Mice lacking the kf-1 gene exhibit increased anxiety- but notdespair-like behavior
Atsushi Tsujimura1, Masato Matsuki1,2, Keizo Takao3,4, Kiyofumi Yamanishi1,2Tsuyoshi Miyakawa3,4 and Tamotsu Hashimoto-Gotoh1,*
1. Department of Biochemistry and Molecular Genetics, RINDG, Kyoto Prefectural University of Medicine, Kyoto, Japan
2. Department of Dermatology, Hyogo College of Medicine, Nishinomiya, Japan
3. Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan
4. Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan
Edited by:Andrew Holmes, National Institute on Alcohol Abuse and Alcoholism, NIH, USA

Reviewed by: David P. Wolfer, University of Zurich, Switzerland
Valerie J. Bolivar, Wadsworth Center, NYSDOH, USA

KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with
Alzheimer’s disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression
is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination andmay modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role ofkf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/−
mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-likebehavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differenceswere observed in exploratory locomotion using the open field test or in behavioral despair using the forced-swim and tail-suspension
tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating
protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually
reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of
psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.
Keywords: anxiety, depression, schizophrenia, ubiquitin ligase, knockout mouse

INTRODUCTION
In the 1990s, a comprehensive search was conducted for thegenes responsible for Alzheimer’s disease (AD). A number ofdominantly inherited causative mutations were then discoveredin the genes encoding β-amyloid precursor protein, presenilin1 (PS1), and presenilin-2 (PS2) (for reviews, see HashimotoGotoh et al., 2006; Lundkvist and Näslund, 2007). At the sametime, we also sought to identify genes that were involved in AD
pathogenesis, and we identified two genes whose transcripts
were found more frequently in the cerebral cortex of an AD
patient than in a non-AD subject. One of these genes, kf-1, was anovel gene that encoded a zinc-binding membrane protein witha RING-H2 finger domain at the C-terminus (Yasojima et al.,1997). The kf-1 gene is conserved in chordates/vertebrates but*Correspondence: Tamotsu Hashimoto-Gotoh, Department of Biochemistry andMolecular Genetics, Research Institute for Neurological Diseases and Geriatrics, Kyoto
Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan. e-mail: thg@
koto.kpu-m.ac.jp
Received: 10 June 2008; paper pending published: 27 June 2008; accepted: 21 August
2008; published online: 08 September 2008.
Citation: Front. Behav. Neurosci. (2008) 2: 4. doi: 10.3389/neuro.08.004.2008
Copyright © 2008 Tsujimura, Matsuki, Takao, Yamanishi, Miyakawa and Hashimoto-Gotoh.
This is an open-access article subject to an exclusive license agreement between theauthors and the Frontiers Research Foundation, which permits unrestricted use, distribution,and reproduction in any medium, provided the original authors and source are credited.

not in invertebrates such as Drosophila melanogaster. Controlof the half-life of kf-1 mRNA may be critical for its biological function, since nucleotide sequences in the 3′-untranslated
region, consisting entirely of a stable two-fold-sheet structure,are highly conserved to an unusual extent between human andmice. This may suggest that the level of KF-1 protein synthesis is regulated strictly in a cell. Northern blot analysis indicatesthat the kf-1 gene is expressed most prominently in the brain but
more or less throughout the body, except in the liver (Yasojima
et al., 1997). KF-1 mediates E2-dependent ubiquitination, andthus may play a role in modulating cellular protein levels as
an E3 ubiquitin ligase (Lorick et al., 1999). Expression of the
kf-1 gene is also elevated in the frontal cortex and hippocampus, but not hypothalamus, of rats after chronic antidepressanttreatments such as the administration of a selective serotonin
re-uptake inhibitor (SSRI) (Yamada et al., 2000), electroconvulsive therapy (Nishioka et al., 2003), and repetitive transcranial
magnetic stimulation (Kudo et al., 2005). Concerning a possible
link of the kf-1 gene expression to other neurodegenerative or
psychiatric diseases, we are not aware of any reports appearing in
the NIH databases (PubMed and GEO profiles). It is known that
depression can be a heralding symptom or prodrome rather thana separate and distinct disorder for AD or dementia (for a review,see Katz, 1998). In fact, it has been reported that depression canoccur early in the course of AD in ps1 mutation carriers unaware
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of their genetic status (Ringman et al., 2004). Taken together,
these observations suggest that KF-1 plays some role or rolesin the regulation of mental disorders such as depression, which
include anxiety and mood disorders as the core symptoms.
Anxiety is an instinct that may have been developed in vertebrates to promote adaptive survival by evasion of unnecessary
danger. However, excessive anxiety is disadvantageous, because
it reduces even behavioral activity that is necessary for adaptation. Thus, there must be underlying mechanisms that havedeveloped as to appropriately regulate anxiety in response toenvironmental circumstances. For example, the amygdala isthought to be important in the expression of anxiety or fear,
and the medial prefrontal cortex plays a key role for fear extinction by regulating amygdala-mediated expression of fear (for areview, see Bishop, 2007).
All the antidepressant drugs developed so far are based on
the monoamine hypothesis, which assumes that the pathogenesis of depression could be caused by depletion of monoamines
like serotonin in the brain, although some recent approaches tothe treatment of depression try to move beyond this hypothesis (Norman, 2006). Nevertheless, there is no convincing evidence of a primary dysfunction of a specific monoamine system
in depression; on the contrary, monoamine depletion does notworsen depression in patients and does not cause depression
in healthy volunteers. Testing systems to measure behavioral
despair have been developed in rats/mice for screening antidepressant compounds. These model systems include the forced
swim test and the tail suspension test, and compounds that elevate monoamine levels in the brain have been found to reducethe immobility time of animals under these conditions of fearand despair (Porsolt et al., 1978; Steru et al., 1985). It is worth
noting that in humans, approximately one-third of patients withdepression do not respond to the drugs developed using thesetests. To develop drugs to help patients who are unresponsive
to currently available antidepressants, it is therefore desirable
to have an animal model that mimics the core symptoms ofdepression, but that does not involve regulation of monoaminelevels in the brain.
In the present study, we conducted a comprehensive behavioral test battery on the mice lacking KF-1 and found that kf-1−/−
mice showed decreased stay time in and number of transitions
to the light box in the light/dark transition test, decreased locomotor activity in the elevated plus maze test, and increased
immediate freezing in the fear conditioning test, indicating
overall increased anxiety-like behavior under stressful situationsin these mutant mice. No significant cognitive abnormalities
were found in the fear conditioning test and in the forced alternation test using the automatic T-maze. Based on the effect of
KF-1 depletion on these anxiety-like behaviors, we propose thatkf-1−/− mice may suppress selectively anxiety under physiologicalconditions, and be useful for both investigating the molecularmechanisms of anxiety/depression and screening novel anxiolytic/antidepressant compounds.

