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Reduced Anxiety and Depression-Like Behaviors in Mice
Lacking GABA Transporter Subtype 1
Guo-Xiang Liu
1,2
, Guo-Qiang Cai
1
, You-Qing Cai
1,2,3
, Zhe-Jin Sheng
1
, Jie Jiang
1
, Zhengtong Mei
1
, Zhu-Gang
Wang
3,4
, Lihe Guo
1
and Jian Fei*
,1,3,4
1
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Model Organism Research Center, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China;
2
Graduate School of Chinese Academy of Sciences, Beijing, China;
3
Shanghai
Nan Fang Model Organism Research Center, Pu Dong, Shanghai, China;
4
Model Organism Division, E-institutes of Shanghai Universities,
Shanghai Jiao Tong University, Shanghai, China
g-Aminobutyric acid (GABA) transporter subtype 1 (GAT1), which transports extracellular GABA into presynaptic neurons, plays an
important regulatory role in the function of GABAergic systems. However, the contributions of the GAT1 in regulating mental status are
not fully understood. In this paper, we observed the behavioral alterations of GAT1 knockout (GAT1
/
) mice using several depression-
and anxiety-related models (eg, the forced-swimming test and the tail-suspension test for testing depression-related behaviors; the open-
field test, the dark–light exploration test, the emergence test, and the elevated plus maze (EPM) test for anxiety-related behaviors). Here
we found that GAT1
/
mice showed a lower level of depression- and anxiety-like behaviors in comparison to wild-type mice.
Furthermore, GAT1
/
mice exhibited measurable insensitivity to selected antidepressants and anxiolytics such as fluoxetine,
amitriptyline, buspirone, diazepam, and tiagabine in the tail-suspension test and/or the EPM test. Moreover, the basal level of
corticosterone was found to be significantly lower in GAT1
/
mice. These results showed that the absence of GAT1 affects mental
status through enhancing the GABAergic system, as well as modifying the serotonergic system and the hypothalamic-pituitary-adrenal
(HPA) activity in mice.
Neuropsychopharmacology (2007) 32, 1531– 1539; doi:10.1038/sj.npp.1301281; published online 13 December 2006
Keywords: GABA transporter 1; depression; anxiety; knockout mouse
INTRODUCTION
Depression and anxiety affect millions of people of different
ages, races, religions, and income. However, the molecular
basis for the development of these disorders remains largely
unknown. Increasing evidence suggests that the g-amino-
butyric acid (GABA) system is important in the pathogen-
esis of psychological disease, including anxiety and
depression. Studies employing magnetic resonance spectro-
scopy suggest that unipolar depression is associated with
reductions in cortical GABA levels. Antidepressant and
mood-stabilizing treatments also appear to raise cortical
GABA levels and ameliorate GABA deficits in patients with
mood disorders (Krystal et al, 2002). GABA receptors,
including GABA
A
receptors and GABA
B
receptors, have
been generally considered as the targets for treatment of
mental illness (Kalueff and Nutt, 1996). However, drugs
used for such treatment have side effects (Johnston, 1996;
Tsang and Xue, 2004). Animal studies with GABA uptake
inhibitors demonstrated marked anxiolytic-like effects,
implying that modulating GABA uptake could be a possible
target for the treatment of mental disorders (Sayin et al,
1992; Schmitt and Hiemke, 1999).
GABA transporters (GATs), located on the plasma
membrane of cells, are key molecules in GABAergic
transmission. Through reuptake of released GABA, GATs
control the duration and intensity of GABAergic activity at
the synapse (Borden, 1996; Radian et al, 1990). Molecular
cloning studies have identified multiple GABA transporter
subtypes, including GAT1, GAT2, GAT3, and GAT4. GAT1
is the major subtype present at both synaptic and
extrasynaptic sites in the brain (Chiu et al, 2002; Guastella
et al, 1990). Our previous work had demonstrated that the
GAT1 gene knockout mice have decreased ethanol aversion
and ethanol reward, and insensitivity to both the sedative/
hypnotic and the motor stimulant effects of ethanol (Cai
et al, 2006). It was also reported that GAT1 deficiency leads
to enhanced extracellular GABA levels and results in an
overactivation of GABA
A
receptors (Jensen et al, 2003).
