slices (in the absence of NO-711) injection
of the GABA
B
antagonist CGP 62349 (⬎8
M) into the bath (arrow) led
to a dramatic increase in the frequency of sIPSCs and smaller increase in
the sEPSCs. For each cell, the event frequency is normalized to the 30 s
preceding the CGP 62349 injection (n⫽4). Events were recorded as shown
in Fig. 6A. B: the increase in sIPSCs frequency was abolished in WT slices
by DNQX (10
M) and APV (50
M), which block ionotropic glutamate
receptors (n⫽4). C: in GAT1 KO slices, no increases in sIPSCs were seen
after CGP 62349 application. There were minor changes in the sEPSC
frequency (n⫽5). D: under blockade of glutamatergic excitation by
kynurenic acid (3 mM) in GAT1 KO slices, the frequency of sIPSCs was
also unchanged after CGP 62349 (n⫽5). E: summary of the effects of CGP
62349 on the normalized sIPSC and sEPSC frequencies in WT and GAT1
KO slices (E: sEPSC; I: sIPSC). The reported event frequency was mea-
sured during a 10-s-long window starting 20 s after the CGP 62349
application. *P⬍0.05, unpaired t-test.
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levels of [GABA]o alter the presynaptic receptor function and
quantal transmitter release in the brain.
Generation of GAT1-deficient mice
The GAT1 KO strain described in this paper differs from a
previously described mGAT1-GFP knock-in mouse: the
present strain contains a floxed 2.2-kb neo selection cassette in
intron 14, which precedes the final coding exon of mGAT1
(Chiu et al. 2002). A large insertion in an intron often interferes
with splicing so that the resulting protein is expressed at
subnormal levels (Labarca et al. 2001; Single et al. 2000; Wang
et al. 1999). This paper exploits the unusual, but not unprece-
dented, fact that the insertion virtually eliminates protein func-
tion: GAT1-mediated GABA-transport was absent in hip-
pocampal synaptosomes of GAT1 KO mice (⬍2% of control).
Interestingly, in the absence of GAT1-mediated NO-711-sen-
sitive GABA transport, other GABA transport (insensitive to
NO-711) did not become upregulated in GAT KO mice.
Increased GABA
A
tone in GAT1-deficient mice
Previous electrophysiological studies showed that the decay
of electrically evoked inhibitory postsynaptic currents and po-
tentials are prolonged by GAT1 inhibition in hippocampal and
neocortical brain slices (Engel et al. 1998; Ling and Benardo
1998; Roepstorff and Lambert 1994; Thompson and Ga¨hwiler
1992). On the other hand, the kinetics of mIPSCs, which reflect
the quantal GABAergic transmission, are normally not shaped
by GABA uptake (Isaacson et al. 1993; Overstreet et al. 2000).
Thus the GABA uptake process influences synaptic transmis-
sion when several nerve terminals are concurrently stimulated.
Recent studies using dual recordings suggest that the activation
of GAT1 after evoked transmission depends on the specific
GABAergic pathway and requires a close anatomical spacing
between the inhibitory boutons (Overstreet and Westbrook
2003).
When we examined the spontaneous GABAergic activity in
brain slices, we found that GAT1 deficiency led to a tonic
GABA
A
receptor-mediated background conductance in the
hippocampus. This finding is mimicked in WT slices by acute
blockade of GAT1 with GAT1 blockers such as NO-711 (Bai
et al. 2001; Frahm et al. 2001; Nusser and Mody 2002; Over-
street and Westbrook 2001). In the GAT1 KO animals, we can
exclude the possibility that in whole cell recordings the tonic
current was caused by locally enhanced GABA levels through
the reverse operation of GAT1 (Wu et al. 2003). The reverse
FIG. 8. Immunocytochemical data showing little or no change in the number of structures immunoreactive for GABA
A
receptors, GABA
B
receptors, GAD65, and vGAT in GAT1 KO mice. Aand B: immunocytochemistry using antibodies against the
␣
1-subunit of the GABA
A
receptor in hippocampal CA1 region of WT and GAT1 KO mice. Cand D: immunocytochemistry using
antibodies against the GABA
B
-R1 receptor in the CA1 region of WT and GAT1 KO. Eand F: glutamic acid decarboxylase 65 kDa
(GAD65) immunocytochemistry in the CA1 region. Gand H: vesicular GABA transporter (vGAT) immunocytochemistry in the
CA1 of WT and GAT1 KO. I: quantification of GABA
B
-R1-, GAD65- or vGAT-positive structures in the immunocytochemical
images. The criteria for counting positive structures were: fluorescence intensity ⬎2.5 times the average intensity, and area ⬎20
or 25 pixels (⬃0.2–0.24
m
2
).
