Ubiquitin-specific peptidase 46 (Usp46) regulates mouse immobile behavior in the tail suspension test through the GABAergic system.

Abstract

The tail suspension test (TST) is widely recognized as a useful experimental paradigm for assessing antidepressant activity and depression-like behavior. We have previously identified ubiquitin-specific peptidase 46 (Usp46) as a quantitative trait gene responsible for decreasing immobility time in the TST in mice. This Usp46 mutation has a 3-bp deletion coding for lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system. However, it is not known precisely how the immobile behavior is regulated by the GABAergic system. Therefore, in the present study, we examined whether the immobility time is influenced by drugs affecting the action mediated by GABA(A) receptor using both 3-bp deleted (the Usp46 mutant) and null Usp46 (Usp46 KO) mice. Nitrazepam, an agonist at the benzodiazepine-binding site of the GABA(A) receptor, which potentiates the action of GABA, produced a dose-dependent increase in TST immobility time in the Usp46 mutant mice without affecting general behaviors. The Usp46 KO mice exhibited short immobility times comparable to the Usp46 mutant mice, which was also increased by nitrazepam administration. The effects of nitrazepam in the Usp46 mutant and KO mice were antagonized by flumazenil. These results indicate that the 3-bp deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABA(A) receptor might participate in the regulation of TST immobility time.

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Ubiquitin-Specific Peptidase 46 (Usp46) Regulates Mouse
Immobile Behavior in the Tail Suspension Test through
the GABAergic System
Saki Imai1, Takayoshi Mamiya2, Akira Tsukada3, Yasuyuki Sakai1, Akihiro Mouri2, Toshitaka Nabeshima2,
Shizufumi Ebihara1*
1Division of Biomodeling, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan, 2Department of Chemical Pharmacology, Graduate School of
Pharmaceutical Sciences, Meijo University, Nagoya, Japan, 3Division of Applied Genetics and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University,
Nagoya, Japan
Abstract
The tail suspension test (TST) is widely recognized as a useful experimental paradigm for assessing antidepressant activity
and depression-like behavior. We have previously identified ubiquitin-specific peptidase 46 (Usp46) as a quantitative trait
gene responsible for decreasing immobility time in the TST in mice. This Usp46 mutation has a 3-bp deletion coding for
lysine in the open reading frame, and we indicated that Usp46 is implicated in the regulation of the GABAergic system.
However, it is not known precisely how the immobile behavior is regulated by the GABAergic system. Therefore, in the
present study, we examined whether the immobility time is influenced by drugs affecting the action mediated by GABAA
receptor using both 3-bp deleted (the Usp46 mutant) and null Usp46 (Usp46 KO) mice. Nitrazepam, an agonist at the
benzodiazepine-binding site of the GABAAreceptor, which potentiates the action of GABA, produced a dose-dependent
increase in TST immobility time in the Usp46 mutant mice without affecting general behaviors. The Usp46 KO mice exhibited
short immobility times comparable to the Usp46 mutant mice, which was also increased by nitrazepam administration. The
effects of nitrazepam in the Usp46 mutant and KO mice were antagonized by flumazenil. These results indicate that the 3-bp
deleted Usp46 mutation causes a loss-of-function phenotype, and that the GABAAreceptor might participate in the
regulation of TST immobility time.
Citation: Imai S, Mamiya T, Tsukada A, Sakai Y, Mouri A, et al. (2012) Ubiquitin-Specific Peptidase 46 (Usp46) Regulates Mouse Immobile Behavior in the Tail
Suspension Test through the GABAergic System. PLoS ONE 7(6): e39084. doi:10.1371/journal.pone.0039084
Editor: Georges Chapouthier, Universite ´ Pierre et Marie Curie, France
Received March 17, 2012; Accepted May 18, 2012; Published June 14, 2012
Copyright: ? 2012 Imai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported in part by a Grant-in-Aid for Scientific Research (A), Grant-in-Aid for challenging Exploratory Research, Takeda Science
Foundation, Life Science Foundation of Japan to S.E., and Grant-in-Aid for JSPS Fellows to S.I. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ebihara@agr.nagoya-u.ac.jp
Introduction
The tail suspension test (TST) is widely used for assessing
antidepressant activity and depression-like behavior. In this test,
mice are subjected to the short-term, inescapable stress of being
suspended by their tails. Under such a condition, mice rapidly
adopt a characteristic immobile posture that has been named
‘‘behavioral despair’’ on the assumption that the mice have given
up hope of escaping. Because antidepressant treatments decrease
the immobility time, this test is frequently used for screening drugs
for antidepressant activity [1,2,3,4].