Received 30 March 2006; revised 29 September 2006; accepted 2
October 2006
*Correspondence: Dr J Fei, Laboratory of Molecular Cell Biology,
Institute of Biochemistry and Cell Biology, Model Organism Research
Center, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, No 7 Building, 319 Yue Yang Road, Shanghai 200031, China,
Tel: + 86 21 54920376, Fax: +86 21 58951005, E-mail: jfei@sibs.ac.cn
Neuropsychopharmacology (2007) 32, 1531 –1539
&
2007 Nature Publishing Group All rights reserved 0893-133X/07
$
30.00
www.neuropsychopharmacology.org
Behavioral tests showed that GAT1-deficiency causes
tremor, ataxia, and nervousness (Chiu et al, 2005). Never-
theless, there is little direct evidence to show the role of
GAT1 in the pathogenesis of depression and anxiety. In our
present study, we employed six behavioral tasks that are
sensitive to clinically efficacious antidepressants or anxio-
lytics to investigate anxiety- and depression-like behaviors
of the GAT1 gene knockout mice. These behavioral tasks
include tail-suspension test, forced swim test, open-field
test, dark–light exploration test, emergence test, and
elevated plus maze (EPM) (Borsini, 1995; Porsolt et al,
1977b; Steru et al, 1985). As hypersecretion of corticoster-
one is associated with depression (Seckl et al, 1990), the
change of plasma corticosterone level in GAT1 knockout
mouse was also examined. Our results repeatedly confirm
that GAT1 plays an important role in the pathogenesis of
anxiety and depression, and suggest that GAT1 mutant mice
represent a useful genetic animal model to understand the
relationship between etiology of depression and anxiety and
GABAergic system function.
MATERIALS AND METHODS
Subjects
GAT1 null mutant mice were generated in our laboratory
(Cai et al, 2006). In the mutant mice, a 1.57 kb DNA
fragment that contains the exon2 and exon3 of the mouse
GAT1 gene was replaced by a 1.37 kb neomycin-resistant
gene cassette (neo) to eliminate the GAT1 gene activity.
Chimeric male mice, generated by injecting the recombi-
nant ES cells into C57BL/6J blastocysts, were bred to C57BL/
6J females to establish germline transmission of the mutant.
GAT1 knockout heterozygotes (GAT1
+/
) were crossed
with wild-type C57BL/6J mice for another two generations.
These heterozygotes were then intercrossed to generate
homozygous, heterozygotes, and wild-types mice for further
experimentation. Mice were group housed (three to five per
cage) under specific pathogen-free conditions with a 12-h
light/dark cycle (lights on at 0700) and provided ad libitum
access to food and water until the age of 12–18 weeks.
Age- and body weight-matched male mice were used for
behavioral experiments. The experiments were conducted
in an isolated behavioral testing room in the animal facility
to avoid external distractions. Investigators observed
animal behaviors through a videomonitor in another room
without any information about the genotype or drug
treatment of the mice. In order to facilitate adaptation to
the experimental environment, mice were housed in the
testing room for at least 1 week before the experiment. Mice
were used only for one experiment. These animal experi-
ments described were approved by the Institutional Animal
Care and Use Committee.
Drugs
Amitriptyline, imipramine, fluoxetine, and buspirone were
purchased from Sigma-Aldrich. Tiagabine was a kind gift
from Shanghai Celstar Research Center for Biotechnology.
Diazepam was purchased from Shanghai Jiufu Pharmaceu-
tical Co., Ltd. These drugs were dissolved in saline before
administration.
Forced-Swimming Test
Mice were placed in a Plexiglas cylinder (10 cm internal
diameter, 50 cm high) filled with 25–261C water (10 cm
height). The behavior of the animals was evaluated
manually and the immobility time was measured during
5 min experimentation. A mouse was judged to be immobile
when it remained floating in the water, making only those
movements necessary to keep its head above water (Bilkei-
Gorzo et al, 2002; Porsolt et al, 1977a).
Tail-Suspension Test
Animals were injected intraperitoneally with a volume of
10 ml/kg of vehicle (saline) or antidepressant. Amitripty-
line (10 mg/kg), imipramine (5 mg/kg), and fluoxetine
(20 mg/kg) were used as the antidepressants. Forty minutes
later, mouse was suspended individually by its tail from a
metal rod. The rod was fixed 50 cm above the surface of a
table covered with soft cloth in a sound-proof room. The
tip of the mouse’s tail was fixed on the rod using adhesive
Scotch tape. The duration of the test was 5 min. The
immobility time of the tail-suspended mice was measured
as previously described (Bilkei-Gorzo et al, 2002; Steru
et al, 1985).
Open-Field Test
The open-field test was conducted in accordance with
published reports (Holmes et al, 2003a, b; Mathis et al,
1994). The open field was a square arena (50 50 50 cm
3
)
with clear Plexiglas walls and floor, brightly illuminated
by overhead fluorescent lighting (1.5 mmol/m
2
/s). Mice
were placed in the center of the box and allowed to freely
explore for a 10-min period. Mice were videotaped using a
camera fixed above the floor and analyzed with a video-
tracking system (morris maze analyzer, V 1.1 by BGB).
