Human Molecular Genetics, 2011, Vol. 20, No.

23 4666–4683
doi:10.1093/hmg/ddr400
Advance Access published on September 8, 2011

Behavioral alterations associated with targeted

disruption of exons 2 and 3 of the Disc1 gene
in the mouse
Keisuke Kuroda 1,5,{, Shinnosuke Yamada 2,5,{, Motoki Tanaka 3,{, Michiro Iizuka 1,6, Hisashi Yano 1,
Daisuke Mori 1,5, Daisuke Tsuboi 1,5, Tomoki Nishioka 1,5, Takashi Namba 1,5, Yukihiko Iizuka 1,5,
Shimpei Kubota 1, Taku Nagai 2,5, Daisuke Ibi 2,5, Rui Wang 2,5, Atsushi Enomoto 4,

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Mayu Isotani-Sakakibara 4, Naoya Asai 4, Kazushi Kimura 6, Hiroshi Kiyonari 7, Takaya Abe 7,
Akira Mizoguchi 6, Masahiro Sokabe 3,8, Masahide Takahashi 4, Kiyofumi Yamada 2,5
and Kozo Kaibuchi 1,5,∗
1
Department of Cell Pharmacology, 2Department of Neuropsychopharmacology and Hospital Pharmacy, 3Department
of Physiology and 4Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya,
Aichi 466-8550, Japan, 5JST, CREST, Kawaguchi, Saitama 322-0012, Japan, 6Department of Neural Regeneration
and Cell Communication, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan, 7Laboratory for
Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047,
Japan and 8FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya, Aichi 466-8550,
Japan

Received June 27, 2011; Revised and Accepted August 31, 2011

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric

disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration,
axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein
by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized
antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we gen-
erated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-ter-
mini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as
disorganization of the brain’s cytoarchitecture, were observed. Western blot analysis revealed that the
DISC1-specific antibodies recognize a protein with an apparent molecular mass of ∼100 kDa in brain
extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies
demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons
and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term
potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as
assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of
full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest
that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the
pathophysiological functions of DISC1.

∗
To whom correspondence should be addressed at: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai,
Showa, Nagoya, Aichi 466-8550, Japan. Tel: +81 527442075; Fax: +81 527442083; Email: kaibuchi@med.nagoya-u.ac.jp
†
These three authors contributed equally to this work.

# The Author 2011. Published by Oxford University Press. All rights reserved.
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INTRODUCTION
Although the etiological mechanisms of psychiatric disorders,
such as schizophrenia, remain largely esoteric, multiple
hypotheses (e.g. dysfunction of dopamine, glutamate or sero-
tonin, neurodevelopmental disorders, stress during pregnancy
and viral infection) have been proposed (1,2). The dissection of
molecular pathways centered on promising risk factors, such as
Disrupted-In-Schizophrenia 1 (DISC1), may help us achieve a
better understanding of the pathogenesis of these disorders.
The DISC1 gene locus was originally identified at the breakpoint
of a balanced chromosomal translocation t(1;11)(q42.1;q14.3)
that co-segregated with schizophrenia, bipolar disorder and re-
current major depression in a large Scottish family (3,4).
Further analysis has revealed that the inheritance of the transloca-
tion is causal and increases the risk of these psychiatric disorders
50-fold (5). Genetic association studies of several ethnic groups
indicate that DISC1 is a major risk factor for psychiatric disor-
ders, including schizophrenia and mood disorders (6). In add-
ition, clinical studies have shown that genetic variations in
DISC1 influence brain function and anatomy (7).
Several groups have isolated DISC1-interacting proteins.
These proteins include nuclear distribution gene E homolog-like
1 (NDEL1), lissencephaly-1 (LIS1), fasciculation and elong-
ation protein zeta 1 (FEZ1), phosphodiesterase 4B (PDE4B),
kinesin family member 5B (KIF5B), growth factor receptor-
bound protein 2 (GRB2), glycogen synthase kinase 3b
(GSK3b), girdin, kalirin, TRAF2 and NCK-interacting kinase
(TNIK) and DIX domain-containing 1 (DIXDC1) (8 – 19).
These studies suggest that DISC1 is involved in neurogenesis,
neuronal migration, axon/dendrite formation and synapse
formation through interactions with the abovementioned pro-
teins (20). DISC1 is thus considered to be a scaffold protein
that regulates specific molecular pathways (21).
To aid our understanding of the physiological function of
DISC1, mouse models with various alterations in DISC1
have been developed. These models include mice with mis-
sense mutations (22), overexpression of truncated forms of
DISC1 (23 – 26) and mice that have a 25 bp deletion in exon
6 of the Disc1 gene [hereafter termed Disc1 (D6) mice]
(27,28). Anatomical and behavioral analyses of these mice
have revealed abnormal phenotypes, such as schizophrenia
and/or depression-related phenotypes (21). However, the
pathophysiological and behavioral functions of DISC1 are
controversial, and possible unknown splice variants of Disc1
hinder efforts to resolve this problem (29). To further under-
stand the physiological role of DISC1 and its relationship to
psychiatric disorders, Disc1 knockout mice and a well-
characterized antibody to DISC1 are required.
In this study, we generated mice lacking exons 2 and 3 of
the Disc1 gene by homologous recombination in embryonic
stem (ES) cells. These mice are hereafter to be termed Disc1
(D2-3) mice. We also made new antibodies against DISC1
for use in evaluating the Disc1 (D2-3) mice and analyzing
DISC1 function. Western blot analysis revealed that the
DISC1 antibodies recognize a polypeptide with a molecular
mass of 100 kDa in wild-type mice but not in the Disc1
(D2-3) mice. Immunochemical analysis revealed that these
DISC1 antibodies recognized endogenous DISC1 that was
mainly localized to the vicinity of the Golgi apparatus in
Human Molecular Genetics, 2011, Vol. 20, No. 23 4667
hippocampal neurons and astrocytes. Gross anatomical abnor-
malities were not evident in the Disc1 (D2-3) mice, but elec-
trophysiological analyses revealed that the threshold for the
induction of long-term potentiation (LTP) was significantly
higher than in normal mice. Additionally, behavioral tests
revealed a phenotype characterized by gender-independent
higher impulsivity as well as gender-dependent deficits of pre-
pulse inhibition (PPI) and increased responsiveness to meth-
amphetamine (METH).
RESULTS
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Generation of mice lacking exons 2 and 3 of Disc1
Although many splice variants of Disc1 at the protein and mRNA
levels have been reported (3,15,30–32), the major form of DISC1
appears to have a molecular mass of 100 kDa, which corre-
sponds to the size of the mouse full-length Disc1 mRNA.
Because each of the putative isoforms contain exons 2 and 3
(30), exons 2 and 3 of Disc1 were replaced with a loxP/
PGK-Neo-pA/loxP cassette, and an in-frame stop codon was
inserted between the remaining exon 2 and the loxP site in
mouse TT2 ES cells from an F1 embryo between a C57BL/6
and CBA cross and HK3i ES cells from a C57BL/6N embryo
(Fig. 1A and B; http://www.cdb.riken.jp/arg/ES_cells.html).
Many proteins, including PDE4B, KIF5B, GSK3b, girdin and
TNIK, associate with DISC1 through the region encoded by
exons 2 and 3 (8,10,12,13,17). Thus, the function of DISC1 as a
scaffold protein that interacts with these proteins is lost in mice
lacking exons 2 and 3 of Disc1. Homologous recombination
was confirmed by southern blot analysis (Fig. 1C) and genomic
polymerase chain reaction (PCR) (Fig. 1D). Mice heterozygous
for the Disc1 mutation (hereafter termed Disc1 +/D2-3 mice)
were backcrossed onto the C57BL/6JJmsSlc background for at
least five generations to generate Disc1 +/D2-3 mice with a pre-
dominantly C57BL/6J genetic background (100/100 marker at
N5). The Disc1 +/D2-3 mice appeared normal and were healthy
and fertile. Mice homozygous for the Disc1 mutation (hereafter
termed Disc1 D2-3/D2-3 mice) were produced at the expected Men-
delian ratio by intercrossing Disc1 +/D2-3 mice. The Disc1 D2-3/D2-3
mice were also viable and fertile and showed no obvious differ-
ences in physical characteristics from the Disc1 +/D2-3 mice.
Reverse transcription (RT)–PCR analysis revealed that the
mRNA derived from exons 2 and 3 of Disc1 were absent in the
Disc1 D2-3/D2-3 mice, whereas exons 7–10 of Disc1 were detected
in both wild-type (hereafter termed Disc1 +/+ ) and Disc1 D2-3/D2-3
mice (Fig. 1B and E). Because both commercial and previously
generated antibodies to DISC1 showed high background levels
when used for western blot analysis, two different polyclonal anti-
bodies were developed (Fig. 1B). The N-terminal antibody
detected two major protein bands with molecular masses of
100 and 45 kDa as assessed by western blot analysis (Fig. 1F
and Supplementary Material, Fig. S1A). The 100 kDa band was
absent in the Disc1 D2-3/D2-3 mouse brain lysate but not in the
Disc1 +/+ mouse brain lysate. In contrast, the 45 kDa band was
present in lysates from both Disc1 +/+ and Disc1 D2-3/D2-3 mice.
This result indicated that the 45 kDa band may represent a non-
specific signal. The C-terminal antibody detected a protein
band with a molecular mass of 100 kDa. Similarly, the
100 kDa band was absent in the Disc1 D2-3/D2-3 mouse brain
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Figure 1. Targeted disruption of Disc1. (A) Summary of the strategy to target the Disc1 locus. The Neo cassette was inserted by homologous recombination,
replacing the region spanning exons 2– 3 of Disc1. Arrows indicate primers used for PCR-based genotyping; asterisk indicates stop codon; the probe used for
southern blot analysis is shown as a bar. (B) Schematic representation of the mouse Disc1 transcript. The gray portion indicates a deleted region. Regions targeted
by RT –PCR and anti-DISC1 antibodies are shown as bars. (C) Southern blot analysis of tail genomic DNA from wild-type (Disc1 +/+ ; +/+), heterozygous
mutant (Disc1 +/D2-3; +/D) and homozygous mutant (Disc1 D2-3/D2-3; D/D) mice. Genomic DNA was digested with ApaI and subjected to hybridization with
the probe. DNA fragments of 14.5 and 24 kbp correspond to the wild-type and the Disc1 (D2-3) mutant alleles, respectively. (D) PCR genotyping of the
Disc1 (D2-3) mice. PCR products of 547 and 619 bp correspond to the wild-type and the Disc1 (D2-3) mutant alleles, respectively. (E) RT– PCR analysis
of the Disc1 (D2-3) mice. Total RNA prepared from P7 brain tissue was reverse transcribed and used for PCR amplification with primers specific for exons
2– 3 and exons 7 –10 of Disc1 and a housekeeping gene (Hprt). An amplification product for exons 2– 3 was not observed in the Disc1 D2-3/D2-3 mice.
However, exons 7– 10 were detected in both Disc1 +/+ and Disc1 D2-3/D2-3 mice. (F and G) Western blot analysis of the Disc1 (D2-3) mice. P7 brain lysates
were blotted with antibodies specific for the DISC1 N-terminal (F) and C-terminal (G) regions. The arrowhead indicates the position of full-length DISC1.
A 45 kDa protein band observed with the N-terminal antibody may be a nonspecific signal.