The CS mouse is an inbred strain originally established by
crossing the NBC and SII strains (both now extinct) at Nagoya
University in Japan. CS mice exhibit several distinct phenotypes of
circadian behavioral rhythms and sleep properties. For example,
CS mice show long free-running periods of over 24 hr (most
inbred strains of mice exhibit free-running periods shorter than
24 hrs), spontaneous rhythm splitting and entrainment of circa-
dian rhythms in response to a daily-restricted feeding schedule
under constant darkness conditions [5,6,7,8]. In general, abnormal
rhythms and sleep patterns are believed to be associated with
mental illness. Therefore, we characterized several behavioral
phenotypes of CS mice and found that their immobility time in
both TST and FST is extremely low (almost no immobility). To
identify the gene responsible for this phenotype, we first performed
quantitative trait locus (QTL) genetic analysis to map the
responsible gene on a chromosome. Subsequently, we produced
several congenic or subcongenic strains to narrow the QTL
interval, and focused on a candidate gene. To determine the
causative gene, we finally rescued the phenotype using bacterial
artificial chromosome transgenic mice. Consequently, we identi-
fied Usp46 encoding ubiquitin-specific peptidase as one of the
genes responsible for the short immobility time [9].
The Usp46 mutation that we identified has a 3-bp deletion
coding for lysine in the open reading frame. This mutation
shortened the duration of loss of righting reflex caused by
muscimol (GABAAreceptor agonist) administration, and reduced
the muscimol-induced GABAA current in hippocampal CA1
pyramidal neurons. In addition, hippocampal expression of the
67-kDa isoform of glutamic acid decarboxylase is decreased in
mice with the Usp46 mutation [9]. Thus, this mutation appears to
be implicated in the regulation of the GABAergic system.
However, it is not yet known clear exactly how the immobile
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behavior is regulated by the GABAergic system. In addition, it is
not clear whether this 3-bp deletion in Usp46 is a loss-of-function
or gain-of-function mutation. Therefore, in the present study, we
addressed these issues using the 3-bp deleted (designated as the
Usp46 mutant mice) and the null Usp46 mice (the Usp46 KO
mice).
Materials and Methods
Animals
We purchased C57BL/6J (B6) mice from CLEA Japan Inc. The
animals were housed under a 12 hr light/dark cycle (LD 12:12,
7:00 on, 19:00 off) with free access to food and water in our animal
facility, at a temperature maintained at approximately 24uC. Only
male mice aged between 8 and12 weeks were used. For all
experiments, the animals were treated in accordance with the
guidelines issued by Nagoya University and Faculty of Pharma-
ceutical Sciences of Meijo University.
Usp46 Mutant Mice
The Usp46 mutant mice were developed as congenic strains on
a B6 genetic background using a marker-assisted breeding
strategy. These mice (B6.CS-Ngu1053) contained chromosome 5
regions harboring the Usp46 of the CS mice [9].
Usp46 KO Mice
The Usp46 knockout (KO) mice (17-13906 Usp46 gene trapped
mice, TG Resource Bank #6072) were descendants of the mouse
strain generated by Trans Genic Inc. (Kumamoto, Japan) using
the gene trap technique (Fig. 1) [10]. Because the original Usp46
KO mice were generated from B6 and CBA mice, these mice were
backcrossed to B6 a minimum of 9 times in order to remove any
possible phenotypic variations caused by a different genomic
background. To determine their genotypes, ear biopsies were
performed at 4 weeks of age for DNA detection using PCR. F1
heterozygous and B6 alleles were amplified using a neo primer
pair: 59-CTGAATGAACTGCAGGACGAG-39 and 59-GTCCA-
GATCATCCTGATCGAC-39, and lox-SA primer pair: 59-
AGGTCGAGGGACCTATACCG-39
GAGGCCGCTTGTCCTCTTTG-39.
mice were crossed between themselves to obtain the F2
generation: wild-type, heterozygous, and homozygous. To de-
termine theirgenotypes,
GACTCTGCTGTTTCTCCTATGCTCC-39, RP1 primer 59-
AATGTTGAGGCAAAGCTGCCAAGCTCAC-39, and SA6AS
primer 59-CCGGCTAAAACTTGAGACCT-39 were used.
and
F1
59-
Theheterozygous
FP3primer59-ATA-
Drugs
Nitrazepam (Wako) and flumazenil (SIGMA) were suspended in
saline with 0.3% carboxymethylcellulose sodium (SIGMA).