The defined ‘margin’ of the arena is 8 cm wide along the
wall and the ‘center’ field is defined as the central
20 20 cm
2
area of the open field, approximately 16% of
the total area.
Light–Dark Exploration Test
The light–dark exploration test was conducted as
described previously (Crawley, 1981; Holmes et al, 2003a, b).
The apparatus consisted of a polypropylene cage
(32 16 16 cm
3
) separated into two compartments (dark
and light) by a partition with a small opening (8 5cm
2
)at
floor level, and equipped with infrared sensor that was used
to monitor the number of transitions and the time spent in
each chamber for each mouse. Ceiling lights were turned off
during these experiments. The light half of the shuttle box
was open-topped, transparent, and illuminated by a desk
lamp (100 W). Mice were individually placed in the center of
the light compartment, facing away from the partition, and
allowed to freely explore the apparatus for 5 min. The
number of light–dark transitions between the two compart-
ments and the total time spent in the dark compartment
were recorded.
Mice with reduced anxiety and depression-like behaviors
G-X Liu et al
1532
Neuropsychopharmacology
Emergence Test
The emergence test was conducted as described previously
(Holmes et al, 2003b; Smith et al, 1998; Takahashi et al,
1989). The apparatus consisted of an opaque black Plexiglas
cube (16 16 19 cm
3
) with an exit (6 4cm
2
) on one side
at floor level. This ‘shelter’ was placed within an open field
(50 50 50 cm
3
), brightly illuminated by overhead fluor-
escent lighting, with the exit facing out into the open field
(parallel with one wall of the open field). Mice were first placed
inside the shelter for a 5-min habituation period with the exit
closed. After that, the exit was opened to allow the mouse leave
the shelter and explore the open field for 5 min. For each
mouse, the latency to emerge from the shelter, time spent out
of the shelter and shelter open-field transitions were recorded.
EPM
The EPM was conducted as described previously (File, 2001;
Holmes et al, 2003a; Lister, 1987). The apparatus comprised
of two open arms (25 8cm
2
) and two closed arms
(25 812 cm
3
) that extended from a common central
platform (8 8cm
2
). A small raised lip (0.5 cm) around the
edges of the open arms prevented animals from slipping off.
The apparatus was constructed from polypropylene and
Plexiglas (white floor, clear walls) and elevated to a height of
50 cm above the floor. Mice were allowed to habituate to the
testing room for 2 days before the test, and pretreated with
gentle handling two times per day to eliminate their
nervousness. For drug treatment experiments, mice were
injected intraperitoneally with a volume of 10 ml/kg of
vehicle (saline) or drugs, buspirone (1 mg/kg), diazepam
(1 mg/kg), and tiagabine (8 mg/kg), 40 min before the EPM
test was performed. Mice were individually placed on the
center square, facing an open arm, and allowed to freely
explore the apparatus under even overhead fluorescent
lighting (1.5 mmol/m
2
/s) for 10 min. Mice were videotaped
using a camera fixed above the maze and analyzed with
a video-tracking system (morris maze analyzer. V 1.1 by
BGB). Open and closed arm entries (all four paws in an
arm) were scored by an experienced observer.
Stress Hormone Measurements
Blood was collected from mice by decapitated bleeding
immediately after 10 min of physical restraint (15 min
restraint in a 50 ml conical tube) or from rest in the home
cage (0900–1000), spun through a serum separator tube
at 1000 gfor 15 min, and stored at 201C until use.
Corticosterone measurements were performed on serum
using a Corticosterone immunoassay (R&D Systems)
according to the manufacturer’s instructions.
Statistical Analysis
Mean values and SE were calculated for each group, and
groups were compared using one-way ANOVA followed by
t-test. po0.05 denotes a statistically significant difference.
Wild-type, heterozygous, and homozygous GAT1-deficient
mice are designated as + / + , + /, and /; and the
number of mice used are designated as n
+/+
,n
+/
, and
n
/
, respectively.
RESULTS
Reduced Depression-Like Behaviors in GAT1-Deficient
Mice
The forced-swimming test (Porsolt et al, 1977a, b) and the
tail-suspension test (Bilkei-Gorzo et al, 2002; Steru et al,
1985) were employed in this study to evaluate the tendency
towards depression-like behavior in the genotypically
different mice groups. Both experimental schemes are
based on the observation that rodents, when forced into
an aversive situation from which they cannot escape, will
cease attempts to escape and become immobile. Antide-
pressants reduce the immobility time in these tests (Porsolt,
2000; Steru et al, 1987) indicating that these methods can be
used to test for depression-related behavior in mice. In the
forced-swimming test, as shown in Figure 1a, immobility
time for GAT1
/
mice was significantly lower than that of
GAT1
+/
and GAT1
+/+
animals (F(2,33) ¼63, po0.001)
(Figure 1a). This phenomenon was also observed in the tail-
suspension test, which showed significantly reduced im-
mobility time in GAT1
/
mice compared to GAT1
+/+
mice (F(2,16) ¼16, po0.001) (Figure 1b). Treatment of
mice with the selective serotonin reuptake inhibitor,
fluoxetine and a tricyclic uptake inhibitor, amitriptyline
significantly reduced immobility time in GAT1
+/+
and
GAT1
+/
mice, but not in GAT1
/
mice. However, a
similar response to another tricyclic uptake inhibitor,
imipramine was observed in all three groups (Figure 1b).