lysate but present in the Disc1 +/+ mouse brain lysate (Fig. 1G and
Supplementary Material, Fig. S1B). To further examine whether
the antibody recognized DISC1, DISC1 was immunoprecipitated
from mouse brain extracts using both the N-terminal and C-
terminal antibodies and the peptides derived from exons 5 and
6 of Disc1 were detected in the immunoprecipitate by the liquid
chromatography tandem mass spectrometry analysis. Taken to-
gether, these results indicate that the Disc1 D2-3/D2-3 mice do not
express DISC1 isoforms containing exons 2 and 3. However,
we cannot dismiss the possibility that truncated forms of DISC1
lacking exons 2 and 3 were produced from unidentified start
sites in Disc1 mRNA.
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Figure 2. No apparent morphological defects during brain development in the Disc1 D2-3/D2-3 mice. (A) Gross overview of brain tissue from P56 Disc1 +/+ and
Disc1 D2-3/D2-3 mice showed no apparent decrease in brain volume in the Disc1 D2-3/D2-3 mice. (B and C) Nissl-stained brain sections of P56 Disc1 +/+ and
Disc1 D2-3/D2-3 mice showed no deficit in the development of the prefrontal cortex (B), the neocortex, the ganglia and the hippocampus (C) in the Disc1 D2-3/
D2-3
mice. The regions within the boxes are shown at a higher magnification in adjacent panels. CC, corpus callosum; AC, anterior commissure; PC, posterior
commissure; CP, caudate putamen; TH, thalamus; DG, dentate gyrus. Asterisks indicate the pia mater.

Morphological defects in brain development were Evaluation of commercially available anti-DISC1