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operation of another GABA transporter, e.g., GAT3 can also be
excluded, as NO-711-insensitive GABA transport remained
unaltered in GAT1 KOs. Instead, similar to cerebellar granule
cells, the tonic conductance most likely arises from extrasyn-
aptic receptors activated by ambient GABA levels (Brickley et
al. 1996, 2001).
In our experiments, the majority of the tonic current was
blocked by furosemide, while the IPSCs were not affected by
this drug. Because furosemide preferentially blocks
␣
4- and
␣
6-containing receptors (but the latter are not expressed in
hippocampus) (Korpi et al. 2002), the furosemide-sensitive
tonic current in CA1 pyramidal cells could be mediated by
␣
4-containing receptors, which may form
␣
-channels or
␣␥
2 channels (Bencsits et al. 1999). The furosemide-insen-
sitive portion of the tonic current could arise from
␣
5-contain-
ing receptors, which are strongly expressed in the CA1 pyra-
midal cells (Pirker et al. 2000) and are thought to be localized
extrasynaptically (Brunig et al. 2002; Crestani et al. 2002).
What are the GABA levels in the slice?
Given that the tonic current is driven by [GABA]
o
, what are
then the approximate levels of GABA in the slice surface
where we record? Perfusion of 0.8
M GABA and 10
M
NO-711 yielded a similar tonic current in WT slices and in
untreated GAT1 KO slices. Thus if the postsynaptic sensitivity
is similar in these animals, we can suggest that [GABA]o in
GAT1 KO slices is ⬃1
M. Processes in addition to GAT1
might control GABA levels near receptors. We found that
NO-711-sensitive uptake accounts for 75–80% of the uptake in
crude synaptosomes from WT tissue, implying that nonGAT1
uptake may produce 20–25% of WT uptake in the GAT1 KO
brain. It is unnecessarily pessimistic to assume that this re-
maining activity produces an error of ⬎20–25% in our esti-
mate of 0.8–1
M GABA.
At such steady-state [GABA]
o
, the data of Overstreet et al.
(2000) led one to expect that 15–20% of GABA receptors
would desensitize at the synapse, producing a corresponding
decrease in s- or mIPSC amplitudes (Overstreet et al. 2000).
We detected no change in IPSC amplitudes when GABA
uptake was blocked, either chronically in the GAT1 KO ani-
mals or acutely during perfusion of NO-711. Similar to the
results obtained here an increased [GABA]o subsequent to
vigabatrin administration was found to decrease mIPSC fre-
quency without changing their amplitudes (Wu et al. 2003).
The discrepancy between our data and those of Overstreet et al.
(2000) may be due to species differences or to another un-
known factor that renders the synaptic receptors in our mouse
preparation less sensitive to desensitization. Even if synaptic
GABA receptors do not desensitize, one might expect GAT1
block or knockout to reduce apparent IPSC amplitude via a
second mechanism. In this second mechanism, synaptic recep-
tors are again exposed to low levels of GABA contributing to
the generation of the large tonic conductance, and synaptic
receptors are locally saturated in response to a quantum of
GABA, so that the mIPSC grows from an altered baseline to a
constant peak. That clearly elevated [GABA]
o
led to no sig-
nificant differences in the amplitudes of s- and mIPSCs is best
explained by the idea that synaptic GABA receptors have low
affinity and are therefore not activated by the low ambient
concentrations of [GABA]
o
in the slice (Stell and Mody 2002).