These findings indicate that prolonged changes in
GABAergic homeostasis affect the depression-related beha-
vioral tendency of mice with possible involvement of the
serotonergic system.
Reduced Anxiety-Like Behaviors in GAT1 Knockout
Mice
Next, we examined the differences in behavior of wild-type,
heterozygous, and homozygous GAT1-deficient mice in the
open field, which is widely used in laboratories to quantify
anxiety-like and locomotor behaviors in mice (Crawley,
1999). Mice prefer to move around the periphery of an
apparatus when they are placed in an open field of a novel
environment. It is thought that the time spent in the central
area of the open field is inversely correlated to their level of
anxiety-related proneness. As shown in Table 1, GAT1
/
mice spent longer time in central area and made more
entries (F(2,51) ¼3.79, p¼0.029) into the central area
than other two genotype mice. Meanwhile, GAT1
/
mice
displayed hyperactivity and enhanced locomotion. This
phenotype manifested as a significant increase in move
time (F(2,51) ¼26.3, po0.001), velocity (F(2,51) ¼9.23,
po0.001), and distance traveled (F(2,51) ¼8.46, po0.001)
in GAT1
/
mice. There was also a significantly higher
number of rearings for GAT1
/
mice (F(2,51) ¼7.63,
p¼0.002).
As the results of the open-field experiments suggested
that GAT1 knockout leads to a reduction in anxiety-like
behaviors, we decided to confirm these observations using
additional tests.
The light–dark exploration paradigm, which is based on
the innate aversion of rodents to brightly illuminated areas
Mice with reduced anxiety and depression-like behaviors
G-X Liu et al
1533
Neuropsychopharmacology
and on the spontaneous exploratory behavior of the
animals, is used primarily to detect anxiogenic behavior
(Hascoet et al, 2001). GAT1
/
mice spent significantly
less time in the dark chamber than wild-type mice
(F(2,43) ¼8.38, po0.001) (Figure 2b), and made more
transitions (F(2,43) ¼6.97, p¼0.002) (Figure 2a), suggest-
ing a reduction in anxiety-like behavior.
The results of emergence test are presented in Figure 2c–e.
There was a significant genotypic effect on shelter open-field
transitions and the percentage time spent out of
the shelter, but not for the latency to initially exit the
shelter (F(2,18) ¼0.45, p¼0.65). One-way ANOVA analysis
showed that GAT1
/
mice made more shelter open-field
transitions (F(2,18) ¼6.52, p¼0.007) and spent more time out
of the shelter (F(2,18) ¼10.9, po0.001) than wild-type mice.
High levels of anxiety are thought to be associated with a
reduced activity of mice in the open compartments in the
EPM test (Shepherd et al, 1994). The results showed
that GAT1
/
mice spent more time in the open arm
(F(2,35) ¼42, po0.001) (Figure 3a), more entries into the
open arm (F(2,35) ¼17, po0.001) (Figure 3b), and more
time on the center platform (F(2,34) ¼3.4, p¼0.04)
(Figure 3d), but not in closed arm entries (F(2,35) ¼0.54,
p¼0.58)(Figure 3c). Treatment of wild-type animals with
anxiolytic agents (buspirone, diazepam), or a GAT1
inhibitor (tiagabine) significantly increased their activity
in the open arms and reduced their activity in the close
arms (Figure 3). However, the same treatment on GAT1
/
mice had less effect on its behavior in elevated plus maze
test (Figure 3).
Stress Hormone Measurements
Reduced anxiety-like behavior in mice can be associated
with low levels of serum corticosterone (Smith et al, 1998).
To determine if the reduced anxiety correlates with low
levels of hormones in GAT1
/
mice, we measured serum
corticosterone levels from naı
¨ve mice and from mice after
10 min of physical restraint-induced stress. Wild-type and
GAT1
/
mice that had undergone restraint-induced stress
exhibited a significantly higher corticosterone levels
than naı
¨ve mice. There were no differences between wild-
type and GAT1
/
mice in this respect (Figure 4). However,
baseline corticosterone levels were significantly lower
in GAT1
/
mice than in wild-type mice (F(2,12) ¼
8.1, p¼0.007). These data suggest that the activity of
the hypothalamic-pituitary-adrenal (HPA) is reduced in
the GAT1 knockout mice under general conditions in the
home cage.