not observed in the Disc1 D2-3/D2-3 mice antibodies

Despite the results of the western blot analysis, which
showed abundant expression of DISC1 in the brain during
early development (Supplementary Material, Fig. S3B and
C), Disc1 D2-3/D2-3 embryos appeared normal upon examin-
ation (data not shown), indicating that DISC1 is probably
dispensable for embryogenesis. The Disc1 D2-3/D2-3 mice
develop and reproduce normally with no apparent morpho-
logical effects in the examined organs, including the brain,
through postnatal (P) day 56, which may reflect the ability
of other proteins to compensate for the absence of DISC1.
No appreciable abnormalities in the volume of the cerebrum,
the cerebellum or the olfactory bulb were observed in the
brains of the Disc1 D2-3/D2-3 mice in comparison to their
Disc1 +/+ littermates during the postnatal period or in adult-
hood (Fig. 2A). An examination of brain structure using
Nissl staining revealed a nearly normal cytoarchitecture of
the neocortex, the basal ganglia and the hippocampus in
the Disc1 D2-3/D2-3 mice at P56 (Fig. 2B and C). Interestingly,
the Disc1 D2-3/D2-3 mice exhibited no reduction in the size of
the white matter tracts, such as the corpus callosum and an-
terior and posterior commissures (Fig. 2C). In addition, the
volume of the prefrontal cortex (Fig. 2C), which is a struc-
ture that has been implicated as a locus of dysfunction in
schizophrenia (33,34), did not appear to be reduced.
Several DISC1 antibodies are commercially available. During
the course of generating Disc1 (D2-3) mice, we noticed that
none of the commercial antibodies recognized the 100 kDa
protein band that was absent in the Disc1 D2-3/D2-3 mice (Sup-
plementary Material, Fig. S1C). Of note, one antibody strong-
ly reacted with a protein band with a molecular mass of
100 kDa. However, this immunoreactivity was not lost in
brain extracts from the Disc1 D2-3/D2-3 mice, although the
antigen recognized by this antibody was included in exon
2. Because the expression level of endogenous DISC1 is
low, even in the brain (Supplementary Material, Fig. S3B
and C), it was difficult to detect endogenous DISC1 using
the commercially available antibodies under our conditions.
Mouse strains defective in DISC1 expression
Gogos and associates identified a 25 bp deletion in exon 6 of
Disc1 in the 129 strains of mice [Disc1 (D6) mice] (27,35).
These researchers claimed that this mutation interfered with
the production of full-length DISC1 (27,28). We determined
whether full-length DISC1 was expressed in brain extracts
from 129X1/SvJJmsSlc mice using our C-terminal antibodies
(these mice, which are homozygous for the deletion of the
allele, are hereafter termed Disc1 D6/D6 mice). The 100 kDa
4670 Human Molecular Genetics, 2011, Vol. 20, No. 23
protein band was not detected in the extract from Disc1 D6/D6
mice using western blot analysis using the exact conditions
under which our antibody recognized DISC1 in extracts from
wild-type C57BL/6J mice (Disc1 +/+ ) (Supplementary Mater-
ial, Fig. S2), thereby indicating that the 129 strains do not
express full-length DISC1. Furthermore, we examined
whether other outbred mice, such as ddY and ICR, harbor the
same deletion as the 129 strains. Genomic PCR showed that
77.5% of the ICR mice examined had homozygous deletion
alleles (Disc1 D6/D6), 22.5% of the ICR mice had one
non-deletion allele and one deletion allele (Disc1 +/D6) and
none of the ICR mice had homozygous wild-type alleles
(Disc1 +/+ ). The C-terminal antibody recognized the 100 kDa
band in the Disc1 +/+ mice but not in the Disc1 D6/D6 mice of
the ddY and ICR strains (Supplementary Material, Fig. S2).
Spatiotemporal expression pattern of DISC1 in mice
In situ hybridization and northern blot analysis have shown
that Disc1 mRNA is chiefly expressed in the mouse cerebrum,
cerebellum and heart during development (3,14,36– 39). To
obtain an overview of the spatiotemporal expression pattern
of the DISC1 protein in mice, a western blot analysis was per-
formed using the C-terminal antibody. Expression of DISC1
was detected in various tissues of prenatal mice (Supplemen-
tary Material, Fig. S3A). DISC1 was highly expressed in the
cerebellum and the lung and was moderately expressed in
the cerebrum, the brainstem, the heart and the kidney. The de-
velopmental profile of DISC1 expression in the cerebrum and
the cerebellum was further analyzed (Supplementary Material,
Fig. S3B and C). The expression of DISC1 peaked during the
postnatal period and gradually declined thereafter. The amount
of DISC1 protein in the cerebrum and the cerebellum was
roughly calculated by quantitative western blot analysis
using the recombinant DISC1 protein as a standard. The
amount of DISC1 in the whole cerebrum and the whole cere-
bellum was estimated as 0.0002% and 0.0003% of the total
protein, respectively, suggesting that DISC1 is a
low-abundance protein.
Immunochemical analysis of DISC1 in hippocampal
neurons and astrocytes
To further examine whether our antibodies recognized en-
dogenous DISC1 in vivo and in vitro, immunofluorescent
staining was performed with the C-terminal antibody in
brain sections from P2 mice and in cultured hippocampal
neurons. In brain sections, DISC1 immunoreactivity was loca-
lized in the pyramidal cell layer of the hippocampal CA1 and
CA3 regions (Fig. 3A and B), which was consistent with
the results of previous studies using in situ hybridization
(14,36 –39). Immunostaining with the DISC1 antibody and
organelle-specific antibodies revealed that DISC1 immunor-
eactivity was mainly localized to the vicinity of the Golgi
apparatus (Fig. 3C and D). The immunoreactivity of DISC1
was diminished in the Disc1 D2-3/D2-3 mice (Fig. 3B and D).
To analyze the subcellular distribution of DISC1 further,
immunoelectron microscopic analysis was performed in hip-
pocampal CA1 pyramidal cells stained with the C-terminal
antibody. DISC1 immunoreactivity was detected in the
vicinity of the Golgi apparatus at the base of the apical den-
drites (Fig. 3E – G).
In cultured hippocampal neurons and astrocytes, DISC1 was
mainly localized to the vicinity of the Golgi apparatus
(Fig. 4A – D). In the immunoelectron microscopic analysis of
hippocampal neurons labeled with the C-terminal antibody,
the DISC1 immunoreactivity was associated with the mem-
brane structures of the Golgi apparatus (Fig. 4E).
Electrophysiological analyses in the Disc1 D2-3/D2-3 mice
Because Disc1 mRNA is highly expressed in the dentate gyrus
of the hippocampus (14,36– 39), the effects of the disruption
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of exons 2 and 3 of the Disc1 gene were investigated on the
synaptic properties of neurons in the dentate gyrus in mouse
hippocampal slices using an extracellular recording technique.
First, the strength of basal synaptic transmission was com-
pared at medial perforant path-dentate granule cell synapses
between Disc1 +/+ and Disc1 D2-3/D2-3 mice. Basal synaptic
transmission was estimated by measuring the amplitude of a
presynaptic fiber volley (PSFV) and the initial slope of the
field excitatory postsynaptic potentials (fEPSPs) over a range
of stimulus intensities. As seen in Supplementary Material,
Figure S4A, the stimulus – PSFV relationships in both
Disc1 +/+ and Disc1 D2-3/D2-3 mice were almost linear, and
the PSFV amplitude in both groups was nearly identical at
every stimulus intensity that was tested [two-way analysis of
variance (ANOVA), F(1,126) ¼ 0.59, P . 0.1, n ¼ 10). In
the stimulus-fEPSP relationships, the mean value of the
fEPSP slope in the Disc1 D2-3/D2-3 mice was smaller than in
the Disc1 +/+ mice over the stimulus range, but the difference
was not significant [two-way ANOVA, F(1,126) ¼ 1.46,
P . 0.1, n ¼ 10, Supplementary Material, Fig. S4B]. The
mean value of the fEPSP slope evoked by test stimuli
(50% of the maximum response) was 20.51 + 0.14 mV/ms
in the Disc1 +/+ mice (n ¼ 10) and 20.48 + 0.12 mV/ms in
the Disc1 D2-3/D2-3 mice (n ¼ 10).
The synaptic responses to weaker high-frequency stimula-
tion (HFS, 50 Hz/100 pulses), standard HFS (100 Hz/100
pulses) and stronger HFS (200 Hz/100 pulses) were subse-
quently examined in the Disc1 +/+ and Disc1 D2-3/D2-3 mice.
In hippocampal slices from the Disc1 +/+ mice, weaker
HFS resulted in an enhancement of the fEPSP slope that per-
sisted for over 60 min, which was indicative of LTP
(123.3 + 2.0% of baseline 60 min after weaker HFS, n ¼ 4,
P , 0.001, paired t-test, Fig. 5A and D). However, in hippo-
campal slices from the Disc1 D2-3/D2-3 mice, the same proto-
col did not induce LTP but instead produced a phenomenon
similar to short-term depression (109.7 + 8.1% of baseline
after weaker HFS, n ¼ 5, P ¼ 0.056, paired t-test, Fig. 5A
and D). In contrast, standard HFS-induced LTP in slices
from the Disc1 +/+ and Disc1 D2-3/D2-3 mice with a signifi-
cantly smaller magnitude of LTP in the Disc1 D2-3/D2-3
mice at 60 min after standard HFS (Disc1 +/+ , 130.8 +
6.1% of baseline 60 min after standard HFS, n ¼ 4, P ¼
0.002, paired t-test; Disc1 D2-3/D2-3, 117.7 + 7.0% of baseline
60 min after standard HFS, n ¼ 5, P ¼ 0.005, paired t-test,
Fig. 5B and D). Notably, the form and magnitude of the
standard HFS-induced LTP in the Disc1 D2-3/D2-3 mice were
similar to those of the weaker HFS-induced LTP in the
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Figure 3. Immunostaining of endogenous DISC1 in vivo. (A– D) Immunofluorescent analysis of DISC1 was performed with anti-DISC1 C-ter antibody (green)
in P2 mouse hippocampus prepared from Disc1 +/+ (A and C) and Disc1 D2-3/D2-3 (B and D) mice. The Golgi apparatus and nuclei were stained with anti-GM130
antibody (red) and Hoechst 33342 (blue), respectively. (A and B) DISC1 immunoreactivity was detected in pyramidal cell layer of the hippocampal CA1 and
CA3 region. (C and D) The boxed regions in (A) and (B) were shown at higher magnification. DISC1 immunoreactivity was mainly localized to the vicinity of
the Golgi apparatus in the hippocampal CA1 pyramidal cells. (E–G) Immunoelectron microscopic analysis of DISC1 was performed with anti-DISC1 C-ter
antibody in CA1 pyramidal cells of P2 mouse hippocampus prepared from the Disc1 +/+ mice. Golgi apparatus (arrow) was composed of stacks of membrane-
bound structures known as cisterna and localized at the base of apical dendrites (AD). DISC1 immunoreactivity (arrowhead) was detected in the vicinity of the
Golgi apparatus near the nucleus (N). The scale bar represents 100 mm (A and B), 10 mm (C and D), 3 mm (E), 500 nm (F) and 100 nm (G).

Disc1 +/+ mice. When a stronger HFS was used to induce HFS, n ¼ 5, P , 0.001, paired t-test; Disc1 D2-3/D2-3, 134.9 +
LTP, the magnitude of the LTP was significantly larger in 4.0% of baseline 60 min after a stronger HFS, n ¼ 4, P ,
the Disc1 D2-3/D2-3 mice than in the Disc1 +/+ mice 0.001, paired t-test, Fig. 5C and D). However, the magnitude
(Disc1 +/+ , 121.2 + 5.3% of baseline 60 min after a stronger of the stronger HFS-induced LTP in the Disc1 D2-3/D2-3 mice
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Figure 4. Immunostaining of endogenous DISC1 in vitro. (A– D) Immunofluorescent analysis of DISC1 was performed with anti-DISC1 C-ter antibody (green)
in cultured hippocampal neurons at DIV3 (A and B) and astrocytes at DIV8 (C and D) prepared from the Disc1 +/+ (A and C) and the Disc1 D2-3/D2-3 (B and D)
mice. DISC1 was localized to the vicinity of the Golgi apparatus, which was stained with anti-GM130 antibody (red). (E) Immunoelectron microscopic analysis
of DISC1 was performed with anti-DISC1 C-ter antibody in cultured hippocampal neurons at DIV5. DISC1 immunoreactivity (arrowhead) was detected in the
vicinity of the Golgi apparatus (arrow) near the nucleus (N). The broken lines represent the cell margins. The scale bar represents 5 mm (A– D) and 500 nm (E).

was similar to that of the standard HFS-induced LTP in the
Disc1 +/+ mice. The frequency dependencies of LTP
magnitude in the Disc1 +/+ and Disc1 D2-3/D2-3 mice were
summarized by plotting the normalized fEPSP slope at
55– 60 min after HFS delivery against HFS frequency as
shown in Figure 5D. In the Disc1 D2-3/D2-3 mice, a higher
frequency of HFS was needed to produce an equivalent
LTP magnitude to that induced in the Disc1 +/+ mice.
These results suggest that LTP can be induced in the synap-
ses of Disc1 D2-3/D2-3 mice but that the threshold for LTP in-
duction is increased by the disruption of exons 2 and 3 of
the Disc1 gene.