Instead, it is likely that extrasynaptic GABA
A
receptors gen-
erate the tonic conductance (Mody 2001), but the molecular
identity of these receptors in various cell types remains to be
determined. It is interesting to note that interneurons them-
selves may have a substantial tonic conductance, albeit of
different pharmacological properties than that of principal cells
(Semyanov et al. 2003). In light of the possible excitatory and
inhibitory actions of GABA on interneurons (Chavas and
Marty 2003), it is difficult to speculate how an enhancement of
the tonic conductance on interneurons might impact the overall
network excitability in GAT1 KOs. An excitatory GABA
action on some interneurons may thus explain the normal
sIPSC frequency in GAT KOs when extracellular GABA lev-
els are elevated.
Decreased GABA
B
tone in GAT1-deficient mice
Extracellular GABA concentrations sufficient to activate the
high-affinity GABA
A
receptors responsible for generating the
tonic current did not activate postsynaptic GABA
B
receptors in
both WT and GAT1 KO mice. Yet, functional GABA
B
recep-
tors were clearly present on pyramidal cells in both genotypes
because baclofen induced a robust outward current, known to
be mediated by GIRK2-containing channels (Luscher et al.
1997). It has been reported that 1
M GABA activates
postsynaptic GABA
A
receptors and strongly activates presyn-
aptic GABA
B
receptors to depress GABA release, while it does
not activate postsynaptic GABA
B
receptors (Yoon and Roth-
man 1991). These results fit with our observation that ambient
GABA levels in the slices, even in the absence of a GAT1-
mediated uptake, preferentially activate presynaptic but not
postsynaptic GABA
B
receptors. However, these data are in
contrast with findings in expressed GABA
B
R1 and R2 sub-
units, which couple efficiently to GIRK channels, and as little
as 200 nM GABA is sufficient to induce a robust K
⫹
current
(Jones et al. 1998). Thus it is plausible that postsynaptic
GABA
B
receptors in brain slices are under some modulatory
control that will reduce their responsiveness to GABA.
We have also studied the tonic activation of presynaptic
GABA
B
receptors that could contribute to lowering the fre-
quency of sIPSC and sEPSC in pyramidal cells. This presyn-
aptic GABA
B
tone was detected in WT slices where [GABA]
o
is low, possibly in the range of a few hundred nanomolars.
Because ultrastructural studies have demonstrated a high den-
sity of presynaptic GABA
B
receptors on excitatory terminals
(Kulik et al. 2002), the activation of presynaptic GABA
B
receptors is expected to alter the release of both glutamate and
GABA. Indeed, in WT animals, we found an increased fre-
quency of sEPSCs and sIPSCs after application of a GABA
B
receptor antagonist. However, it has to be kept in mind that the
GABA
B
antagonist might affect sIPSC frequency via two
distinct mechanisms: a direct effect on the inhibitory terminals
and soma and an indirect action by changing the excitatory
drive onto interneurons. A relief from the GABA
B
tone would
cause an increased glutamate release onto interneurons, leading
to increased interneuronal firing. In support of this possibility,
CGP 62349 did not have an effect when the WT interneurons
were pharmacologically isolated by DNQX and APV. The
direct glutamatergic inputs onto pyramidal cells were less
influenced by presynaptic GABA
B
receptors. Therefore at least
in our preparation, ambient GABA may predominantly stimu-
2699GABA RECEPTOR ACTIVATION IN GAT1 KO MICE
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late GABA
B
heteroreceptors located on glutamatergic connec-
tions onto GABAergic interneurons (McBain et al. 1999).
Elucidating these connections and their regulation requires
direct recordings from interneurons (Lei and McBain 2003),
followed by a comprehensive anatomical classification (Ha´jos
and Mody 1997).