Nitrazepam (0.1, 0.3, and 1 mg/kg) was administered intraperi-
toneally at a volume of 0.1 mL/10 g body weight 30 min before
the open-field test (OFT) was conducted. Flumazenil (10 mg/kg)
was administered subcutaneously 5 min prior to nitrazepam
treatment. Control animals received the same volume of vehicle
(saline in 0.3% carboxymethylcellulose).
Open Field Test (OFT)
Before the behavioral test, mice were moved to the animal room
adjacent to the test room, where their behaviors were assayed. The
mice were kept there for 1 week under the same conditions as
those before being moved. We assayed mice during the light phase
(11:00–16:00) after at least a 2 hr adaptation to the test room.
Each mouse was placed in the center of a gray plastic box
(40640640 cm) with the floor divided into 64 compartments
(565 cm each), and was allowed to freely explore for 5 min under
40 to 50-lx fluorescent light. During the test, the number of
grooming, rearing, and climbing (standing on hind legs with
forefeet on the wall) behaviors was scored. Because very few
instances of rearing were observed, rearing and climbing were
combined. After the OFT, the total distance of moving within the
box was calculated using SMART software (version 2.0, Panlab,
Spain). At the end of the test, the mouse was returned to its home
cage, and all apparatus was cleaned with 70% ethanol.
Tail Suspension Test (TST)
Immediately after the OFT, TST was performed. The pro-
cedure for TST was the same as that used in our previous study
[9]. Briefly, mice were suspended by their tails using an elastic
band attached to the tails by adhesive tape, and the elastic band
was hooked onto a horizontal rod. The distance between the tip of
the nose of the mouse and the floor was approximately 20 cm. The
mice were suspended for a period of 7 min, and the time spent
immobile during the last 6 min of the 7 min was recorded for each
individual, by an observer blinded to the genotype.
Northern Blot Analysis
Male mice were killed by decapitation and the brains were
collected, frozen in liquid nitrogen, and stored at 280uC until
RNA extraction was conducted. Total RNA was extracted using
TRIZol Reagent (Invitrogen, Carlsbad, USA) and quantified by
absorbance at 260 nm (1 optical density unit=40 mg RNA/mL).
Total RNA (1 mg) was reverse transcribed into complementary
DNA (cDNA) using the Moloney murine leukemia virus (M-
MLV) reverse transcriptase and oligo(dT) primer. The cDNA
was amplified by 45 cycles of PCR using mouse Usp46 specific
primers (F514: 59-AACACTATTGCGGACATCCTG-39 and
R1982: 59-AAAGCCACGTTTCTGGAAAAT-39). The PCR
cycle consisted of 10 s of denaturation at 98uC, 30 s of annealing
at 60uC, and 2 min of extension at 72uC. The amplified product
was cloned into the pGEM-T easyTMplasmid vector and
sequenced by the dideoxy chain termination method using
a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems). The DNA fragments were used as probes for
northern blot analyses. The location of these probes on the
Usp46 cDNA is shown in Figure 2A. Total RNA samples were
separated on 1% (w/v) agarose gel containing 2.2 M formalde-
hyde. The separated RNA samples were blotted onto a Hybond+
nylon membrane (GE Healthcare, BKM, UK) and hybridized to
the mouse Usp46 cDNA probe labeled with [a-32P] dCTP. After
hybridization, the membrane was washed sequentially in 26SSC
containing 0.1% SDS at 62uC for 30 min, 0.16SSC containing
0.1% SDS at 62uC for 30 min twice. The amount of mRNA was
determined by a Fuji BAS 2000 Bioimaging Analyzer (Fujifilm,
Tokyo, Japan).
Statistics
The data are expressed as mean + S.E.M. Significant
differences between the 2 groups were determined using
Student’s t-tests. One-way ANOVA with the Tukey-Kramer
test, or two-way ANOVA with Bonferroni’s test were used for
multiple comparisons.
Results
Usp46 KO Mice
The trap vector was inserted into an intron of Usp46
approximately 16 kbp downstream from exon 1 (Fig. 1A). A
Usp46 Regulates Immobility in the TST
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PCR product of 772 bp was detected in the wild-type mice, but
not in the KO mice. Instead, 1.4 kbp products were detected in
the KO mice. In the heterozygous mice, as expected, both
products were observed (Fig. 1B). To confirm the absence of
Usp46 mRNA expression in the brains of the KO mice, we
performed northern blot analysis. The results showed the
absence, and approximately half the level of Usp46 mRNA in
the brains of KO mice and heterozygous mice, respectively
(Fig. 2B, C). We also confirmed that the KO mice showed
significantly shorter immobility times than the wild-type mice in
TST (Fig. 1C).