Figure 5. Synaptic transmission and plasticity in the Disc1 D2-3/D2-3 mice.
(A) Induction of LTP by 100 pulses at 50 Hz in Disc1 +/+ and Disc1 D2-3/
D2-3
mice. LTP was induced in the Disc1 +/+ mice, whereas was not in the
Disc1 D2-3/D2-3 mice. (B) Induction of LTP by 100 pulses at 100 Hz in both
Disc1 +/+ and Disc1 D2-3/D2-3 mice. The magnitude of the LTP in the
Disc1 D2-3/D2-3 mice was smaller than in the Disc1 +/+ mice. (C) Induction
of LTP by 100 pulses at 200 Hz in both Disc1 +/+ and Disc1 D2-3/D2-3 mice.
The magnitude of the LTP in the Disc1 D2-3/D2-3 mice was larger than in the
Disc1 +/+ mice. (D) Summary of the LTP magnitude induced by 100 pulses
at 50, 100 and 200 Hz at 55– 60 min after HFS in both Disc1 +/+ and
Disc1 D2-3/D2-3 mice. In the Disc1 D2-3/D2-3 mice, the higher frequency of HFS
than the Disc1 +/+ mice was needed to produce an equivalent LTP magnitude
to that in the Disc1 +/+ mice. Comparisons between Disc1 +/+ and Disc1 D2-3/
D2-3
mice were made using a two-tailed independent t-test.
Human Molecular Genetics, 2011, Vol. 20, No. 23 4673
Behavioral analyses of the Disc1 D2-3/D2-3 mice
There were no apparent differences between juvenile
Disc1 +/+ and Disc1 D2-3/D2-3 mice (4 –8 weeks old) in most
of the behavioral tests conducted. In the open field test,
there was a marginal but significant increase in the locomotor
distance traveled within the inner circle of the open field in
the female (128 + 11%) but not the male (109 + 16%)
Disc1 D2-3/D2-3 mice compared with the activity of the
Disc1 +/+ mice (Table 1).
Abnormalities in emotional and social behavior were
observed in adult Disc1 D2-3/D2-3 mice (12 – 24 weeks old).
These behaviors were assessed using the elevated plus-maze
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test, the cliff-avoidance test and the social interaction test
(Table 2). In the elevated plus-maze test, open arm time was
significantly increased, whereas closed arm time was signifi-
cantly decreased in the Disc1 D2-3/D2-3 mice compared with
the Disc1 +/+ mice (P , 0.01, Fig. 6A and B). The
Disc1 D2-3/D2-3 mice also exhibited a marked increase in open
and closed arm entries compared with the Disc1 +/+ mice
(P , 0.05, Supplementary Material, Fig. S5A and B). In the
cliff-avoidance test, the number of jumping events during a
10 min observation period was significantly increased in the
Disc1 D2-3/D2-3 mice compared with the Disc1 +/+ mice (P ,
0.05, Fig. 6C). Furthermore, the cumulative frequency of the
first jumping off of the platform during a 3 min observation
period was significantly increased in the female Disc1 D2-3/
D2-3
mice compared with the Disc1 +/+ mice (Supplementary
Material, Fig. S5C – E). In the social interaction test, a slight
but significant increase in social interaction time was evident
in the Disc1 D2-3/D2-3 mice compared with the Disc1 +/+ mice
[F(1,30) ¼ 4.54, P , 0.05] (Fig. 6D and Supplementary
Material, S5F –H). There was no difference in performance
in the open field or forced swim tests between the Disc1 D2-3/
D2-3
mice and the Disc1 +/+ mice (Table 2).
With respect to cognitive function, adult Disc1 D2-3/D2-3
mice, especially females, exhibited an impairment of sensori-
motor gating as indicated by PPI deficits (P , 0.05, Fig. 6E –
G) without changes in the acoustic startle amplitude (Supple-
mentary Material, Fig. S6I). Adult Disc1 D2-3/D2-3 mice were
generally normal with respect to various learning and
memory tests, including the Y-maze test, the novel object rec-
ognition test and the working memory test using the radial arm
maze (Table 2 and Supplementary Material, Fig. S5J – L).
Context-dependent conditioned fear freezing was significantly
enhanced in the Disc1 D2-3/D2-3 mice compared with the
Disc1 +/+ mice (P , 0.05, Fig. 6H), although no significant
difference in tone-dependent freezing time (Fig. 6I) or sensi-
tivity to an electric footshock was observed between the two
groups (0.19 + 0.01 mA in the Disc1 +/+ mice, 0.18 +
0.01 mA in the Disc1 D2-3/D2-3 mice). Latent inhibition of tone-
dependent freezing was also normal in adult Disc1 D2-3/D2-3
mice (Table 2).
Finally, sensitivities to METH, which is an indirect
dopaminergic agonist, and MK-801, a non-competitive N-
methyl-D-aspartate (NMDA) receptor antagonist, were deter-
mined in adult Disc1 D2-3/D2-3 mice. In the Disc1 D2-3/D2-3
mice, especially the females, METH-induced hyperactivity
was significantly potentiated compared with the Disc1 +/+
4674 Human Molecular Genetics, 2011, Vol. 20, No. 23

Table 1. Summary of phenotypes of the Disc1 D2-3/D2-3 mice in juvenile group

Domain of function Test Parameter n Male+ n Male n Female

Female
+/+ D/D +/+ D/D +/+ D/D

Psychosis-related Locomotor Locomotor activity 12 15 ¼ 5 7 ¼ 7 8 ¼

behavior
Affective behavior Open field Total movement 12 15 ¼ 5 7 ¼ 7 8 ¼
Inner movement 12 15 ¼ 5 7 ¼ 7 8  (P ¼ 0.048)
Social interaction Interaction time 12 15 ¼ 5 7 ¼ 7 8 ¼
Cognition/learning and PPI Prepulse (69, 73, 77 18 20 ¼ 8 11 ¼ 10 9 ¼
memory and 81 dB)
Y-maze Alternation 18 20 ¼ 8 11 ¼ 10 9 ¼
Nobel object Exploratory preference 12 15 ¼ 5 7 ¼ 7 8 ¼

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recognition Exploration time 12 15 ¼ 5 7 ¼ 7 8 ¼

n, number of animals; , higher than +/+; ¼, no difference.

mice [F(1,18) ¼ 9.83, P , 0.05] (Fig. 6J – L). There was no
difference between the Disc1 D2-3/D2-3 and Disc1 +/+ mice in
their sensitivity to MK-801 (Table 2).
Effect of clozapine treatment on the Disc1 D2-3/D2-3 mice
Because the behavioral analysis of the Disc1 D2-3/D2-3 mice
demonstrated an apparent impairment in performance in the
elevated plus-maze test, the effect of acute administration of
clozapine [1 mg/kg, intraperitoneally (i.p.)] was analyzed in
the Disc1 D2-3/D2-3 mice. A two-way ANOVA revealed a
significant interaction between drug treatment and genotype
with respect to the time spent in the open arms [Fig. 7A;
drug treatment– genotype interaction F(1,33) ¼ 4.81, P ,
0.05] and the time spent in the closed arms [Fig. 7B; drug
treatment – genotype interaction F(1,33) ¼ 5.84, P , 0.05]. A
multiple-comparison test with Tukey – Kramer post hoc tests
indicated that the increase in the time spent in the open
arms by saline-treated Disc1 D2-3/D2-3 mice was significantly
ameliorated by treatment with clozapine (P , 0.05,
Fig. 7A). Treatment with clozapine also rescued the decrease
in the time spent in the closed arms by the Disc1 D2-3/D2-3 mice
(P , 0.05, Fig. 7B). Clozapine treatment had no effect on the
performance of Disc1 +/+ mice in the elevated plus-maze test
(Fig. 7 and Supplementary Material, S6).
Monoamines and their metabolite levels in the brains
of adult Disc1 D2-3/D2-3 mice
In general, no differences between the Disc1 D2-3/D2-3 mice and
the Disc1 +/+ mice regarding monoamine levels or metabolite
contents were observed in any of the various brain regions that
were examined. In one exception, a slight but significant re-
duction in serotonin content was observed in the cerebellum
of the Disc1 D2-3/D2-3 mice compared with the Disc1 +/+ mice
(Table 3).
DISCUSSION
We generated mice lacking exons 2 and 3 of Disc1 [Disc1
(D2-3) mice] and made specific antibodies to the N- and
C-termini of DISC1. Western blot analysis using either
antibody detected a protein band with a molecular mass of
100 kDa that was absent in brain lysates from the
Disc1 D2-3/D2-3 mice. Mass spectral analysis determined that
the N-terminal and C-terminal antibodies recognize the en-
dogenous mouse DISC1. We also confirmed that the full-
length DISC1 was not expressed in brain extracts of mice
with a 25 bp deletion in exon 6 of Disc1 [Disc1 (D6) mice].
These results indicate that the 100 kDa protein is the major
isoform of DISC1. However, we cannot neglect the possibility
of the existence of other isoforms, although the expression
levels of other isoforms, if present, are much lower than that
of the 100 kDa DISC1.
DISC1 interacts with many proteins, including NDEL1,
LIS1, FEZ1, PDE4B, KIF5B, GRB2, GSK3b, girdin, kalirin,
TNIK and DIXDC1 (8 – 19). A number of these proteins inter-
act with DISC1 through the region encoded by exons 2 and 3
(21). Therefore, the function of DISC1 as a scaffold protein,
which requires interaction with these proteins, should be lost
in Disc1 (D2-3) mice.
To understand the physiological significance of DISC1,
many laboratories, including our own, have knocked down
the expression of DISC1 using small interfering RNA or
short hairpin RNA (8,10– 12,16– 19,40– 47). Although the
acute knockdown of DISC1 impairs neuronal migration,
axon elongation, dendritic maturation and neurogenesis, the
Disc1 D2-3/D2-3 mice showed nearly normal cytoarchitecture
of the neocortex, the basal ganglia and the hippocampus
and no reduction in the size of the white matter tracts or
the volume of the prefrontal cortex. Thus, a detailed analysis
will be required to examine the structural changes that may
occur in the Disc1 D2-3/D2-3 mice. Chronic depletion of
DISC1 may induce molecular compensation during develop-
ment. To overcome this complicating factor, the generation
of a conditional Disc1 knockout mouse is necessary.
Our immunohistochemical studies revealed that DISC1 is
localized to the vicinity of the Golgi apparatus in hippocam-
pal neurons and astrocytes. Previous studies have shown that
both NDEL1 and LIS1 are localized in the Golgi apparatus
(48,49). DISC1 can associate with ARFGEF2, PACS1 and
GM130 (17,50), all of which are involved in vesicle traffick-
ing from the Golgi apparatus. Thus, it is possible that DISC1
localizes to the peripheral region of the Golgi apparatus by
virtue of its association with these proteins. DISC1 may
 Human Molecular Genetics, 2011, Vol. 20, No. 23 4675