[GABA]
o
is likely to be many times higher in GAT1 KO
slices compared with WT. Yet a presynaptic GABA
B
tone
could not be revealed in the GAT1 KO animals. Previous
studies showed that 21 days of oral treatment with the GAT1
blocker tiagabine causes no changes in the GABA
A
or GABA
B
receptor binding in the mouse hippocampus (Thomsen and
Suzdak 1995). Similarly, our immunocytochemical studies in
mice lacking GAT1-mediated GABA uptake revealed no
changes in the numbers of GABA
A
and GABA
B
receptors or in
the level of vGAT- or GAD65-containing inhibitory nerve
terminals. Our data thus indicate that prolonged high extracel-
lular GABA concentrations decreased the function of the path-
way underlying presynaptic GABA
B
control of sIPSC fre-
quency, most likely without a change in receptor number. The
molecular basis for this effect is unknown, but there are several
precedents for the idea that prolonged activation of other
G-protein-coupled receptors results in altered signaling path-
ways controlled by the receptors. G-protein-coupled receptors
desensitize during prolonged exposure to agonist then recover
over days subsequent to agonist removal (Wetherington and
Lambert 2002). It is established that desensitization can in-
volve phosphorylation, receptor endocytosis, and/or reduced
gene expression (Ferguson 2001). Another possibility is that
levels of endogenous RGS proteins, which determine the mag-
nitude of G-protein-mediated presynaptic inhibition (Chen and
Lambert 2000), may have been altered by the prolonged ago-
nist exposure. Regardless of the mechanism, our findings are
consistent with a novel plasticity of the function of presynaptic
GABA
B
receptors.
Decreased quantal GABA
A
-mediated transmission
The reduced frequency of mIPSCs in GAT1 KOs was an
unexpected finding, particularly in light of their unaltered
amplitudes, and kinetics, and the lack of change in postsynaptic
GABA
A
receptors and presynaptic GABAergic markers. The
decreased frequency is unlikely to have been caused by acti-
vation of GABA
B
receptors because sIPSCs recorded with
CsCl electrodes were not depressed by a presynaptic GABA
B
tone (Fig. 7D), and mIPSCs are usually quite resistant to
GABA
B
activation (Otis and Mody 1992; Overstreet and West-
brook 2001). Moreover, a chronic elevation in extracellular
GABA levels by pretreatment of slices with the GABA
transaminase inhibitor vigabatrin reduces the frequency of
mIPSCs in a GABA
B
receptor-independent manner (Overstreet
and Westbrook 2001). Because the number of GABAergic
boutons, as inferred from the vGAT staining, did not change in
GAT1 KOs, a likely explanation for the reduction in mIPSC
frequency is a reduced rate of quantal release at individual
nerve terminals. This could be due to a reduction in the number
of docked vesicles, or to upstream mechanisms, such as a
reduced spontaneous Ca
2⫹
release from intracellular stores
(Llano et al. 2000). To test these possibilities further, it is
necessary to examine the Ca
2⫹
sensitivity of mIPSCs and to
perform quantitative ultrastructural studies on subpopulations
of GABAergic terminals. Reductions in mIPSC frequency also
occur in experimental epilepsy (Hirsch et al. 1999) and may
represent a general type of plasticity when ambient GABA
levels are high.
Conclusions
The deficiency of GABA uptake in GAT1 KO mice chron-
ically elevates ambient GABA levels. In the hippocampus, this
leads to an increase in a GABA
A
receptor-mediated tonic
conductance, which may control cell excitability, chloride ho-
meostasis, or cell volume (Mody 2001). Future studies will
have to determine how prolonged increases in extracellular
GABA levels, achieved here by GAT1 deficiency, decrease
quantal GABA release, and presynaptic GABA
B
receptor func-
tion, as these changes are likely to have profound influences on
hippocampal network function.
Present addresses: K. Jensen, Wallenberg Neuroscience Center, Lund Uni-
versity, S-221 84, Sweden; and C.-S. Chiu, Dept. of Neurobiology, Merck &
Co., West Point, PA 19486.
DISCLOSURES
This work was supported by National Institutes of Health Grants DA-09121,
DA-010509, NS-11756, MH-49176, MH-61468, NS-30549, DA-14947, Na-
tional Science Foundation Grant 0119493, and a Della Martin Fellowship to
C.-S. Chiu.
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