MATERIALS AND METHODSTARGETING VECTOR

Mouse kf-1 genomic clones were isolated from a 129/Sv λ Fix
III genomic DNA library (Stratagene) using mouse kf-1 cDNA
as a hybridization probe. Gene fragments were subcloned into
pKF4 and pHSG396 for further analysis and manipulation(Hashimoto-Gotoh, 1995; Takeshita et al., 1987). The essential

portion of the mouse kf-1 gene was sequenced, including all fourexons (Ex) and three introns (In). The DNA segment used toconstruct the targeting vector was ∼14.7 kb long and was downstream of the AseI site at position 15,600 in In 3. The position
numbers for restriction sites are based on the GenBank database(Accession no. AB012161). Schematic representations of themouse kf-1 gene, targeting vector, and recombinant and deletion alleles are shown in Figure 1A. To delete the critical coding
region, i.e., Ex 4, a conditional knockout (KO) procedure using
Cre-lox recombination was employed (Sakai and Miyazaki,1997). The resulting targeting DNA was cloned into pHSG396,
yielding pHSG396mkf-1Ex4.
SELECTION OF TARGETED ES CLONES AND
GENERATION OF kf-1−/− MICE

The selection of targeted ES cells (line R1; Nagy et al., 1993)
and generation of kf-1−/− mice were done essentially as described
previously (Matsuki et al., 1998). Briefly, ten million ES cells
were transfected by electroporation with 25 µg of NotI-linearized pHSG396mkf-1Ex4 DNA. After selection with 150 µg/ml
of neomycin, 432 clones were analyzed, among which fiveclones were positive, giving a 2,428-bp PCR product using
the forward primer: 5′-CCCTGTCTACCGTTTCTACCCCT3′ and the reverse primer: 5′-GAATGGGCTGACGCTTCCTCGTG-3′ (Figure 1A). One of the clones was expanded andmicroinjected into BDF1 blastocysts. Male chimeras derived
from the clone were mated with C57BL/6N females. Germline transmission of the targeted recombination was verified bySouthern blot analysis of tail DNA against 32P-labeled mouse
kf-1 DNA of the Ex 4 coding region. The resulting kf-1+/lox malemice were mated with CAG (Cytomegalovirus immediate earlyenhancer + chicken β-actin hybrid promoter + rabbit β-globin
polyA signal containing region) promoter-driven Cre gene containing transgenic female mice (Sakai and Miyazaki, 1997). F1
heterozygotes, kf-1+/−, were then crossed to C57BL/6N, and theresulting F2 heterozygotes were backcrossed to C57BL/6N. Thiswas repeated for eight generations, and then male and female
F9 heterozygotes were intercrossed to generate a homozygous
kf-1−/− line. The lack of kf-1 gene was verified by Southernhybridization of BspHI-digested tail DNA using the sameprobe as above (Figure 1B). The absence of kf-1 mRNA wasconfirmed by Northern blot analysis using 10 µg of total RNA
from the whole-brain against the same probe (Figure 1B). The
kf-1−/− mouse, called the Hicky mouse, may be obtained fromthe Riken BioResource Center, Japan (BRC No. 01916).
ANIMALS AND EXPERIMENTAL DESIGN

Prior to behavioral studies, kf-1−/− males obtained from thecrossing between male and female F9 heterozygotes weremated with C57BL/6N females (kf-1+/+). The resulting heterozygotes were then intercrossed to produce homozygous kf-1+/+
and kf-1−/− littermates. The behavioral tests were performed
with male mice of this generation, which were 10 weeks old at
the start of the behavioral studies (n = 20 for both groups). Mice
were reorganized as soon as they were genotyped at the timeof weaning, and group-housed with four mice per cage, two
kf-1+/+ and two kf-1−/− littermates. Housing conditions included
a 12-h light/dark cycle, with lights on at 7:00 a.m. and accessto food and water ad libitum. Behavioral testing was performed
between 9:00 a.m. and 6:00 p.m., excepting for that of T-mazeforced alternation task that was performed between 7:00 p.m.
and 5:00 a.m. After the tests, all apparatus were cleaned with

