World Journal of Neuroscience, 2011, 1, 1-8 WJNS
doi:10.4236/wjns.2011.11001 Published Online May 2011 (http://www.SciRP.org/journal/wjns/).
Published Online May 20 11 in SciRes. http://www.scirp.org/journal/wjns
Analyses of fear memory in Arc/Arg3.1-deficient mice: intact
short-term memory and impaired long-term and remote
memory
Kaz u yu ki Yama da 1*, Chihiro Homma1, Kentaro Tanemura2, Toshio Ikeda3,
Shigeyoshi Itohara3, Yoshi ko Na ga oka 1
1Support Unit for Animal Resources Development, Research Resources Center, Brain Science Institute, RIKEN, Saitama, Japan;
2Division of Cellular & Molecular Toxicology, Biological Safety Research Center, National Institute of Health Sciences,
Tokyo, Japan;
3Laboratory for Behavioral Genetics, Brain Science Institute, Saitama, Japan.
Received 25 April 2011; 23 May 2011; 26 May 2011
ABSTRACT
Activity-regulated cytoskeleton-associated protein
(Arc/Arg3.1) was originally identified in patients
with seizures. It is densely distributed in the hip-
pocampus and amygdala in particular. Because the
expression of Arc/Arg3.1 is regulated by nerve in-
puts, it is thought to be an immediate early gene. As
shown both in vitro and in vivo, Arc/Arg3.1 is in-
volved in synaptic consolidation and regulates some
forms of learning and memory in rats and mice
[1,2]. Furthermore, a recent study suggests that
Arc/Arg3.1 may play a significant role in signal
transmission via AMPA-type glutamate receptors
[3-5]. Therefore, we conducted a detailed analysis
of fear memory in Arc/Arg3.1-deficient mice. As
previously reported, the knockout animals exhib-
ited impaired fear memory in both contextual and
cued test situations. Although Arc/Arg3.1-defic ient
mice showed almost the same performance as
wild-type littermates 4 hr after a conditioning trial,
their performance was impaired in the retention
test after 24 hr or longer, either with or without
reconsolidation. Immunohistochemical analyses showed
an abnormal density of GluR1 in the hippocampus
of Arc/Arg3.1-deficient mice; however, an applica-
tion of AMPA potentiator did not improve memory
performance in the mutant mice. Memory impair-
ment in Arc/Arg3.1-deficient mice is so robust that
the mice provide a useful tool for developing treat-
ments for memory impairment.
Keywords: Activity-Regulated Cytoskeleton-Associated
Protein (Arc/Arg3.1); Knockout (Ko) Mouse;
Short-Term Memory; Long-Term Memory; Reconsoli-
da tio n; AM PA Rece pto r
1. INTRODUCTION
Activity-regulated cytoskeleton-associated protein (Arc/
Arg3.1) is encoded by an effector immediate early gene
and is selectively localized in neuronal dendrites [6].
Arc/Arg3.1 and its encoded protein are thought to play a
role in activity-dependent plasticity of dendrites [7].
Arc/Arg3.1 mRNA is greatly increased by long-term
potentiation (LTP)-inducing electrical stimuli [8,9]; ad-
ministration of psycho-stimulant drugs such as cocaine
[10], amphetamines/methamphetamines [11-13], and
phencyclidine [14]; insulin [15], middle cerebral artery
occlusion [16], electroconvulsive shock [17,18], olfac-
tory inputs [19], mating [20], stress [21], and other stim-
uli that prompt neuronal activity [22,23]. The mRNA is
then rapidly delivered to the dendrites [9]. Furthermore,
intense synaptic activity induces selective localizatio n of
Arc/Arg3.1 mRNA to activated synapses [9,24]. There-
fore, Arc/Arg3.1 is believed to be related to synaptic
plasticity, and thus many electrophysiological and bio-
chemical studies have been conducted to investigate this
possibility [7-9,25]. These studies have revealed that
Arc/Arg3.1 may be a key molecule involved in late-
phase LTP, during which long-term memories (LTM)
are thought to be established.
Guzowski et al. [1] found that rats in which Arc/
Arg3.1 antisense oligonucleotides were infused into the
hippocampus perform relatively poorly in a water-maze
probe test. This result strongly suggested that Arc/Arg3.1
plays an important role in the formation of some forms
of spatial LTM. Furthermore, Plath et al. [2] developed
Arc/Arg3.1-deficient mice and demonstrated learning
and memory impairment in these mice in the Morris

K. Yamad a et al . / World Journal of Neuroscience 1 (2011) 1-8 2
water maze task, contextual and cued fear conditioning,
novel object recognition, and the conditioned taste-
-aversion test. These results indicate that Arc/Arg3.1
regulates a wide range of learning and memory function.
According to both behavioral and electrophysiological
data, they concluded that Arc/Arg3.1 is essential for
LTM. Although the authors concluded that Arc/Arg3.1
does not play a significant role in synaptic potentiation
and early LTP, they did not show adequate behavioral
evidence to conclude the role of Arc/Arg3.1 in the fear
memory (they only showed intact short-term memory
(STM) 10 min after the initial trial, and intact novel ob-
ject recognition test). LTM deficiency in Arc/Arg3.1-
deficient mice was evident about 120 min after high-
frequency stimuli. Thus, the addition al memory tests are
necessary that should be conducted at about 120 min or
later after the training and/or conditioning.