Dose-dependent Effects of Nitrazepam on TST
Immobility Time in Usp46 Mutant Mice
Nitrazepam, an agonist of the benzodiazepine-binding site of
GABAAreceptors that causes an enhanced binding of GABA to
these receptors, produced a dose-dependent increase in TST
immobility times in the Usp46 mutant mice (Fdose (3,40)=19.09,
P,0.01; Fgenotype(1,40)=73.56, P,0.01; Fdose6genotype(3,40)=5.36,
P,0.01; two-way ANOVA) (Fig. 3). However, such a dose-
dependent increase was not apparent in the wild-type mice,
although a significant difference between doses of 0.1 mg/kg and
1 mg/kg was observed. Because nitrazepam is known to have
Figure 1. Generation of the Usp46 KO mice. (A) Integration site of the pU-17 trap vector. The trap vector is inserted approximately 16 kbp
downstream from exon 1. The trap vector contains a splice acceptor (SA), the b-galactosidase/neomycin-resistance fusion (b-geo) gene,
a polyadeylation signal (pA) and pSP73 vector sequences [10]. The white arrows (FP3, RP1, and SA6AS) indicate the primers used for genotyping. (B)
Genotyping by the polymerase chain reaction. DNA fragments of 772 bp from the wild type allele and 1.4 kbp from the inserted allele were amplified
by the primer pairs FP3-RP1 and FP3-SA6AS, respectively. (C) Immobility time in wild-type (WT) and Usp46 KO mice (KO) in the tail suspension test
(TST). The KO mice showed a significantly shorter immobility time than the WT mice in the TST. Data represent the mean + S.E.M. for 5–6 mice in each
group. **P,0.01 (Student’s t-test).
doi:10.1371/journal.pone.0039084.g001
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sedative effects, we measured general behaviors in OFT.
However, an administration of this drug did not affect general
behaviors (total activity, frequency of climbing + rearing, and
grooming for 5 min in OFT) (P.0.05; one-way ANOVA with
Tukey-Kramer post-hoc test) (Table 1), while nitrazepam affected
TST immobility times in the Usp46 mutant mice.
Effects of co-administration of Nitrazepam and
Flumazenil on TST Immobility Time in Usp46 Mutant
and KO Mice
As seen in Fig. 3, nitrazepam (1 mg/kg) completely restored
the immobility times to a level of wild-type in the Usp46 mutant
mice. Therefore, we used this dose to ascertain whether the
restored immobility time is blocked by flumazenil, an antagonist
at the benzodiazepine-binding site of the GABAAreceptor [11].
Prior to this test, we examined the effects of a single
administration of flumazenil on TST immobility time and
general behaviors (total activity, frequency of grooming, and
climbing + rearing for 5 min in OFT) in both the Usp46 mutant
(n=6) and the wild-type mice (n=6) (all mice used were naive),
and found that there were no significant effects of flumazenil on
these behaviors in both types of mice (data not shown).
To investigate whether the GABAAreceptor is involved in the
regulation of immobility time in TST, the antagonizing effects of
flumazenil on the nitrazepam-induced increase of immobility
time were examined. As a result, we observed that the enhanced
TST immobility time elicited by nitrazepam was blocked by
flumazenil in both Usp46 mutant and KO mice (Ftreatment
(2,42)=28.52, P,0.01; Fgenotype(2,42)=35.35, P,0.01; Ftreatment6
genotype(2,42)=3.47, P,0.05; two-way ANOVA with Bonferroni’s
test; Fig. 4). Co-administration of these drugs had mild effects on
total activity levels in wild-type mice, and on the frequency of
rearing + climbing behavior in KO mice (Table 1).
Figure 2. Expression of Usp46 mRNA in the brain analyzed by northern blot analysis. (A) Schematic diagram of cDNA and probes for
Usp46. Probes A and B were used for the northern blot analysis. Black arrowhead indicates the EcoR1 site. F514 and R1982 are a primer pair for PCR.
(B) Northern blot from brain total RNA hybridized with the mouse Usp46 cDNA probes A and B, labeled with [a-32P] dCTP. The blot was hybridized
with a cDNA probe for S18 as an internal control (the middle panel). The bottom panel shows a photograph of an RNA gel, indicating that equivalent
amounts of total RNA were used in each lane. (C) Relative values of Usp46 mRNA. PSL (Photo stimulated luminescence) was measured. Signals from
the Usp46 KO mice (KO) were not detected (N.D.). The differences between the wild-type mice (WT) and the heterozygotic mice (H) were significant
(**P,0.01, n=6, Student’s t-test).
doi:10.1371/journal.pone.0039084.g002
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Discussion
In the present study, we addressed 2 primary questions: (1)
whether Usp46 KO mice show a similar phenotype to Usp46 (3-bp
deleted) mutant mice in TST; and (2), whether the immobility
time is regulated by the GABAergic system.