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Figure 1 | Schematic presentation of the mouse kf-1 gene structure, targeting vector, and floxed and knockout alleles. (A) For construction of the targeting vector, the diphtheria toxin A (DT-A) and neomycin resistance (neo) genes were inserted in reverse orientation at AseI and StuI sites at positions 15,600 and
17,515, respectively, and loxP cassettes were inserted using StuI and BglII sites at positions 17,515 in In 3 and 20,081 in Ex 4, respectively. Closed and open
boxes indicate the approximate positions of coding and non-coding exons, respectively. P with an arrowhead indicates the 49-bp promoter region identified by
the CAT assay (data not shown). BspHI restriction fragments, 5,861 and 4,759 bp in size, are indicated by open bidirectional-arrows above the kf-1 (+) and kf-1(lox) genomes, respectively. The position corresponding to the 2,428-bp PCR product (see Materials and Methods) used to verify homologous recombination
between the targeting vector and the kf-1 gene is shown below the kf-1 (lox) genome. Restriction sites for AseI, BspHI, BglII, and StuI were shown by A, B, Bg,
and S, respectively. (B) Southern blot of BspHI-digested genomic DNA using the mouse kf-1 probe. The DNA bands indicated by arrows (5,861 and 4,759 bp)
correspond to the BspHI-fragments shown in (A). Lanes 1, 2, 3, and 4 are kf-1+/+, kf-1lox/lox, kf-1+/−, and kf-1−/−, respectively. Note that the intensity of the DNA
band in lane 3 is half as intense as in lane 1. (C) Northern blot analysis of kf-1 gene expression. Experimental procedures are as described in Sections ‘Targeting
vector’ and ‘Selection of targeted ES clones and generation of kf-1−/− mice’. Lanes are as the same in (B).

super hypochlorous water to prevent a bias based on olfactory
cues with the apparatus. The order of each testing is as indicated in Table 1. All animal experiments, including the production and maintenance protocols, were reviewed and approvedby the Animal Care and Use Committee of the Kyoto Prefectural
University of Medicine. Behavioral studies were performed inaccordance with the guidelines for animal experimentation of
Graduate School of Medicine, Kyoto University.
GENERAL HEALTH AND NEUROLOGICAL SCREEN

To compare the physical characteristics of kf-1−/− mice and their
kf-1+/+ littermates, we first conducted the neurological screening as previously described (Miyakawa et al., 2001). The righting, whiskers touch, and ear twitch reflexes were evaluated, anda number of physical features including body weight and temperature, and the presence of whiskers or bald hair patches wererecorded.
NEUROMUSCULAR EXAMINATIONThe neuromuscular strength was examined by the grip strength
and wire-hanging tests. The grip strength meter (O’Hara & Co.)was used to assess forelimb grip strength. Mice were lifted andheld by their tail so that their forepaws could grasp a wire grid.
Mice were then gently pulled backward by the tail with their
posture parallel to the surface of the table until they released thegrid. The peak force applied by mouse forelimbs was recorded innewtons (N). Each mouse was tested three times and the greatest value measured was used for statistical analysis. In the wirehanging test, mice were placed on a wire mesh, which was theninverted and waved gently, so that the subject gripped the wire.
Latency to fall was recorded, with a 60 s cut-off time.

LIGHT/DARK TRANSITION TESTThe apparatus used for light/dark transition test consisted of acage (21 cm × 42 cm × 25 cm) divided into two sections of equal
size by a partition with door (O’Hara & Co., Tokyo). One chamber was brightly illuminated (390 lx), whereas the other chamber
was dark (2 lx). Mice were placed into the dark side, and allowed
to move freely between the two chambers with door open for
10 min. The total number of transitions, time spent in eachcompartment, first latency to light side, and distance traveled
were recorded automatically. On-line material describing thismethod is available visually (Takao and Miyakawa, 2006).
OPEN FIELD TEST

Locomotor activity was measured using the open field test.
Each subject was placed in the center of open field apparatus(40 cm × 40 cm × 30 cm; Accuscan Instruments, Columbus,
OH, USA). Total distance traveled (centimeters), vertical activity (rearing measured by counting the number of photobeam
interruptions), time spent in the center, the beam-break counts
for stereotypic behaviors, and the number of fecal boli wererecorded. Data were collected for 120 min. Photo beams werepositioned 1 cm inside apart from walls at the edge of the open
field. If any of these beams was not interrupted, mice were considered to be in the center area.
ELEVATED PLUS MAZE TESTThe elevated plus-maze consisted of two open arms
(25 cm × 5 cm) and two enclosed arms of the same size, with
15 cm high transparent walls. The arms and central square weremade of white plastic plates, and were elevated to a height of
55 cm above the floor. In order to minimize the likelihood of
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Table 1 | Comprehensive behavioral test battery and phenotypes of kf-1−/− mice.
Tests
General health
Whisker, coat, reflexes
Physical examination
Body temperature (ºC)
Body weight (g)Wire hanging time (sec)
Grip strength (N)Light/dark transition
AnxietyOpen field
Exploratory locomotion
Elevated plus maze
Anxiety
Hot platePain sensitivity (sec)Social interaction
Total duration of contacts (sec)
Number of contacts (times)
Total duration of active contact (sec)Mean duration/contacts (sec)Distance traveled (cm)Rotarod
Motor coordination
Prepulse inhibition
Sensorimotor gating
Porsol forced swim
Immobility time (behavioral despair)T-maze forced alternation task
Spatial working memory (cognitive function)
Contextual and cued fear conditioning
Contextual memory (cognitive function)
Tail suspension
Immobility time (behavioral despair)

Phenotypes

Measurements kf-1+/+, kf-1−/−

p and F values

Days

≈

n/a

n/a

1

≈
≈
↑
≈

36.99 ± 0.17, 37.15 ± 0.10
26.35 ± 0.43, 26.70 ± 0.57
44.55 ± 4.21, 55.55 ± 2.27
0.83 ± 0.03, 0.86 ± 0.04

p = 0.4146, F1,38 = 0.681
p = 0.6254, F1,38 = 0.242
p = 0.0270, F1,38 = 5.295
p = 0.4060, F1,38 = 0.706