DISCUSSION
GABA is the most important inhibitory neurotransmitter in
the central nervous system (CNS), and GABAergic systems
play an important role in the pathophysiology of depression
and anxiety. It has been observed by magnetic resonance
+/+
+/+
+/-
+/-
-/-
-/-
180
160
140
120
100
80
60
40
20
0
***
***
*** *** ***
***
Immobility time [s]Immobility time [s]
200
150
100
50
0
*
*
**
Saline
Amitriptyline
Imipramine
Fluoxetine
##
###
a
b
Figure 1 GAT1
/
mice showed reduced depression-like behaviors in
forced-swimming test and tail-suspension test. (a) Forced-swimming test
(n¼12 per group). The behavior of the genotypically different mice was
measured during a 5 min forced swim test. The immobility time for wild-
type, heterozygous, and homozygous mice were 159.478.7 s; 98.377.6 s;
13.675.1 s, respectively. *** po0.001 (one-way ANOVA followed by t-
test). (b) Tail-suspension test. Mice were injected with saline (n
+/+
¼6,
n
+/
¼7, n
/
¼6), or 10 mg/kg amitriptyline (n
+/+
¼7, n
+/
¼6, n
/
¼8),
or 5 mg/ kg imipramine (n
+/+
¼7, n
+/
¼6, n
/
¼8), or 20 mg/kg
fluoxetine (n
+/+
¼5, n
+/
¼5, n
/
¼5) as described in Materials and
methods. The immobility time was measured during a 5 min tail-suspension
test.
##
po0.01,
###
po0.001, vs wild-type mice treated with vehicle.
*po0.05, **po0.01, ***po0.001, vs the same genotype treated with
vehicle (one-way ANOVA). Values are presented as the mean7SEM.
Table 1 Performance of GAT1
/
Mice in Open Field
Genotype
Parameter +/+ (n¼13) +/(n¼20) /(n¼21)
Move time (s) 287717.4 361712.1*** 429713.0***
Rest time (s) 314717.4 239712.1*** 171713.0***
Distance (cm) 22377214.0 2459+141.4* 34777298.6***
Velocity (cm/s) 3.770.3 4.270.2* 6.070.5***
Margin distance (cm) 18447204.5 20477114.6** 28967274.6***
Margin time (s) 53779.50 511717.5 503722.2
Center distance (cm) 114715.5 136729.9 181743.3
Center time (s) 16.572.90 37.4710.0 26.973.47
Center entries 6.571.1 6.771.4 1172.1*
Rears 15.671.90 16.073.20 44.279.10**
Velocity (cm/s) ¼distance (cm)/total time (s).
***po0.001; **po0.01; *po0.05, mean7SEM, vs wild-type mice.
Mice with reduced anxiety and depression-like behaviors
G-X Liu et al
1534
Neuropsychopharmacology
spectroscopy that unipolar depression is associated with
reductions in cortical GABA levels in patients (Krystal et al,
2002). In addition, tiagabine, a GAT1 inhibitor, has been
used for the treatment of anxiety disorders (Crane, 2003;
Lydiard, 2003; Schaller et al, 2004; Schmitt and Hiemke,
1999). These observations provide a rationale to examine
mood-related behavior changes in GAT1
/
mice.
The forced-swimming and tail-suspension tests are the
most widely used animal models for antidepressant drug
screening. Immobility, a posture to reflect a state of
+/+ +/- -/- +/+ +/- -/-
0
2
4
6
8
10
12
14
16
Transitions
**
0
10
20
30
40
50
60
70
% Time in dark compartment
**
ab
cd
e
0
1
2
3
4
5
6
Shelter-open field transitions
***
+/+ +/- -/-
+/+ +/- -/-
0
10
20
30
40
50
60
% Time out of shelter
**
+/+ +/- -/-
0
5
10
15
20
25
The latency to exit the shelter (sec)
Figure 2 GAT1
/
mice showed reduced anxiety-like behaviors in the light–dark exploration and emergence tests. (a) In the light–dark exploration test
(n
+/+
¼17, n
+/
¼15, n
/
¼14). GAT1
/
mice made more light–dark transitions. (b) GAT1
/
mice spent less time in the dark compartment than
GAT1
+/+
mice. (c) In the emergence test (n
+/+
¼6, n
+/
¼8, n
/
¼7). GAT1
/
mice made more shelter open-field transitions. (d) GAT1
/
mice
spent more time out of the shelter than GAT1
+/+
mice. (e) The first time to exit the shelter of GAT1
/
mice is longer than GAT1
+/+
mice, but the
difference is not significant. **po0.01; ***po0.001 vs GAT1
+/+
mice.