Table 2. Summary of phenotypes of the Disc1 D2-3/D2-3 mice in adult group

n Male+Female n Male n Female

Domain of function Test Parameter +/+ D/D +/+ D/D +/+ D/D

Psychosis-related Locomotor Locomotor activity 26 26 ¼ 9 12 ¼ 17 14 ¼

behavior MK-801-induced Locomotor activity 17 19 ¼ 8 8 ¼ 9 11 ¼
hyperactivity
METH-induced Locomotor activity 18 20 ¼ 8 10 ¼ 10 10  (P ¼ 0.0057)
hyperactivity
Affective behavior Open field Total movement 14 15 ¼ 7 7 ¼ 7 8 ¼
Inner movement 14 15 ¼ 7 7 ¼ 7 8 ¼
Elevated Open arm entries 16 20  (P ¼ 0.00014) 8 10  (P ¼ 0.0031) 8 10  (P ¼ 0.024)
plus-maze Closed arm entries 16 20  (P ¼ 0.010) 8 10 ¼ 8 10  (P ¼ 0.037)
Time in open arms 16 20  (P ¼ 0.00013) 8 10  (P ¼ 0.0018) 8 10  (P ¼ 0.028)

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Time in closed arms 16 20  (P ¼ 0.00056) 8 10  (P ¼ 0.022) 8 10  (P ¼ 0.015)
Cliff-avoidance Frequency of 26 20  (P ¼ 0.037) 13 11 ¼ 13 9 ¼
jumping
Forced swim Immobility time 14 16 ¼ 8 10 ¼ 6 6 ¼
Social interaction Interaction time 12 20  (P ¼ 0.042) 6 8 ¼ 6 12 ¼
Cognition/learning PPI Prepulse (69, 73, 77 12 16  (P ¼ 0.027) 6 8 ¼ 6 8  (P ¼ 0.022)
and memory and 81 dB)
Latent inhibition Tone-dependent 15 17 ¼ 8 8 ¼ 7 9 ¼
freezing
Y-maze Alternation 14 16 ¼ 7 8 ¼ 7 8 ¼
Radial-maze Working memory 12 17 ¼ 7 12 ¼ 5 5 ¼
errors
Fear conditioning Context-dependent 14 16  (P ¼ 0.042) 7 8 ¼ 7 8 ¼
freezing
Tone-dependent 14 16 ¼ 7 8 ¼ 7 8 ¼
freezing
Novel object Exploratory 14 20 ¼ 7 10 ¼ 7 10 ¼
recognition preference
Exploration time 14 20 ¼ 7 10  (P ¼ 0.037) 7 10 ¼

n, number of animals; , higher than +/+; , lower than +/+; ¼, no difference.

participate in vesicle movement from the Golgi apparatus.
DISC1 is also known to interact with KIF5B, NDELl and
LIS1 and to act as a cargo adapter linking kinesin and
dynein to cargo proteins, such as NDEL1/LIS1 and GRB2
(16,17). Although DISC1 has been reported to be localized
in the mitochondria (46,51,52), we did not detect immunor-
eactivity of DISC1 in association with these organelles.
This discrepancy may be explained by the fact that the pre-
vious studies were performed using a commercial antibody.
However, we cannot dismiss the possibility that under the
conditions used, our antibody did not detect mitochondria-
associated DISC1.
Electrophysiological analyses of the dentate gyrus of
mouse hippocampal slices were conducted to examine the
effect of the disruption of exons 2 and 3 of the Disc1 gene
on synaptic functions. There was no difference in basal syn-
aptic transmission at the medial perforant path-granule cell
synapses in the Disc1 +/+ and Disc1 D2-3/D2-3 mice. In con-
trast, though HFS-induced LTP could be observed at these
synapses in the Disc1 D2-3/D2-3 mice, the threshold for LTP in-
duction was significantly higher than in the Disc1 +/+ mice.
Such a threshold shift in the LTP induction is observed in
metaplasticity, which has been proposed to relate to higher
order brain functions (53). The factors that contribute to the
induction of metaplasticity are diverse. For example, glial-
derived D-serine affects NMDA receptor activity in such a
way that it shifts the threshold for LTP in the rat
hypothalamic supraoptic nucleus (54). Studies of the hippo-
campus have revealed that pregnenolone sulfate, which is
one of the most abundant neurosteroids, induces a modulatory
metaplasticity in an L-type voltage-gated calcium channel-
dependent manner (55) and that brain-derived neurotrophic
factor (BDNF) is likely to alter the capacity for plastic
changes in synaptic efficacy (56). Interestingly, it has been
reported that serum levels of D-serine, neurosteroids and
BDNF are altered in schizophrenia (57– 62). The relationship
between these factors and DISC1 function represents an intri-
guing subject for study that may lead to an understanding of
the mechanisms through which DISC1 affects synaptic trans-
mission and higher order brain functions.
Adult, but not juvenile, Disc1 D2-3/D2-3 mice displayed
abnormal emotional behavior as assessed by the elevated
plus-maze test and the cliff-avoidance test. This observation
suggests that disruption of exons 2 and 3 of the Disc1 gene
may cause behavioral effects that are dependent upon neurode-
velopment and that result in lower anxiety and/or higher
impulsivity in adult mice. The Disc1 D2-3/D2-3 mice also exhib-
ited increased social interaction with an unfamiliar intruder
mouse during the social interaction test and increased explor-
ation time of novel objects in the novel object recognition test;
both of these behaviors may be associated with lower anxiety/
higher impulsivity. The higher impulsivity observed in the
Disc1 D2-3/D2-3 mice is consistent with the clinical features of
patients with schizophrenia (63,64).
4676 Human Molecular Genetics, 2011, Vol. 20, No. 23

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Figure 6. Behavioral analyses of the Disc1 D2-3/D2-3 mice. (A and B) Performance of adult Disc1 D2-3/D2-3 mice in the elevated plus-maze test. Time spent in the
open and closed arms as characterized by gender. Values indicate the mean + SE [Disc1 +/+ (+/+) (n ¼ 16) and Disc1 D2-3/D2-3 (D/D) (n ¼ 20) in female +
male, Disc1 +/+ (n ¼ 8) and Disc1 D2-3/D2-3 (n ¼ 10) in female, Disc1 +/+ (n ¼ 8) and Disc1 D2-3/D2-3 (n ¼ 10) in male]. (C) Performance of adult Disc1 D2-3/
D2-3
mice in the cliff-avoidance test. Number of jumping events during a 10 min observation categorized by gender. Values indicate the mean + SE
[Disc1 +/+ (n ¼ 26) and Disc1 D2-3/D2-3 (n ¼ 20) in female + male, Disc1 +/+ (n ¼ 13) and Disc1 D2-3/D2-3 (n ¼ 9) in female, Disc1 +/+ (n ¼ 13) and
Disc1 D2-3/D2-3 (n ¼ 11) in male]. (D) Performance of adult DISC1 Disc1 D2-3/D2-3 mice in the social interaction test. Accumulated time of social interaction cate-
gorized by gender over five trials. Values indicate the mean + SE [Disc1 +/+ (n ¼ 12) and Disc1 D2-3/D2-3 (n ¼ 20) in female + male, Disc1 +/+ (n ¼ 6) and
Disc1 D2-3/D2-3 (n ¼ 12) in female, Disc1 +/+ (n ¼ 6) and Disc1 D2-3/D2-3 (n ¼ 8) in male]. (E– G) Performance of adult Disc1 D2-3/D2-3 mice in the PPI test.
PPI (%) at four different prepulse intensities (69, 73, 77 and 81 dB) categorized by gender. Values indicate the mean + SE [Disc1 +/+ (n ¼ 12) and
Disc1 D2-3/D2-3 (n ¼ 16) in female + male, Disc1 +/+ (n ¼ 6) and Disc1 D2-3/D2-3 (n ¼ 8) in female, Disc1 +/+ (n ¼ 6) and Disc1 D2-3/D2-3 (n ¼ 8) in male].
(H and I) Performance of adult DISC1 Disc1 D2-3/D2-3 mice in the fear conditioning test. Context- and tone-dependent memory during the fear conditioning
test categorized by gender. Values indicate the mean + SE [Disc1 +/+ (n ¼ 14) and Disc1 D2-3/D2-3 (n ¼ 16) in female + male, Disc1 +/+ (n ¼ 7) and
Disc1 D2-3/D2-3 (n ¼ 8) in female, Disc1 +/+ (n ¼ 7) and Disc1 D2-3/D2-3 (n ¼ 8) in male]. (J–L) Performance of adult Disc1 D2-3/D2-3 mice in the METH-induced
hyperactivity test. Locomotor activity was measured for 180 min immediately after METH treatment categorized by gender. Single beam breaks were summar-
ized as ‘counts’. Data are shown as 5 min increments. Values indicate the mean + SE [Disc1 +/+ (n ¼ 18) and Disc1 D2-3/D2-3 (n ¼ 20) in female + male,
Disc1 +/+ (n ¼ 10) and Disc1 D2-3/D2-3 (n ¼ 10) in female, Disc1 +/+ (n ¼ 8) and Disc1 D2-3/D2-3 (n ¼ 10) in male]. ∗ P , 0.05, ∗∗ P , 0.01 versus Disc1 +/+ mice.