1
1
1
2

↑

Figure 9

Figure 93≈

Figure 3

Figure 3

7,8↑

Figure 10

Figure 109≈9.7 ± 0.67, 10.5 ± 0.64

p = 0.4045, F1,38 = 0.711

10

≈
≈
≈
≈
≈

85.9 ± 4.95, 85.0 ± 6.22
53.2 ± 4.85, 51.3 ± 3.76
16.0 ± 1.81, 14.9 ± 1.34
1.7 ± 0.15, 1.7 ± 0.133275 ± 140, 3323 ± 172

p = 0.9101, F1,38 = 0.002
p = 0.7604, F1,38 = 0.096
p = 0.6153, F1,38 = 0.261
p = 0.9609, F1,38 = 0.002
p = 0.8310, F1,38 = 0.047

11
11
11
11
11

≈

Figure 4

Figure 414, 15↑Figure 11

Figure 11

16≈

Figure 5

Figure 517, 18≈Figure 7

Figure 7

41–71≈

Figure 8

Figure 8

77–78≈

Figure 6

Figure 679≈, No significant differences between kf-1+/+ and kf-1−/−; ↑, Increased in kf-1−/− compared to kf-1+/+. The testing order was from the 1st to 79th day as indicated in the right column.

animals falling from the apparatus, 3-mm high plexiglas ledges
were provided for the open arms. Arms of the same type werearranged at opposite sides to each other. Each mouse was placedin the central square of the maze (5 cm × 5 cm), facing one of
the closed arms. Mouse behavior was recorded during a 10 min
test period. The number of entries into, and the time spenton open and enclosed arms were recorded. For data analysis,
we employed the following four measures: the percentage ofentries onto open arms, the stay time on open arms (seconds),
the number of total entries, and total distance traveled (centimeters). Data acquisition and analysis were performed automatically, using Image EP software.

performed automatically using Image SI software. Number ofcontacts, duration of contacts, and total distance traveled were
measured.

HOT PLATE TESTSTARTLE RESPONSE/PREPULSE INHIBITION TESTThe hot plate test was used to evaluate the nociception or thesensitivity to a painful stimulus. Mice were placed on a hot plate
at 55.0 ± 0.3°C (Columbus Instruments, Columbus, OH, USA),and latency to the first hind-paw response was recorded. The
hind-paw response was either a foot shake or a paw lick.

A startle reflex measurement system was used (O’Hara & Co.,Tokyo). A test session began by placing a mouse in a plexiglas
cylinder where it was left undisturbed for 10 min. The durationof white noise that was used as the startle stimulus was 40 ms
for all trial types. The startle response was recorded for 140 ms(measuring the response every 1 ms) starting with the onset ofthe prepulse stimulus. The background noise level in each chamber was 70 dB. The peak startle amplitude recorded during the
140 ms sampling window was used as the dependent variable. A
test session consisted of six trial types [i.e., two types for ‘startle-stimulus-only’ trials, and four types for prepulse inhibition(PPI) trials]. The intensity of startle stimulus was 110 or 120 dB.

SOCIAL INTERACTION TEST IN A NOVEL ENVIRONMENT

Two mice of identical genotypes, which were previously
housed in different cages, were placed into a box together(40 cm × 40 cm × 30 cm) and allowed to explore freely for
10 min. Social behavior was monitored by a CCD camera,
which was connected to a Macintosh computer. Analysis was

ROTAROD TEST

Motor coordination and balance were tested with the rotarod
test. We performed the rotarod test using an accelerating rotarod(UGO Basile Accelerating Rotarod) by placing a mouse on arotating drum (3 cm diameter) and measuring the time during
which each animal was able to maintain its balance on the rod as
latency time to fall (seconds). Speed of the rotarod was accelerated from 4 to 40 rpm over a 5-min period.

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The prepulse sound was presented 100 ms before the startle
stimulus, and its intensity was 74 or 78 dB. Four combinationsof prepulse and startle stimuli were employed (74–110, 78–110,
74–120, and 78–120 dB). Six blocks of the six trial types were
presented in pseudo-random order such that each trial type waspresented once within a block. The average inter-trial interval
was 15 s (range: 10–20 s).
PORSOLT FORCED SWIM TESTThe Porsolt forced swim test was done essentially as described
previously (Miyakawa et al., 2001). The apparatus consistedof four plexiglas cylinders (20 cm height × 10 cm diameter).
The cylinders were filled with water at 23°C, up to a height of
7.5 cm. Mice were placed into the cylinders, and their behavior was recorded over a 10-min test period (days 1 and 2). Dataacquisition and analysis were performed automatically, using
Image PS software (see Section ‘Image analysis’).
T-MAZE FORCED ALTERNATION TASK TESTThe forced alternation task test was conducted using an automatic T-maze (Figure 2). It was constructed of white plastics
runways with walls 25-cm high. The maze was partitioned
off into six areas by sliding doors that can be opened downward. The stem of T was composed of area S2 (13 cm × 24 cm)
and the arms of T were composed of area A1 and A2(11.5 cm × 20.5 cm). Area P1 and P2 were the connecting passage way from the arm (area A1 or A2) to the start compartment (area S1). The end of each arm was equipped with a pellet
dispenser that could provide food reward. The pellet sensors
were able to record automatically pellet intake by the mice. One
week before the pre-training, mice were deprived of food until
their body weight was reduced to 80–85% of the initial level.
Mice were kept on a maintenance diet throughout the course of
all the T-maze experiments. Before the first trial, mice were subjected to three 10-min adaptation sessions, during which they
were allowed to freely explore the T-maze with all doors open
and both arms baited with food. On the day after the adaptation session, mice were subjected to a forced alternation protocol for 16 days (one session consisting of 10 trials per day;cutoff time, 50 min). Mice were given 10 pairs of training trials
per day. On the first (sample) trial of each pair, the mouse wasforced to choose one of the arms of the T (area A1 or A2), andreceived the reward at the end of the arm. Choosing the incorrect arm resulted in no reward and confinement to the arm for
10 s. After the mouse consumed the pellet or the mouse stayed>10 s without consuming the pellet, the door that separated thearm (area A1 or A2) and connecting passage way (area P1 or
P2) would be opened and the mouse could return to the starting compartment (area S1), via connecting passage way. The
mouse was then given 3 s delay there and a free choice between
both T arms and rewarded for choosing the other arm thatwas not chosen on the first trial of the pair. The location of thesample arm (left or right) was varied pseudo-randomly acrosstrials using Gellermann schedule so that mice received equal
numbers of left and right presentations. A variety of fixed extramaze cues surrounded the apparatus. On the 17th to 20th day,delay (10, 30, or 60 s) was applied after the sample trial.
CONTEXTUAL AND CUED FEAR CONDITIONING