Mice with reduced anxiety and depression-like behaviors
G-X Liu et al
1535
Neuropsychopharmacology
‘behavioral despair’ in which animals no longer escape, is
thought to be related to depression, and drugs with
antidepressant activity reduce the time that the animals
remain immobile (Bilkei-Gorzo et al, 2002; Borsini et al,
1988; Porsolt et al, 1977a). In our present study, we found a
significantly reduced immobility time for GAT1
/
mice in
both tests. These behavioral effects are similar to those that
we and other investigators have observed for wild-type
animals treated with antidepressant drugs, such as ami-
triptyline, imipramine, and fluoxetine (Figure 1b) (Bilkei-
Gorzo et al, 2002; Borsini et al, 1988; Porsolt et al, 1977a).
Fluoxetine, a selective serotonin reuptake inhibitor, has
high affinity for the 5-HTT. The time spent immobile in the
tail-suspension test of wild-type mice was significantly
reduced by acute administration of fluoxetine, which occurs
via increased availability of 5-HT following 5-HTT
blockade. In marked contrast, GAT1
/
mice were totally
insensitive to the anti-immobility effects of fluoxetine in the
same test. Amitriptyline and imipramine, being tricyclic
antidepressants, have high affinities for the 5-HTT and
norepinephrine transporter (NET), and demonstrate sig-
nificant effects on wild-type mice in tail-suspension tests.
Amitriptyline, like fluoxetine, also showed no behavioral
effects on GAT1
/
mice, however, it is interesting that
imipramine had similar effects on GAT1
/
mice as on
wild-type mice. These findings indicate serotonergic and
adrenergic systems may have been modified in GAT1
mutant mice secondary to the modification in the
GABAergic system. This hypothesis is weakly supported
by the report that mice lacking the serotonin transporter
exhibit similar pharmacological effects to fluoxetine
(Holmes et al, 2002). Amitriptyline and imipramine are
antidepressants with more mixed pharmacological profiles.
They have high affinity for both 5-HTT and NET, and
+/+ +/- -/- +/+ +/- -/-
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
##
###
***
***
***
***
***
***
***
*
***
*
% open arm entries
% open arm time
#
###
Saline
Buspirone
Diazepam
Tiagabine
ab
cd
20
18
16
14
12
10
8
6
4
2
0
Close arm entries
**
**
+/+ +/- -/-
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
Centre time (s)
##
#
*
****
**
+/+ +/- -/-
Figure 3 GAT1
/
mice showed reduced anxiety-like behaviors in EPM. The anxiolytic agents, buspirone (1 mg/kg) (n
+/+
¼8, n
+/
¼7, n
/
¼6) and
diazepam (1 mg/kg) (n
+/+
¼5, n
+/
¼5, n
/
¼6), and the GAT1 inhibitor tiagabine (8 mg/kg) (n
+/+
¼6, n
+/
¼8, n
/
¼9), produced anxiolytic-like
effects in GAT1
+/+
mice but not GAT1
/
mice. GAT1
/
mice (a) spent more open arm time, (b) made more open arm entries and (d) spent more time
on the center platform than GAT1
+/+
mice. (a) Increased percent open arm time, (b) percent open arm entries and (d) time spent on the center platform
in GAT1
+/+
and GAT1
+/
mice was seen at all doses tested, with no effect in GAT1
/
mice. There was no difference in (c) closed arm entries between
GAT1
+/+
and GAT1
/
control mice. There were 12 mice per group treated with the vehicle. Tiagabine decreased closed arm entries in GAT1
+/+
and
GAT1
+/
mice but not in GAT1
/
mice.
#
po0.05,
##
po0.01,
###
po0.001, vs wild-type mice treated vehicle. *po0.05, **po0.01, ***po0.001, vs the
same genotype treated with vehicle (one-way ANOVA).
Mice with reduced anxiety and depression-like behaviors
G-X Liu et al
1536
Neuropsychopharmacology
similar effects on wild-type mice. However, they have
different pharmacological effects on GAT1
/
mice in the
tail-suspension test. These data indicate that the action of
amitriptyline and imipramine in the mouse CNS could be
different. It has been reported that imipramine has a higher
affinity for 5-HTT and NET than amitriptyline (Frazer,
1997) and a relatively higher affinity for H
1
histamine,
a
1
-adrenergic, and cholinergic receptors than fluoxetine
(Frazer, 1997). Our results support the idea that the GAT1 is
involved in the pathophysiology of depression. Such a role
was also suggested by a recent clinical study that demon-
strated the efficacy of the GAT1 antagonist tiagabine in
patients with a major depressive disorder (Crane, 2003).