Clozapine is an atypical antipsychotic that has been shown
to attenuate a broad range of symptoms in subjects with
schizophrenia (65). In our analysis of the effects of acute ad-
ministration of clozapine to Disc1 D2-3/D2-3 mice on perform-
ance in the elevated plus-maze test, clozapine-treated
Disc1 D2-3/D2-3 mice no longer exhibited deficits in the time
spent in the open arms and closed arms, which indicated
that the emotional deficits in these mice, such as lower
anxiety and/or higher impulsivity, were attenuated by
treatment with clozapine. These results suggest that deficiency
of DISC1 function can be rescued by treatment with anti-
psychotic drugs; this finding may reflect the therapeutic
effects of such drugs on psychotic symptoms in patients
with schizophrenia.
Interestingly, the Disc1 D2-3/D2-3 female mice exhibited more
severe deficits in some behavioral tests, such as the PPI test,
the cliff-avoidance test and the METH-induced hyperactivity
test than did the male Disc1 D2-3/D2-3 mice. Consistent with the

Figure 7. Effect of clozapine treatment on performance in the elevated
plus-maze test. (A and B) Effect of clozapine treatment on time spent in
open and closed arms. Values indicate the mean + SE [saline-treated Disc1 +/+
(n ¼ 11), saline-treated Disc1 D2-3/D2-3 (n ¼ 10), clozapine-treated Disc1 +/+
(n ¼ 9) and clozapine-treated Disc1 D2-3/D2-3 (n ¼ 7)]. ∗ P , 0.05 versus saline-
treated Disc1 +/+ mice, #P , 0.05 versus saline-treated Disc1 D2-3/D2-3 mice.
observed gender-dependent changes in behavior, our in vivo
dialysis study revealed gender-dependent alterations of
METH-induced dopamine release in the nucleus accumbens
of the Disc1 D2-3/D2-3 mice (data not shown). Although the
reason for this gender difference remains unclear, sex hormones
may play a role. For instance, it has been demonstrated that an
increase in the plasma level of female hormones (e.g. estrogen
and progesterone) impairs sensorimotor gating (66 –68).
In this study, we demonstrated that the Disc1 D2-3/D2-3 mice
showed PPI deficits, which are a well-known phenomenon
observed in patients with schizophrenia and other neuro-
psychiatric disorders (69). Such deficits have been observed
in most genetically engineered mouse models with mutated
DISC1 (Table 4). In contrast, our newly developed
Disc1 D2-3/D2-3 mice displayed the potentiation of context-
dependent fear memory, whereas other memory functions, in-
cluding cue-dependent fear memory and working memory, did
not differ from those of wild-type mice. Therefore, it seems
unlikely that DISC1 plays an indispensable role in learning
and memory in mice.
Previous reports have demonstrated that genetically engi-
neered mouse models with mutated DISC1 show decreased
social interaction and memory impairment (22 – 25). The
present findings are somewhat inconsistent with these
reports; this discrepancy may be due to the presence of differ-
ent amounts and/or functions of DISC1 in different genetically
engineered mice. The present study generated mice with tar-
geted deletions in DISC1; in these mice, intact DISC1 was
not expressed in the brain, while other previously reported
mouse models express intrinsic and/or extrinsic DISC1
(Table 4) (22– 25,27,28,45,70,71).
Altered monoaminergic neurotransmission has been
reported in patients with schizophrenia (72), and these altera-
tions may be associated with behavioral abnormalities (73).
There were few differences in the tissue content of monoamines
Human Molecular Genetics, 2011, Vol. 20, No. 23 4677
and their metabolites in the brain tissue of Disc1 D2-3/D2-3 and
wild-type mice. Therefore, it seems reasonable to infer that
the development of monoaminergic neurons is largely unaffect-
ed by the loss of DISC1 in mice. However, because
METH-induced hyperactivity and dopamine release in the
nucleus accumbens were potentiated in the Disc1 D2-3/D2-3
mice, further studies that examine potential alterations in dopa-
minergic neurotransmission in the Disc1 D2-3/D2-3 mice are war-
ranted.
MATERIALS AND METHODS
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Generation of Disc1 (D2-3) mice
Disc1 (D2-3) mice (Acc Nos CDB0583K and CDB0678K:
http://www.cdb.riken.jp/arg/mutant%20mice%20list.html)
were generated by gene targeting in TT2 ES cells (74) and
HK3i ES cells (75) as described previously (http://www.cdb.
riken.jp/arg/protocol.html). The targeting vector was designed
to replace most of exon 2 and all of exon 3 with a loxP/
PGK-Neo-pA/loxP cassette (http://www.cdb.riken.jp/arg/ca
ssette.html) and to insert an in-frame stop codon between
the remainder of exon 2 and the loxP site. Successful homolo-
gous recombination was confirmed in two clones of the TT2
ES cells (#5, #33) and two clones of the HK3i ES cells
(#24, #146) by PCR (forward primer, 5′ -GTA CTC GGA
TGG AAG CCG GTC TTG TC-3′ ; reverse primer, 5′ -GGT
TTC AGA GGA TCC TCC GTT CAC C-3′ ) and southern
blot analysis with a 5′ probe and a 3′ probe; the clones
yielded highly chimeric mice. The heterozygous offspring
were intercrossed to obtain homozygous mutants, which
were genotyped by southern blot analysis with a 5′ probe
and PCR using a mixture of three primers (forward primer
‘Wt’, 5′ -GGT CAG AGC TCA GAG AAC TCC TTG
TGG-3′ , forward primer ‘Neo’, 5′ -CAT CGC CTT CTA
TCG CCT TCT TGA CG-3′ ; reverse primer ‘Rev’, 5′ -GCT
TCC TAC AGA GCA GAC TCC ACA CC-3′ ). Primers Wt
and Rev amplified the wild-type allele (547 bp), and primers
Neo and Rev amplified the mutated allele (619 bp). Four
strains of Disc1 (D2-3) mice derived from four different
clones of ES cells showed identical phenotypes. For the ana-
lysis of the mutant mice, a marker-assisted selection congenic
strategy was employed. Clone #33-derived mice, which had
75% C57BL/6 and 25% CBA background in the N1 gener-
ation, were backcrossed with C57BL/6JJmsSlc more than
five times such that all 100 markers corresponded to C57BL/
6 alleles. Histological and behavioral analyses were performed
using littermates (both males and females) generated by cross-
breeding of the backcrossed clone #33 mice. C57BL/
6JJmsSlc, 129X1/SvJJmsSlc, Slc:ddY and Slc:ICR mice
were obtained for breeding from SLC (Hamamatsu, Shizuoka,
Japan). Mice were genotyped for the 25 bp deletion in Disc1
(D6) using PCR primers (forward primer, 5′ -GCT GTG
ACC TGA TGG CAC T-3′ ; reverse primer, 5′ -GCA AAG
TCA CCT CAA TAA CCA-3′ ) as described previously (35).
All of the animal protocols were approved by the Animal
Care and Use Committee of the Nagoya University Graduate
School of Medicine, and the Principles for the Care and Use
of Laboratory Animals, which were approved by the Japanese
Pharmacological Society and the National Institutes of Health
4678 Human Molecular Genetics, 2011, Vol. 20, No. 23

Table 3. Monoamines and their metabolite contents in each region of the Disc1 D2-3/D2-3 mouse brain

NE MHPG DA DOPAC HVA 5-HT 5-HIAA

Prefrontal cortex +/+ 618.9 + 41.5 204.9 + 12.4 83.3 + 5.9 34.1 + 3.6 87.6 + 7.2 1085.7 + 82.8 188.5 + 5.1
D/D 625.2 + 35.1 220.0 + 13.0 86.8 + 7.6 32.9 + 2.0 110.9 + 13.6 1038.5 + 59.1 181.1 + 12.7
Striatum +/+ 124.2 + 18.3 221.3 + 14.9 27141.5 + 952.8 1044.3 + 32.0 2522.2 + 142.5 1187.7 + 53.1 693.6 + 40.6
D/D 105.3 + 10.3 204.3 + 19.4 25055.4 + 1029.7 980.8 + 40.0 2464.6 + 180.7 1086.1 + 74.8 629.2 + 26.9
Hippocampus +/+ 433.4 + 39.0 160.9 + 16.6 13.7 + 2.6 18.5 + 1.3 36.1 + 3.8 1046.0 + 116.4 374.5 + 35.2
D/D 427.0 + 18.1 169.6 + 12.6 17.1 + 3.2 17.7 + 1.3 40.9 + 3.9 997.9 + 50.3 343.1 + 20.8
Midbrain +/+ 497.6 + 27.9 134.2 + 18.0 186.8 + 125.0 56.0 + 10.1 130.7 + 21.4 1045.0 + 71.6 308.3 + 26.3
D/D 503.9 + 18.9 154.5 + 4.8 343.2 + 19.9 67.6 + 2.9 157.8 + 8.2 1029.6 + 63.8 301.4 + 18.6
Cerebellum +/+ 403.1 + 22.5 171.5 + 8.7 15.7 + 4.0 18.0 + 2.5 18.7 + 2.2 227.5 + 14.7 98.1 + 8.2
D/D 363.0 + 8.6 186.3 + 13.2 11.3 + 2.2 17.3 + 1.9 48.3 + 18.0 191.2 + 7.6 ∗ 86.0 + 5.5

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Values are expressed as nanogram per gram of wet tissue (mean + SE). +/+ (n ¼ 7) and D/D (n ¼ 9). ∗ P , 0.05 versus +/+.
NE, norepinephrine; DA, dopamine; 5-HT, 5-hydroxytryptamine; MHPG, 3-methoxy-4-hydroxyphenylglycol; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA,
homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic acid.