Each mouse was placed in a test chamber (26 cm × 34 cm × 29 cm)
inside a sound-attenuated chamber (O’Hara & Co., Tokyo) andallowed to explore freely for 2 min. A 60 dB white noise, which
served as the conditioned stimulus (CS), was presented for 30 s,
followed by a mild (2 s, 0.5 mA) foot shock, which served as theunconditioned stimulus (US). Two more CS–US pairings were
presented with 2 min inter-stimulus interval. To examine shock
sensitivity, we measured distance traveled when the foot shock
were delivered sensitivity (from 2 s before the shock to 2 s after
the shock, total 6 s). Context testing was conducted 1 day, 1 week,
and 1 month after conditioning in the same chamber. Cued testing with altered context was conducted 1 day after conditioning
using a triangular box (35 cm × 35 cm × 40 cm) made of white
opaque plexiglas, which was located in a different room. Dataacquisition, control of stimuli (i.e., tones and shocks), and data
analysis were performed automatically, using Image FZ software.
Images were captured at one frame per second. For each pairof successive frames, the amount of area (pixels) by which themouse moved was measured. When this area was below a certain
threshold (i.e., 20 pixels), the behavior was judged as ‘freezing’.
When the amount of area equaled or exceeded the threshold, thebehavior was considered as ‘non-freezing’. The optimal threshold(amount of pixels) to judge freezing was determined by adjusting it to the amount of freezing measured by human observation. ‘Freezing’ that lasted less than the defined time threshold(i.e., 2 s) was not included in the analysis.
TAIL SUSPENSION TESTThe tail suspension test was performed for a 10-min test session
according to the procedures described by Steru et al. (1985).
Mice were suspended 30 cm above the floor in a visually isolated area by adhesive tape placed ∼1 cm from the tip of the tail,and their behavior was recorded over a 10-min test period. Dataacquisition and analysis were performed automatically, using
Image TS software (see Section ‘Image analysis’).
IMAGE ANALYSISThe applications used for the behavioral studies (Image LD,
Image EP, Image TM, Image FZ, Image SI, Image TS, andImageHA) were based on the public domain NIH Image program (developed at the U.S. National Institutes of Health andavailable on the Internet at http://rsb.info.nih.gov/nih-image/)and ImageJ program (http://rsb.info.nih.gov/ij/), which weremodified for each test by Miyakawa (available through O’Hara& Co., Tokyo, Japan).
STATISTICAL ANALYSIS
Figure 2 | Apparatus of T-maze forced alternation task test.

Statistical analysis was conducted using Stat View (SAS institute). Data were analyzed by two-way ANOVA, or two-way
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repeated measures ANOVA, unless noted otherwise. Values intables and graphs were expressed as mean ± SEM.

RESULTSSTRUCTURE OF THE MOUSE kf-1 GENE AND
GENERATION OF kf-1−/− MICE

Initially, we analyzed the human kf-1 gene for polymorphisms
and found a G/A transition at the −13 position before the initiation codon in one of 10 subjects. Subsequently, we analyzed themouse kf-1 gene using four independent mouse kf-1 genomic
clones covering an ∼30.3-kb genomic region (see Materials
and Methods). The mouse kf-1 gene lies within an ∼18 kb
segment containing four exons. Ex 4, which codes for the 522
C-terminal amino acids of the 683 amino acid KF-1 protein,
was chosen as a target for deletion (Figure 1A). Ex 4 includes
the coding sequences for the transmembrane regions, the ubiquitination site, and the zinc-binding RING-H2 finger domain.
F1 heterozygotes, kf-1+/−, were obtained as described in Materials
and Methods. After repeated backcrossing to C57BL/6N, the F9heterozygotes kf-1+/− mice were intercrossed and kf-1+/+, kf-1+/−,and kf-1−/− mice were produced with the predicted Mendelian
ratio, implying that there was no embryonic lethal effect due tothe lack of KF-1 in mice. KF-1 was not detectable even in kf-1+/+
mice using immunohistochemistry or Western blotting due tothe low level of KF-1 protein synthesis and also due to KF-1
ubiquitination, although the anti-mKF-1 antibodies we used
could detect mouse KF-1 that was exogenously expressed in
COS cells. Similar to PS1 and PS2, immunostaining showed thatexogenous mouse KF-1 protein was localized to the endoplasmic
reticulum membranes and to Golgi bodies (data not shown).
For the success of immunohistochemistry against native KF-1
protein, one might need monoclonal antibodies with higher
and/or more specific affinity.
EFFECT OF THE kf-1−/− GENOTYPE ON SOMATIC PARAMETERS AND
GENERAL BEHAVIOR