Anxiety-like behavior and exploratory locomotion in
GAT1 null mutant mice were studied using four separate
behavioral paradigms. GAT1
/
mice were more active in
the open field, spent more time in the central area and
showed more rearing postures and center entries than
GAT1
+/+
mice. The phenomenon is consistent with a
reduced anxiety-like behavior (ie, increase center time) and
increased locomotor exploration (ie, increase horizontal
and vertical activities). It has been demonstrated that in
exploration-based tests for anxiety-like behaviors, such as
the light–dark exploration or emergence tests, animals with
anxiety would rather spend more time in the dark
compartment (Blanchard et al, 1990; File, 2001; Holmes
et al, 2003b). In light–dark exploration tests, the GAT1
/
mice showed more transitions between light and dark
compartments and spent more time in the light compart-
ment than GAT1
+/+
mice. These results indicate that the
GAT1
/
mice were prone to exhibit reduced anxiety.
Alternatively, the absence of an anxiety-like preference for
the dark compartment in GAT1
/
mice might indicate that
these animals demonstrate behavioral inhibition because of
placement in the light compartment at the start of the test
session, so that skewed scores might show a false light
compartment preference. In order to avoid drawing false
conclusions, the emergence test was introduced as inter-
pretation is less likely to be confounded by behavioral
inhibition caused by precipitous exposure to an aversive
light stimulus. In this test, the animals were initially placed
within a protected, darkened shelter before the light–dark
conflict phase. Here also, GAT1
/
mice showed increased
exploration and preferred a brightly illuminated arena, as
compared to the GAT1
+/+
mice. These data suggest that
the light compartment preference of GAT1
/
mice could
be a reduced anxiety-like behavior, rather than behavioral
inhibition. The result of the EPM test confirmed that
GAT1
/
mice demonstrate reduced anxiety-like behaviors.
In the EPM, GAT1
+/+
mice showed a greater avoidance of
the aversive open arms than GAT1
/
mice, consistent with
reduced anxiety-like behavior in GAT1
/
mice on this test.
In summary, our results from the four different test
paradigms indicate that GAT1-deficient mice were generally
less anxious than control mice.
To further investigate the role of GAT1 in anxiety
behaviors, three anxiolytic drugs: buspirone, diazepam,
tiagabine that have different molecular targets were were
chosen to study their effects on GAT1 mutant mice using
the behavioral model of the EPM. Diazepam and tiagabine
act on the GABAergic system. Diazepam is an agonist at
BDZ sites to enhance Cl
current of GABA
A
, and is a
benzodiazepine with CNS depressant properties (Banfi et al,
1982; Atack, 2005). Tiagabine hydrochloride, a selective
GABA-reuptake inhibitor, increases GABA tone via GAT1
blockade (Nielsen et al, 1991; Rekling et al, 1990).
Buspirone, a serotonin1A receptor partial agonist, also
displays an antagonist effect on D(2)-dopamine and the a2-
adrenergic receptors (Gobert et al, 1999). Treatment of
wild-type animals with these drugs significantly increased
the activity in the open arms of EPM test. However, mice
lacking the GAT1
/
were insensitive to the behavioral
effects of these drugs.
As expected, GAT1
/
mice were completely insensitive
to tiagabine, the GAT1 inhibitor. The failed effect of
diazepam (the BDZ agonist) on the mutant mice indicates
that GABA
A
receptor function was modulated by dysfunc-
tion of GABA reuptake and the consequential nervous
system adaptation. The pharmacological effects of buspir-
one on GAT1
/
mice together with the results of fluoxetine
and amitriptyline further suggest alterations to the seroto-
nergic system of the mutant mice. These might be explained
by complex interactions between GABAergic and seroto-
nergic systems (Katsurabayashi et al, 2003; Tao and
Auerbach, 2000). Serotonin release could be regulated by
GABA in brain. Muscimol, a GABA
A
receptor agonist,
reduced while bicuculline, a GABA
A
receptor blocker,
produced an approximately three-fold increase in the dorsal
raphe
´nucleus serotonin (Tao and Auerbach, 2000). In
GAT1
/
mice, the deficiency of GABA reuptake in
brain may lead overflow of released GABA to from the
synaptic cleft. We hypothesize that the overflow GABA in
the GAT1
/
mouse brain enhanced the tonic inhibition of
GABAergic interneurons, with subsequent reduction in
released GABA by these interneurons that act on seroto-
nergic systems. Moreover, the excitability of GABAergic
interneurons could also be modified by 5-HT. 8-Hydroxy-
2-dipropylaminotetralin, a 5-HT(1A) agonist, presynapti-
cally decreased electrically evoked GABA release whereas
+/+ +/- -/-
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
Corticosterone [ng/ml]
naive
restraint
**
Figure 4 Normal and stress-induced corticosterone levels in GAT1
/
mice serum. GAT1
+/+
and GAT1
/
mice were removed from
home cage (naive) or exposed to 10 min of physical restraint-induced
stress. Blood was collected from mice by decapitated bleeding immediately
(n
+/+
¼5, n
+/
¼5, n
/
¼5). Columns and bars represent mean7SEM,
respectively. **po0.01 vs GAT1
+/+
mice.