Guide for the Care and Use of Laboratory Animals, were fol-
lowed.
RNA extraction and RT – PCR
Total RNA was extracted from P7 mouse brain tissue samples
using the RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden,
Germany). Reverse transcription was performed with 1 mg
of RNA using the Transcriptor First Strand cDNA Synthesis
Kit (Roche Applied Science, Mannheim, Germany). PCR
amplification was performed using an Ex-Taq system
(Takara Bio, Otsu, Shiga, Japan) with 35 cycles for Disc1
exons 2 – 3 (forward primer, 5′ -GAA TGT GGC ACG GTC
TCC TC-3′ ; reverse primer, 5′ -TAA TCG CCA TCC TCG
ACC AC-3′ ) and Disc1 exons 7– 10 (forward primer,
5′ -TGG CTG TCA GAG AAC TCA CTG CTC AG-3′ ;
reverse primer, 5′ -CCA TGC ACT TCA CAG TGT TTG
CCT TCA-3′ ). PCR amplification for Hprt was also performed
using PCR primers (forward primer, 5′ -CCT GCT GGA TTA
CAT TAA AGC ACT G-3′ ; reverse primer, 5′ -GTC AAG
GGC ATA TCC AAC AAC AAA C-3′ ) as described previous-
ly (26).
Plasmid construction and protein purification
cDNA encoding murine DISC1 (mDISC1) was generously
provided by Dr Akira Sawa (Johns Hopkins University, Balti-
more, MD, USA). The mDISC1 cDNA (amino acid residues
1 – 852 of mDISC1) was inserted into the pGEX-4T-2 vector
(GE Healthcare, Uppsala, Sweden). An mDISC1 C-terminal
fragment (amino acid residues 658 – 852 of mDISC1) was
inserted into the pGEX-4T-2 and pMAL-c2 vectors (New
England Biolabs, Ipswich, MA, USA). The cDNA encoding
full-length dnaK was cloned from BL21 (DE3) Escherichia
coli cells. Next, dnaK was amplified by PCR and subcloned
into the pGEX-6P-3 vector (GE Healthcare). The identity of
each fragment was confirmed by DNA sequencing. The
MBP-mDISC1 C-terminal protein was produced in BL21
(DE3) E. coli cells and was purified on an amylose resin
(New England Biolabs). The GST-mDISC1 C-terminal and
GST-dnaK proteins were produced in BL21 (DE3) E. coli
cells and were purified with glutathione-Sepharose 4B beads
(GE Healthcare). The MBP-mDISC1 C-terminal and
GST-mDISC1 C-terminal proteins were bound to a Mono Q
column (GE Healthcare) and eluted with a linear gradient
(0– 1000 mM NaCl in 20 column volume). A full-length
GST-mDISC1 protein was produced in Rosetta-gami 2
E. coli cells (Merck, Darmstadt, Germany) and purified with
glutathione-Sepharose 4B beads.
Antibodies
Four guinea pig anti-DISC1 N-terminal antibodies were
prepared by Medical & Biological Laboratories Co., Ltd.
(MBL, Nagoya, Aichi, Japan) and purified using specific pep-
tides (amino acid residues 131 – 170 of mDISC1) as antigens.
Two rabbit and two guinea pig anti-DISC1 C-terminal anti-
bodies were prepared by MBL. To generate rabbit and
guinea pig polyclonal antibodies against the C-terminal
region of DISC1, the MBP-mDISC1 C-terminal protein was
used as the antigen. The antiserum was precleared using
GST-dnaK protein immobilized to CNBr-activated Sepharose
4B beads (GE Healthcare). The anti-DISC1 C-terminal anti-
body was affinity-purified from the precleared antiserum
using the GST-mDISC1 C-terminal protein immobilized on
CNBr-activated Sepharose 4B beads. Antibodies against
DISC1 (Mid and C-ter, rabbit polyclonal, Invitrogen, Carls-
bad, CA, USA and N-16, goat polyclonal, Santa Cruz Biotech-
nology, Santa Cruz, CA. USA), GM130 (mouse monoclonal,
BD, Franklin Lakes, NJ, USA) and glyceraldehyde-3-phos-
phate dehydrogenase (mouse monoclonal, Invitrogen) were
purchased.
Tissue lysates
All of tissue lysates were extracted by the addition of RIPA
buffer [20 mM Tris – HCl (pH 7.5), 1 mM EDTA, 150 mM
NaCl, 1.0% NP-40, 0.1% sodium deoxycholate, 0.1%
sodium dodecyl sulfate (SDS) and Complete Protease Inhibi-
tor Cocktail (Roche Applied Science)]. Protein concentrations
were determined using the bicinchoninic acid Protein Assay
Reagent Kit (Pierce, Rockford, IL, USA). The lysates were
boiled with 3× SDS – polyacrylamide gel electrophoresis
(PAGE) sample buffer [187.5 mM Tris – HCl (pH 6.8), 9%
 Human Molecular Genetics, 2011, Vol. 20, No. 23 4679

Table 4. Comparison with other Disc1 mutant mice

Hikida(23) Pletnikov and Li(24) Niwa(48) Clapcote and Koike and Present study
Ayhan (25,85) Lipina(22,86) Kvajo (27,28)
Human1-597 Inducible Inducible Knockdown Q31L L100P D6 (exon 6) D2-3 (exons
expression of DISC1 via in utero 2– 3)
Human1-597 C-terminal gene transfer
fragment
Test Parameter C57BL/6 B6;SJL;CBA C57BL/6N ICR C57BL/6J C57BL/6J C57BL/6J

Locomotor Locomotor activity  (Male)  (Male) ND ¼ ¼  ¼ ¼

MK-801-induced Locomotor activity ND  (Male) ND ND ND ND ND ¼
hyperactivity
METH-induced Locomotor activity ND ND ND  ND ND ND  (Female)
hyperactivity

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AMP-induced Locomotor activity ND  (Male) ND ND ND  (Male) ND ND
hyperactivity
Open field Total movement  (Male) ¼ ND ND ND ND ND ¼
Inner movement ¼ (Male) ¼ ND ND ND ND ND ¼
Elevated plus-maze Open arm entries ¼ (Male) ND ND ND ND ND ND 
Closed arm entries ND ND ND ND ND ND ND 
Time in open arms ¼ (Male) ¼ ND ND ¼ ¼ ND 
Time in closed arms ND ¼ ND ND ND ND ND 
Cliff-avoidance test Frequency of jumping ND ND ND ND ND ND ND 
Forced swim test Immobility time  (Male)  (Female)  ¼  ¼ ND ¼
Social interaction Interaction time ¼ (Male)  (Male)  ND  ¼ ND 
PPI Prepulse (69–90 dB)  (Male) ¼ ND    ¼ 
Latent inhibition Suppression ratio ND ND ND ND   ND ¼
Y-maze Alternation ¼ (Male) ND ND ND ND ND ND ¼
Radial-maze Working memory ND ND  ND ND ND  (Male) ¼
Fear conditioning Context-dependent ND ND ND ND ND ND ¼ (Male) 
freezing
Tone-dependent ND ND ND ND ND ND ¼ (Male) ¼
freezing
Nobel object Exploratory preference ND ND ND  ND ND ¼ (Male) ¼
recognition test Exploration time ND ND ND ND ND ND ND  (Male)

METH, methamphetamine; AMP, amphetamine; , higher than WT; , lower than WT; ¼, no difference, ND, not determined.
(23) Hikida et al., 2007; (25) Pletnikov et al., 2008; (70) Ayhan et al., 2011; (24) Li et al., 2007; (45) Niwa et al., 2010; (22) Clapcote et al., 2007; (71) Lipina et al.,
2010; (27) Koike et al., 2006; (28) Kvajo et al., 2008.