KO mice lacking the kf-1 gene appeared normal and healthy;
there were no evident abnormalities in the physical aspects of
kf-1 KO mice, including reproductive capability. We then measured body temperature and weight, as well as grip strength andwire-hanging time. There were no significant differences inthese measurements between kf-1+/+ and kf-1−/− littermate mice,
excepting for wire-hanging time, namely the kf-1−/− mice exhibited significantly longer wire-hanging time than their kf-1+/+

littermates (Table 1). Because there were no significant differences in grip strength and body weight between kf-1+/+ andkf-1−/− mice, differences in wire-hanging time did not seem tobe rooted in physical differences; instead, this disparity could bea result of psychological differences. In addition, no significant
differences were observed in locomotion (Figures 3A–D), nociception (Table 1), social behavior (Table 1), motor coordination(Figure 4), behavioral despair (Figures 5A,B and 6), or in spatial working memory (Figure 7A). Concerning cognitive functions, no significant differences were found in the T-maze forced
alternation task test (Figures 7A,B) and in context testing or incued testing with an altered context between kf-1+/+ and kf-1−/−,although immediate freezing was significantly enhanced in the
kf-1−/− mice (Figures 8A–C).
LIGHT/DARK TRANSITION AND ELEVATED PLUS MAZE TESTS:
INCREASED ANXIETY IN kf-1−/− MICE

In the light/dark transition test, kf-1−/− mice traveled significantly shorter distances than kf-1+/+ mice in the light but not in
the dark (Figure 9A; p = 0.0005, F1,38 = 14.482 and p = 0.1471,
F1,38 = 2.190, respectively), and their stay-time in the light
was reduced significantly as well (Figure 9B; p = 0.0004,
F1,38 = 15.280). The number of transitions between light anddark was also decreased significantly in kf-1−/− mice (Figure 9C;
p = 0.0392, F1,38 = 4.561). As can be seen in Figures 9A,B, thedistance traveled and stay time of kf-1−/− mice were reduced inparallel against those of kf-1+/+ mice in the light, implying thatmice had equivalent moving capability per second between
the two genotypes. These results suggest an elevated level ofanxiety, but not a reduced level of physical capability, specificto light conditions in kf-1−/− mice, although they did not show
an increased photophobic response as observed in the latency
time for first entering the light compartment from the dark
(Figure 9D; p = 0.8986, F1,38 = 0.016).
The results of elevated plus maze test were similar. The numberof entries into the crossing center from open and closed arms,
and the total distance traveled, were decreased significantly inkf-1−/− mice compared to their kf-1+/+ littermates (Figures 10A,B;
p = 0.0005, F1,38 = 14.758 and p = 0.0016, F1,38 = 11.555, respectively), suggesting increased anxiety in kf-1−/− mice understressful situation on heights. However, no significant differences were observed in % number of entries into and % staytime on the open arms between the two genotypes as observedin Figures 10C,D (p = 0.6548, F1,38 = 0.203 and p = 0.2490,

Figure 3 | No significant differences between kf-1+/+ and kf-1−/− mice in open field test. The kf-1+/+ and kf-1−/− mice were subjected to the open-field test.
(A) Total locomotion distance (p = 0.2879, F1,38 = 1.162). (B) Count of vertical activity (p = 0.7164, F1,38 = 0.134). (C) Time spent on the center of the compartment (p = 0.3930, F1,38 = 0.747). (D) Count of stereotypic behavior (p = 0.4435, F1,38 = 0.600) (see Section ‘Open field test’). WT and KO stand for kf-1+/+ andkf-1−/− mice, respectively.

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A novel anxiety/depression animal model

Figure 4 | Normal motor coordination/learning of kf-1+/+ and kf-1−/− micein the rotarod test. The kf-1+/+ and kf-1−/− mice were tested on an accelerating rotarod and no significant differences were found between the two
genotypes (p = 0.5108, F1,38 = 0.441) (see Section ‘Rotarod test’). WT and KO
stand for kf-1+/+ and kf-1−/− mice, respectively.

Figure 5 | No significant differences in despair-like behavior between
kf-1+/+ and kf-1−/− mice in Porsolt forced swim test. (A) Immobility time(%) in each block for day 1 (p = 0.3525, F1,38 = 0.886) and day 2 (p = 0.1145,
F1,38 = 2.610). (B) Total distance (centimeters) traveled in each block for day
1 (p = 0.1304, F1,38 = 2.390) and day 2 (p = 0.0435, F1,38 = 4.362). No significant differences were found between kf-1+/+ and kf-1−/− mice, althoughsignificant reduction of distance traveled was barely observed in kf-1−/− micecompared to kf-1+/+ littermates in day 2 (see Section ‘Porsolt forced swim
test’). WT and KO stand for kf-1+/+ and kf-1−/− mice, respectively.

F1,38 = 1.371, respectively). No significant differences in theseparameters may be due to a floor effect. Some mice did not visit
in fact the open arms at all in both the genotypes, 3 and 5 micein kf-1+/+ and kf-1−/−, respectively. Alternatively, the discrepancy
between the two tests (Figures 9A–C vs. Figures 10C,D) may
reflect the different aspect of anxiety-like behaviors (see also
Discussion).

Figure 6 | Immobility in the tail-suspension assay. Immobility time is presented as % time in each block. There was no significant differences between
kf-1+/+ and kf-1−/− in immobility in the tail suspension test (p = 0.0855,
F1,38 = 3.118) (see Section ‘Tail Suspension Test’). WT and KO stand for kf-1+/+
and kf-1−/− mice, respectively.

Figure 7 | Spatial working memory in T-maze forced alternation
task test. (A) No significant differences were observed in spatial working memory between kf-1+/+ and kf-1−/− mice as presented by % correct
responses (p = 0.6146, F1,36 = 0.258). (B) On the 17th to 20th trials, thedelay was applied after sample trial for 3 s (p = 0.3829, F1,36 = 0.779),
10 s (p = 0.6371, F1,36 = 0.226), 30 s (p = 0.6267, F1,36 = 0.241), or 60 s
(p = 0.4110, F1,36 = 0.691). Data are given as mean (±SEM; see Section‘T-maze forced alternation task test’). WT and KO stand for kf-1+/+ and kf-1−/−
mice, respectively.