Mice with reduced anxiety and depression-like behaviors
G-X Liu et al
1537
Neuropsychopharmacology
m-chlorophenylbiguanide, a 5-HT(3) agonist, presynapti-
cally facilitated GABA release (Katsurabayashi et al, 2003).
These actions are likely related to its anxiolytic effects. As
these effects were absent in GAT1
/
mice, we supposed
that the dopaminergic and adrenergic systems may also
have been modified secondary to GABAergic system
changes. Another possibility to explain the GAT1
/
mice
insensitive to the behavioral effects of anxiolytic drugs is the
ceiling effect. As they were already showing less anxiety in
normal status.
Jensen et al (2003) recently reported a GAT1-deficient
mouse strain, intron-14-neo-intact-mGAT1, in which a
neomycin resistance cassette (neo) was inserted in intron
14. A green fluorescent protein (GFP) moiety was also fused
to the C-terminus of the mGAT1 coding region in exon 14
(Jensen et al, 2003). This mouse strain was originally
constructed as a genetic intermediate in the eventual
construction of a neo-deleted mGAT1-GFP knockin strain
(Chiu et al, 2002). Although the exact mechanism for gene
knockout by such a manner has not been illustrated,
inhibition of GAT1 expression in mutant mouse brain was
proven. An anxiety-prone behavior, which differed from
our results with the open field and EPM tests, was observed
in these mice (Jensen et al, 2003). These differences may be
attributed to the different genetic background between the
two mutant mice strains (C57BL/6J in the studies of Chiu
et al (2005), whereas, mixed C57BL/6J and 129SvJ genetic
background mice were used in our studies). However, the
different strategy for making GAT1 knockout mice could
contribute to the results of different phenotype. Unlike
Chiu’s model, in our mutant mice the GAT1 gene was
completely deleted in every cell type from the beginning of
development. We show here that GAT1 gene deletion results
in clearly reduced anxiety-like behaviors in mice by various
well-established methods.
Many patients with severe major depressive episodes have
dysregulated circadian cortisol control with significantly
higher blood levels than healthy control subjects (Strickland
et al, 2002; Urani et al, 2005). Elevated cortisol levels in
depressed patients usually return to normal following
successful antidepressant treatment (Wolkowitz et al,
1993, 1999). Previous studies have demonstrated that
classical benzodiazepines decrease HPA cortex axis activity
(Mikkelsen et al, 2005) and a low serum corticosterone
levels could be associated with reduced anxiety (Ferguson
et al, 2004; Smith et al, 1998). We investigated the function
of the HPA axis in GAT1
/
mice by measuring corticos-
terone levels following physical restraint-induced stress and
during normal activity. Corticosterone levels were lower in
the GAT1
/
mice than that in wild-type mice during
normal activity. However, there were no differences between
the different genotypes under stress. This suggests that the
function of the HPA axis in GAT1
/
mice was modified
under normal conditions, but its sensitivity to stress might
not be changed.
In conclusion, our results support the hypothesis that the
regulation of GAT1 function affects anxiety and depression-
like behaviors. Based on our observations in GAT1
/
and
GAT1
+/
mice with absent and low GAT-1 expression,
respectively, our data provide insights to the contribution of
GAT1 expression in the manifestation of these mental
disorders. Therefore, drugs that can regulate the function or
expression of GAT1 with subsequent modulation of the
GABAergic system may have therapeutic value in the
treatment of these mental illnesses. GAT1
/
mice can
provide a useful tool to further delineate the pharmacolo-
gical actions of antidepressants and anxiolytics, and the
pharmacogenetics of depression and anxiety.
ACKNOWLEDGEMENTS
This work was supported in part by Grants from the
National Natural Science Foundation of China (30370447),
Chinese Academy of Sciences, Science, and Technology
Commission of Shanghai Municipality (03DZ14018,
03DJ14088) and E-Institutes of Shanghai Municipal Educa-
tion Commission (Project Number: E03003). We thank
Dr Eroboghene E Ubogu and Dr Gurunathan Murugesan for
their helpful discussions and critical review of the manu-
script. We thank Mrs Xixia Zhou for the care of the animals
used in this study, as well as Mrs Jing Gu and Mrs Hui Gao
from the National Key Laboratory of Medical Neurobiology,
Fu Dan University for their technical help.
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