SDS, 15% glycerol, 6% 2-mercaptoethanol and 0.06% bromo-
phenol blue].
Immunoprecipitation assay and mass spectral analysis
Lysates from P7 mouse brain tissue were extracted by the
addition of lysis buffer (20 mM Tris – HCl, 1 mM EDTA,
50 mM NaCl, 1.0% NP-40 and Complete Protease Inhibitor
Cocktail) and were cleared by centrifugation at 100 000g
for 60 min at 48C. The supernatants were incubated with N-
terminal or C-terminal anti-DISC1 antibodies for 1 h at 48C,
and the immunocomplexes were precipitated using protein
A-Sepharose 4B beads (GE Healthcare). The immunocom-
plexes were washed three times with lysis buffer
and were eluted by boiling in SDS –PAGE sample buffer.
The eluate was separated by SDS – PAGE, and the gel
was silver-stained using the SilverQuest Silver Staining
Kit (Invitrogen). The protein band corresponding to the
100 kDa DISC1 polypeptide was excised from the gel.
Gel pieces were destained, reduced, S-carboxymethylated
and digested with trypsin. The resulting peptides were sub-
jected to nano-electrospray tandem mass spectroscopic ana-
lysis using a Finnigan LTQ Orbitrap XL mass spectrometer
(Thermo Fisher, San Jose, CA, USA) combined with a Para-
digm MS4 HPLC system (Michrom BioResources Inc.,
Auburn, CA, USA).
Cresyl violet (Nissl) staining
Paraffin-embedded brain tissue sections were deparaffinized
and placed in 0.5% cresyl violet in distilled water at 378C
for 10 min. The sections were briefly rinsed twice in 90%
ethanol and were dipped in 100% ethanol three times before
being dehydrated in xylene three times for 5 min each. The
sections were affixed to glass cover slips using Permount
solution and were examined under a light microscope.
Immunostaining
Mice were transcardially perfused with 2% paraformaldehyde
(PFA) in 100 mM phosphate buffer (PB) (pH 7.4) on day P2.
The brains were removed, post-fixed with 2% PFA for 6 h
and sectioned sagittally with a cryostat (Leica Microsystems,
Wetzlar, Germany) at 10 mm. The sections were blocked
using blocking buffer [100 mM PB (pH 7.4), 0.005%
saponin, 1% Block Ace (DS Pharma Biomedical, Suita,
4680 Human Molecular Genetics, 2011, Vol. 20, No. 23

Osaka, Japan) and 5% normal goat serum (Vector Laboratories O2/5% CO2, and its pH was adjusted to 7.4. The slices were
Inc., Burlingame, CA, USA)]. The sections were incubated maintained for at least 1 h at RT (26 – 288C) in an incubation
with primary antibodies overnight at 48C in antibody dilution chamber containing gassed ACSF.
buffer [100 mM PB (pH 7.4), 0.005% saponin, and 1% Block
Ace], washed three times in wash buffer [100 mM PB (pH
7.4) and 0.005% saponin] and incubated with Alexa Fluor
488 goat anti-rabbit IgG, Alexa Fluor 555 goat anti-mouse
IgG (Invitrogen) and Hoechst 33342 (Nacalai, Kyoto, Japan) Electrophysiology
in the antibody dilution buffer for 1 h at room temperature
A single hippocampal slice was transferred to the recording
(RT). Finally, the sections were washed three times in wash
chamber and superfused continuously with gassed ACSF at
buffer.
a rate of 2 – 2.5 ml/min at RT. A stimulating electrode (mono-
Hippocampal neurons were prepared from mouse embryos
polar stimulation) was positioned in the molecular layer
at embryonic day 16 (E16) using papain (76). The neurons

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were seeded on poly-D-lysine- and laminin-coated cover
slips and were cultured in Neurobasal medium (Invitrogen)
supplemented with the B-27 supplement (Invitrogen) and
1 mM glutamine. The hippocampal neurons at day in vitro
(DIV) 3 and DIV8 were fixed with 4% PFA in PB for
10 min and were treated with blocking buffer for 10 min.
The neurons were incubated with primary antibodies in
the antibody dilution buffer overnight at 48C, washed
three times in wash buffer and incubated for 1 h with
Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555
goat anti-mouse IgG in the antibody dilution buffer for
1 h at RT.
Immunofluorescence was performed using a laser scanning
confocal microscope (Model LSM 5 Pascal, Zeiss, Oberko-
chen, Germany and Nikon-A1, Nikon, Tokyo, Japan).
Immunoelectron microscopy
Immunoelectron microscopy using the silver-enhanced
immunogold method was performed as previously described
(77). P2 mouse brains and cultured hippocampal neurons at
DIV5 were fixed with 2% PFA in 100 mM PB (pH 7.4)
and incubated at RT for 5 h with the primary anti-DISC1 C-
terminal antibody described above followed by incubation
with a secondary anti-rabbit antibody coupled to 1.4 nm
gold particles (Nanoprobes Inc., Yaphank, NY, USA). The
sample-bound gold particles were silver-enhanced using the
HQ-silver kit (Nanoprobes Inc.) at RT for 8 min. The
samples were post-fixed with 0.5% osmium oxide in a
buffer (pH 7.3) containing 100 mM cacodylate. The samples
were dehydrated by passage through a graded series of
ethanol (50, 70, 90 and 100%) and propylene oxide and em-
bedded in epoxy resin. From this sample, ultrathin sections
were cut, stained with uranyl acetate and lead citrate and
observed with an electron microscope (JEM-1011 EX;
JEOL, Akishima, Tokyo, Japan).
Hippocampal slice preparation
Electrophysiological analyses were conducted on 3-week-old
male mice. The mice were decapitated under deep anesthesia
with ethyl ether. The brains were quickly removed, and trans-
verse 400 mm thick slices were cut from the hippocampus
using a vibratome in ice-cold artificial cerebrospinal fluid
(ACSF) containing 128 mM NaCl, 5 mM KCl, 1.3 mM
MgSO4, 1.25 mM KH2PO4, 2.41 mM CaCl2, 26 mM NaHCO3
and 10 mM D-glucose. The ACSF was gassed with 95%
30– 50 mm from the granule cell layer in the dentate gyrus
to activate the medial perforant path. Constant-current pulses
(100 ms) were supplied by a stimulator (SEN-3301, Nihon
Kohden, Tokyo, Japan) every 30 s. The intensity of the test
stimuli was adjusted to evoke 50% of the maximum re-
sponse. Using the conventional extracellular recording tech-
nique, fEPSPs were recorded from the middle molecular
layer in the dentate gyrus with a glass pipette filled with
2 M NaCl (2 – 3 MV). Baseline recordings were established
by delivering test stimuli for more than 60 min before the ex-
perimental recordings. LTP was induced by 100 pulses at 50,
100 and 200 Hz. The signals were amplified and filtered at
5 kHz with an amplifier (Axopatch 200B, Axon Instruments,
CA, USA). Data acquisition and analysis were performed
using pCLAMP 9.0 software (Axon Instruments). The data
are expressed as the mean + SD; N indicates the number of
slices tested, and one slice was prepared from each mouse.
The synaptic transmission in the extracellular recordings was
estimated from the rising phase of the fEPSP slope using
linear regression during the first 0.6 ms after the PSFV (78).
The magnitude of LTP was expressed as the mean percentage
change occurring 55 –60 min after HFS with respect to the
final 5 min of the baseline period. Comparisons were made
using a two-tailed t-test for paired or independent samples.
P-values below 0.05 were considered to be statistically signifi-
cant.
Behavioral analyses
Information about the open field test (79), the locomotor activ-
ity test (79), the Y-maze test (80), the PPI test (79), the fear
conditioning test (80), the elevated plus-maze test (81), the
novel object recognition test (82), the social interaction test
(79,80), the forced swim test (81), the latent inhibition
test (83), the cliff-avoidance test (84) and the radial-maze
test (85) is provided in the Supplementary Information
section. Monoamine metabolism was analyzed as described
previously (86). The data are presented as the mean + SE.
Differences between two groups were analyzed using a two-
tailed Student’s t-test. Differences in pre-pulse inhibition,
locomotor activity and results of the social interaction test
were analyzed by a repeated ANOVA. The time-course data
in the cliff-avoidance test were analyzed by the log-rank
test. The effect of clozapine treatment was analyzed by a
two-way ANOVA followed by Tukey – Kramer post hoc tests.

ACKNOWLEDGEMENTS
cDNA encoding mDISC1 was generously provided by
Dr Akira Sawa (Johns Hopkins University). We thank
Dr Mutsuki Amano, Junko Uraguchi-Asaki, Yasutaka
Fujino, Yasuhiro Funahashi, Yusuke Funahashi, Dr Takao
Hikita, Dr Katsuhiro Kato, Takafumi Kinoshita, Ryo
Kuwata, Shinichi Nakamuta, Dr Shinichiro Taya, Dr Shujie
Wang and Dr Takashi Watanabe for helpful discussions and
the preparation of some materials, Yuko Yamashita for her
technical assistance and Takako Ishii and Naoko Hatamoto
for secretarial assistance. We acknowledge the Division of
Medical Research Engineering of the Nagoya University
Graduate School of Medicine for the use of ImageQuant
LAS 4010 (GE), a Leica CM3050 S cryostat and Nikon-A1
microscopy, and we thank the Division for Research of
Laboratory Animals, Center for Research of Laboratory
Animals and Medical Research Engineering (Technical
Staff, Noboru Ogiso, Yasutaka Ohya and Kumiko Yano) for
animal care and use.
Conflict of Interest statement. None declared.
FUNDING
This work was supported by JST, CREST, a Grant-in-Aid for
Scientific Research (S) (20227006) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of Japan
(MEXT) and a Grant-in-Aid for GCOE research from MEXT.
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