PREPULSE INHIBITION TEST: INCREASED SENSORIMOTOR GATINGThe PPI test is used widely to measure deficits in informationprocessing abilities or sensorimotor gating in schizophrenic
patients (Geyer and Ellenbroek, 2003), and can be employed inboth human and animal experiments (Arguello and Gogos, 2006).
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Tsujimura et al.

Figure 8 | Levels of freezing in kf-1−/− mice during conditioning, cued
testing with altered context, and context testing. (A) Immediate freezing during conditioning phase (p = 0.0211, F1,38 = 5.788). (B) Contextual
testing conducted 1 day after conditioning (p = 0.9891, F1,38 = 0.0002). (C)Cued test with altered context (p = 0.2352, F1,38 = 1.455) and pre-tone period(p = 0.0774, F1,38 = 3.294) are shown. There were no significant differences
observed between kf-1+/+ and kf-1−/− mice in contextual and cued fear conditioning test, although significant difference was observed in the immediate
freezing without conditioning (see Section ‘Contextual and cued fear conditioning’). WT and KO stand for kf-1+/+ and kf-1−/− mice, respectively.

Figure 10 | Performance of kf-1−/− mice in the elevated plus maze test. At
the start of the test, mice were placed individually in the crossing center fromopen and closed arms. Data are given as mean (±SEM). (A) Number of entries
into the center crossing between open and closed arms. (B) Total distance(centimeters) traveled. (C) % Number of entries into open arms. (D) % Timespent on open arms. Note that the closed arms were enclosed at both sides
by transparent walls, and therefore mice could recognize visibly their position on heights even on the closed arms. **p < 0.005; ***p < 0.0005 (seeSection ‘Elevated plus maze test’). WT and KO stand for kf-1+/+ and kf-1−/−
mice, respectively.

PPI is defined as the degree (%) to which the acoustic startle
response is reduced when the startle-eliciting stimulus is precededby a brief, low-intensity, non-eliciting stimulus, and can be used inboth human and animal experiments. The kf-1−/− mice had lower
startle amplitudes than their kf-1+/+ littermates at either 110 or
120 dB in ‘startle-stimulus-only’ trials for all stimulus amplitudes(Figure 11A; p = 0.0043, F1,38 = 9.226). Interestingly, the percent
PPI, an index of sensorimotor gating, was significantly greater inkf-1−/− than in kf-1+/+ (Figure 11B; overall ANOVA including all
four conditions, p = 0.0023, F1,38 = 10.676). Although a significant
difference between genotypes was not observed when the startle
stimulus intensity was 120 dB (p = 0.0935, F1,38 = 2.960), the difference between genotypes was significant at the startle stimulus intensity of 110 dB (p = 0.0013, F1,38 = 11.973). The reduced
PPI values at 120 dB than those at 110 dB may be a ceiling effectcaused by the strong intensity of the startle stimulus.

Figure 9 | Increased anxiety-like behavior in kf-1−/− mice in the light/
dark transition test compared to kf-1+/+ littermates. Mice were placed
individually in the dark compartment at the start of the test. Data are given as
mean (±SEM). (A) Distance traveled (centimeters) in the light and dark sides.
(B) Time (seconds) the mice stayed in the light side. (C) Number of transitions
between the light and dark sides. (D) Latency time (seconds) before the firstentry into the light side. *p < 0.05; ***p < 0.0005 (see Section ‘Light/darktransition test’). WT and KO stand for kf-1+/+ and kf-1−/− mice, respectively.

DISCUSSION
The mouse kf-1 gene is expressed throughout the body, except
in the liver; the highest expression is in the brain (Yasojima
et al., 1997). However, the lack of kf-1 gene did not lead to any
obvious abnormalities in appearance or in behaviors such as
exploratory locomotion, nociception, social behavior, motor
coordination, behavioral despair, spatial working memory, andcontext memory (Table 1; Figures 3–8). The exception to this8
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A novel anxiety/depression animal model

Figure 11 | Reduced startle amplitude and increased prepulse inhibition in kf-1−/− mice. (A) Startle amplitude was significantly reduced in ‘startlestimulus-only’ trials (p = 0.0043). (B) % Prepulse inhibition was significantly increased in kf-1−/− compared to that in kf-1+/+ mice (overall ANOVA including all
four conditions, p = 0.0023). Data are given as mean (±SEM; see Section ‘Startle response/prepulse inhibition test’). WT and KO stand for kf-1+/+ and kf-1−/−
mice, respectively.