The KO mice with the insertion of the trap vector, which
contains the b-galactosidase gene in the region between exon 1
and exon 2, did not express Usp46 within the brain. As shown in
Fig. 1, Usp46 KO mice showed significantly shorter immobility
time than wild-type mice. This phenotype in Usp46 KO mice was
similar to Usp46 mutant mice (Fig. 3, 4) [9]. Additionally, we
observed b-galactosidase expression by immunohistochemistry in
the hippocampus, one of the brain regions that strongly expresses
Usp46, in the KO mice (unpublished results). These results indicate
that the 3-bp coding for lysine is important for USP46
deubiquitinating enzyme to be functionally active, and suggests
that this mutation causes a loss-of-function phenotype. Indeed,
recently, it has been reported that this 3-bp deletion decreases
USP46 deubiquitinating enzyme activity [12].
Previously, we have reported that Usp46 is implicated in the
regulation of the neuronal GABAergic system, based on results
showing that the sensitivity to muscimol (a selective GABAA
receptor agonist and a partial agonist for GABACreceptors) is
attenuated in the muscimol-induced righting reflex and the
muscimol-induced inhibitory current in CA1 pyramidal neurons
of the hippocampus of Usp46 mutant mice [9]. However, there is
no direct evidence that attenuation of the GABAergic system alters
immobile behavior in the TST. In this study, therefore, we
addressed this issue and found that the TST immobility time was
increased by nitrazepam in a dose-dependent manner in Usp46
mutant mice, as compared to the wild-type mice, in which the
immobility time slightly increased only at the highest dose (Fig. 3).
This attenuation was blocked by flumazenil in both the Usp46
mutant and in the KO mice (Fig. 4). Additionally, nitrazepam did
not significantly change total activity levels, frequency of
grooming, or climbing + rearing behaviors in the OFT (Table 1).
Furthermore, it is known that a low dose of nitrazepam, which has
no sedative effects, does not induce sleep in mice [13].
Additionally, there are some reports that the GABAergic system
might be involved in the regulation of TST immobility time; the
GABAAreceptor a3 subunit was identified as a candidate gene
affecting immobility time in the TST using QTL analysis [14],
Gabra3 KO mice have exhibited low immobility times in FST
[15], GABAAreceptor a2 subunit KO mice have showed longer
immobility times in the TST [16], and GABA transporter subtype
1 (GAT1) KO mice have showed shorter immobility times in both
the TST and FST [17]. Further, it is reported that GAD65 (a 65-
kDa isoform of glutamate decarboxylase) KO mice exhibit
Figure 3. Effects of nitrazepam administration on tail suspension test (TST) immobility time. Nitrazepam induces dose-dependent
increases of TST immobility time in Usp46 mutant mice (MT). Data are expressed as mean + S.E.M. for 6 mice in each group.##P,0.01 compared with
the wild-type mice for each dose; *P,0.05, **P,0.01 (two-way ANOVA with Bonferroni’s test).
doi:10.1371/journal.pone.0039084.g003
Table 1. Effects of nitrazepam (1 mg/kg) and flumazenil
(10 mg/kg) on general behaviors in OFT.
BehaviorGenotype Saline Nitrazepam
Nitrazepam
+ +
Flumazenil
Total
Activity (cm)
WT (6) 1489.96191.4 1807.76151.21296.7683.8**
MT (6)2142.56115.3 1840.86173.4 1670.66433.6
KO (5) 2417.66223.31763.86194.42421.16440.0
Rearing +
Climbing
WT (6)17.365.632.563.419.062.2
MT (6) 29.364.2 21.363.9 24.666.6
KO (5)33.065.6 29.065.5 39.666.3*
Grooming WT (6) 1.260.3 1.060.42.660.9
MT (6)1.060.3 0.860.22.060.6
KO (5) 1.260.5 1.260.32.860.7
Data are shown as the mean 6 S.E.M. The number of animals is given in
parentheses. **P,0.01 compared with the saline-treated group in wild-type
(WT) mice, *P,0.05 compared with the saline-treated group in the Usp46 KO
mice (KO) (Tukey-Kramer post-hoc test).
doi:10.1371/journal.pone.0039084.t001
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