Arc/Arg3.1 may be involved in the endocytosis of
AMPA-type glutamate receptors [3-5] by inducing the
internalization of AMPA receptors. The morphological
properties of AMPA receptors in Arc/Arg3.1-deficient
mice were not, however, analyzed, and how AMPA re-
ceptor trafficking relates to impaired LTM remains un-
known.
Ube3A regulates Arc/Arg3.1 degradation in synapses,
and mutation of Ube3A may be a cause of autism spec-
trum disorders [26-28] and Angelman syndrome [29,30].
Using Ube3A-deficient mice, Greer et al. [31] recently
reported that an increase in Arc/Arg3.1 expression leads
to a decrease in the number of AMPA receptors. These
results suggest that there may be abnormal expression of
AMPA receptors in Arc/Arg3.1-deficient mice.
In order to elucidate the memory process of Arc
/Arg3.1-deficient mice, in our current study, we used
classical fear conditioning paradigm. First, we confirmed
the LTM impairment in Arc/Arg3.1-deficient mice. Then,
we conducted a time course analysis of the memory im-
pairment of Arc/Arg3.1-deficient mice, and assessed the
remote memory function of the mutant mice. Further-
more, we compared the brain structure between wild-
type and Arc/Arg3.1-deficient mice by immunohisto-
chemically.
2. RESULTS
2.1. Successful Generation of
Arc/Arg3.1-Deficient Mice
We generated Arc/Arg3.1-deficient mice with MS12 ES
cells derived from the C57BL/6 mouse strain (Supple-
mental information). Before conducting fear-condition-
ing experiments, we confirmed the intact pain sensation
of Arc/Arg3.1-deficient mice with a tail-flick and a hot-
plate paradigm in another batch of mice (n = 10 each). In
both tests, they displayed responses indistinguishable
from those of wild-type littermates (data not shown).
These results indicated that Arc/Arg3.1-deficient mice
had normal pain sensation and that they and their wild-
type littermates would respond equivalently to electric
foot shocks. Therefore, a classical fear-conditioning pa-
radigm was appropriate to assess their memory function .
2.2. Ar c/Arg3.1-Deficient Mice Exhibited
Impairment in Both Contextual Fear
and Cued Fear Memory
Classical fear conditioning consists of a conditioning
phase, a context test, and an auditory cued test. Arc/
Arg3.1-deficient mice exhibited a clearly lower occur-
rence of freezing than did wild-type mice in the context
test. The mean percentage of freezing frequency was
significantly lower in Arc/Arg3.1-deficient mice (Mann-
Whitney’s U-test, U = 15, p < 0.01; Figure 1A). The
subsequent auditory cue test consisted of two parts: the
first half was done without a cue to assess the
non-specific and/or generalized fear response to the new
context, and the latter half was done with an auditory
cue to assess the fear response to the cue (Figure 1B).
The mean freezing frequency of Arc/Arg3.1-deficient
mice was also significantly lower than that of wild-type
mice under both conditions (without cue:
Mann-Whitney’s U-test, U = 18, p < 0.05; with cue: U =
0, p < 0.001). The identical tests were replicated five
times with both male and female mice. There were no
differences in impaired memory performance according to
sex. Female Arc/Arg3.1-deficient mice did, however, dis-
play a significantly lower freezing frequency (data not
shown). Arc/Arg3.1-deficient mice showed a significant
decrease in the freezing response to the new context
without an auditory cue. Because the results of the cued
Figure 1. Summary of contextual and cued-fear memory per-
formance of wild-type (WT) and Arc/Arg3.1-deficient (KO)
mice. (A) Results of the context test. (B) Results of the cued
test. Data represent the mean ± S.E.M. Asterisks indicate sta-
tistical significance (**, p < 0.01; *, p < 0.05).

K. Yamad a et al . / World Journal of Neuroscience 1 (2011) 1-8 3
test reflect both generalized contextual fear [32] and
cued fear, cued tests may be redundant. Therefore, only
the contextual test trial was used in the following ex-
periments.