is that kf-1−/− mice displayed a pronounced increase in anxietylike behavior in the light/dark transition test (Figures 9A–C).
In addition, ‘timidity-like’ responses were observed understressful situations such as prolonged wire-hanging time
(Table 1), enhanced immediate freezing with an aversive footshock (Figure 8A), and decreased locomotor activity on heights(Figures 10A,B) in kf-1−/− mice. Since no significant differenceswere observed between kf-1+/+ and kf-1−/− mice in body weightand grip strength (Table 1), and in exploratory locomotion
using the open field test (Figure 3A), these behaviors should
be rooted in psychological, but not physical, alteration inkf-1−/− mice. These responses are consistent with the increased
anxiety-like behavior observed in the light/dark transition test.
However, there was no significant difference regarding the staytime index on open arms between kf-1+/+ and kf-1−/− mice in theelevated plus maze test (Figures 10C,D). How can we explain
this apparent discrepancy (Figures 9A–C and Figures 10A,B
vs. Figures 10C,D)? In the laboratory of T.M., a comprehensive behavioral test battery has been conducted with identical
protocols on >70 different KO and transgenic mice in the past
5 years (Takao et al., 2007). Although there were several strainsof mutant mice that showed increased anxiety-like behavior,kf-1−/− mice were the second greatest mice among them in termsof the size of genotype effect on ‘stay-time in light’ index of
the light/dark transition test {δ = −1.058, where δ = [(averageof mutant stay-time in light) − (average of wild type stay-timein light)]/(total SD)} (Takao and Miyakawa, data not shown).
Considering the large genotype effect observed in the light/darktransition test (Figures 9A–C), we believe that this apparent
discrepancy is due to a floor effect in the elevated plus mazetest or to the difference of the kind of anxiety-like behaviors
that were assessed by these two tests. By a factor analysis using
our large set of data using >5,000 mice, we are aware that these
two tests assesses different aspects of anxiety-like behavior,although there are significant correlations between indices inthese tests (Yamasaki et al., 2006). There are also published data
indicating that different anxiety tests measure different aspectsof anxiety-like behaviors (Holmes et al., 2003a; Ramos andMormède, 1998; Turri et al., 2001), demonstrating the fact thatone but not the other assay could provide evidence of alteredanxiety-like behavior. To distinguish different anxiety-related
behavioral dimensions, kf-1−/− mice would have to be tested
further by another anxiety-related behavioral tests including
elevated plus maze with opaque walls, square maze, and mirror
chamber.
Considering the fact that the level of kf-1 gene expression
is augmented in the frontal cortex and hippocampus, but notin the hypothalamus, after chronic antidepressant treatments(Kudo et al., 2005; Nishioka et al., 2003; Yamada et al., 2000),
these results suggest that kf-1 gene may play a role(s) in theemotional regulation such as the suppression of anxiety or
fear under stressful conditions. In this context, it is worth tonote that roles of prefrontal cortex and hippocampus in modulating amygdala activity have been reported to be responsible for adaptive emotional regulation of context-specificretrieval of anxiety or fear extinction (Corcoran et al., 2005;
Sotres-Bayon et al., 2006). Therefore, our observations suggesting a possible ‘antianxiety’-like effect of KF-1 seem to beconsistent with all these reports. Considering that KF-1 is an
E3 ubiquitin ligase to modulate cellular protein level (Lorick
et al., 1999), it appears to be crucial to identify what is (are)the target protein(s) degraded depending on KF-1 ubiquitination in the future studies. Cellular concentration of thisputative target protein(s) may have played an important role
during the vertebral evolution to determine the anxiety level
to defy or evade potential dangers in various environmental
circumstances.
The kf-1 gene expression is up-regulated in the frontal cortex
of rats treated by chronic SSRI administration (Yamada et al.,2000). It may be possible that the effect of KF-1 depletion on
anxiety-like behavior could be associated with the dysregulationof serotonergic system in these mutant mice. Increased anxietylike behavior has been reported in mice lacking serotonin-related
genes. For example, mice (C57BL/6J) lacking the serotoninreceptor gene (5-HT1A) displayed increased ‘anxiety’ levels in the
open-field, elevated zero maze, and novel-object tests (Heisler
et al., 1998). Similarly, mice lacking the gene (5-HTT) for serotonin transporter were shown to have increased anxiety-likebehavior in the light/dark transition, elevated plus maze, andopen-field tests on the C57BL/6J background (Holmes et al.,9

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Tsujimura et al.

2003b). It has been pointed out that the absence of the 5-HT1A
gene during the postnatal developmental period, but not in
adult mice, affects anxiety-like behavior (Gross et al., 2002). It
would be necessary, therefore, to determine in the future studies
whether the behavioral abnormality of kf-1−/− mice is caused bya developmental impairment by KF-1 depletion or its absence
at adulthood.
Concerning the possible association of the kf-1 gene with AD,
no significant effects of genotype were observed on memory or
cognitive function in the present studies (Table 1). Interestingly,kf-1−/− mice exhibited significantly increased PPI values(Figure 11), which are usually reduced in human schizophrenic
patients (Geyer and Ellenbroek, 2003). PPI deficit has beenreported also in mouse models of schizophrenia (Chen et al.,2008; Clapcote et al., 2007; Miyakawa et al., 2003). Taking thesereports into account, agents that can inhibit the expression orfunction of the kf-1 gene, if any, could possibly ameliorate sensorimotor-gating deficits that are seen in patients of schizophrenia and other psychiatric disorders. Finally, it will be interesting
to see whether or not the G/A transition in front of the initiation
codon of the human kf-1 gene influences the mRNA translation efficiency. In conclusion, the kf-1−/− mice and cells derived
thereof may be useful for elucidating the molecular mechanismof anxiety/depression, and furthermore, the kf-1−/− mice may
provide a useful tool for finding novel anxiolytic/antidepressantcompounds, which may compensate KF-1 by antagonizing its
target protein(s).

CONFLICT OF INTEREST STATEMENT
The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could
be construed as a potential conflict of interest.

ACKNOWLEDGEMENTS
We thank M. Nakagawa for encouragement during this work; J.
Miyazaki for providing CAG-Cre mice; T. Matsuo and K. Itohfor help with immunohistochemistry. T. Hashimoto-Gotoh
thanks T. Mizuno, K. Hayashi, and C. Gross for valuable discussions and comments. This work was supported by a Grant-inAid for Geriatric Research Joint-Projects from Kyoto-fu, and inpart by a Grant-in-Aid for Scientific Research on Priority Areas,
Integrative Brain Research (Shien) from MEXT in Japan, and by
a Grant-in-Aid from the Institute for Bioinformatics Researchand Development (BIRD) at the Japan Science and Technology
Agency.

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