2.3. Arc/Arg3.1-Deficient Mice Exhibited Intact
STM but Impaired LTM
We conducted fear-conditioning tests to compare STM,
LTM, and the retention of LTM in wild-type and Arc/
Arg3.1-deficient mice. In the initial context test (4 hr
after conditioning), the mean percentage of freezing of
Arc/Arg3.1-deficient mice was indistinguishable from
that of wild-type mice (Mann-Whitney’s U-test, U = 42,
n.s.; Figure 2). In the second context test conducted
24 hr after cond ition ing , the mean p ercentag e of freezing
of Arc/Arg3.1-deficient mice was, however, significantly
lower than that of wild-type mice (Mann-Whitney’s
U-test, U = 24, p < 0.05; Figure 2). In the 1-week and
4-week tests, the mean percentage of freezing of Arc/
Arg3.1-deficient mice was also significantly lower than
that of wild-type mice (1-week test: Mann-Whitney’s
U-test, U = 2, p < 0.01; 4-week test: Mann-Whitney’s
U-test, U = 0, p < 0.01; Figure 2). The freezing fre-
quency of wild-type mice increased significantly during
the experiment, contrary to that of Arc/Arg3.1-deficient
mice, whose freezing frequency decreased significantly
(Two-way ANOVA with repeated measures: genotype,
F(79,1) = 58.5, p < 0.001; retention time, F(79,3) = 2.47,
p = 0.07, n.s.; genotype × retention time, F(79,3) = 12.6,
p < 0.001; wild-type 4 hr vs. 4 week, t = 3.33, p < 0.01;
Arc/Arg3.1-deficient mice 4 hr vs. 4 week, t = 4.91, p <
0.01).
2.4. Arc/Arg3.1-Deficient Mice Showed Almost
No Remote Memory of Fear
As in the STM and LTM test, the same mice were used
Figure 2. Summary of the STM and LTM tests of
wild-type (WT) and Arc/Arg3.1-deficient (KO) mice. Da-
ta represent the mean ± S.E.M. Asterisks indicate statisti-
cal significance (**, p < 0.01; *, p < 0.05).
for all experiments, because the retention curve could
have been affected by the repeated exposure to the con-
ditioning context (e.g., reconsolidation, Suzuki et al.,
[33]). Therefore, we conducted a remote memory test.
Mice were conditioned and kept without any treatment
except standard daily care for 4 weeks, and then we
conducted a context test. Wild-type mice showed a high
freezing frequency, but Arc/Arg3.1-deficient mice did
not show any freezing response (Mann-Whitney’s U-test:
U = 0, p < 0.01; Figure 3).
2.5. AMPA Receptors Were Expressed at Higher
Levels in the Hippocampus of
Arc/Arg3.1-Deficient Mice
Immunohistochemical analyses were conducted to clar-
ify the distribution of neuronal processes and synapses.
Although increased immunoreactivity for neuronal
processes (NF-M, MAP1A) was detected in the cerebral
cortex of Arc/Arg3.1-deficient mice, no substantial dif-
ferences were detected in the hippocampal region (Fig-
ure 4). Nevertheless, immunoreactivity for pre- and
post-synaptic proteins (SYP, HOM) was somewhat in-
creased in the hippocampus of Arc/Arg3.1-deficient mice
(Figure 4). We also found increased immunoreactivity
for GluR1 in the CA1 region and dentate gyrus (DG) of
the hippocampus and for SYP in the CA3 region of
Arc/Arg3.1- deficient mice as compared with that of
wild-type mice (Figure 5). In order to confirm the in
creased GluR1 reactivity in hippocampus, we calculated
the ratio of fluorescence intensity and compared between
wild-type and Arc/Arg3.1-deficient mice in a semi- qua-
ntitative manner. Arc/Arg3.1-deficient mice exhibited
Figure 3. Summary of remote memory test of wild-
type (WT) and Arc/Arg3.1-deficient (KO) mice. Data
represent the mean ± S.E.M. Asterisks indicate statis-
tical significance (**, p < 0.01).

K. Yamad a et al . / World Journal of Neuroscience 1 (2011) 1-8 4
Figure 4. Immunohistochemical analysis of hippocampal neurons and synapses of wild-type (WT) and Arc/Arg3.1-deficient (KO)
mice. A–F, Immunohistochemical images of neurofilament-m (NF-M; green) and MAP1A (red). G–R, Immunohistochemical images
of synaptophysin (SYP; green) and homer (HOM; red). A–C, G–I, and M–O, wild-type mice. D–F, J–L, and P–R,
Arc/Arg3.1-deficient (KO) mice. C, F, I, L, O, and R, merged images. A–F, cerebral cortex and hippocampus; scale bar = 500 m.
G–L, hippocampus; scale bar = 200 m. M–R, CA1 region of the hippocampus; scale bar = 20 m.
KOWT
SYP/ GluR1_CA1
SYP/ GluR1_DG
ABC DEF
GHI JKL
B ar = 200 u m
B ar = 20 um
B ar = 200 u m
B ar = 20 u m
Figure 5. Immunohistochemical analysis of the distribution of AMPA-type glutamate receptors (GluR1) in wild-type and
Arc/Arg3.1-deficient (KO) mice. Immunohistochemical images of synaptophysin (SYP, green) and AMPA-type glutamate receptors
(GluR1, red). A–C and G–I, wild-type mice. D–F and J–L, Arc/Arg3.1-deficient (KO) mice. C, F, I, and L, merged images. Scale bars
are identical as those of Figure 4.
statistically significant increase of GluR1 immunoreac-
tivity in CA1 and CA3 (both p<0.01, Figure 6).
3. DISCUSSION
In our current study, we demonstrated impaired fear
memory in Arc/Arg3.1-deficient mice. In addition, im-
munohistochemical analysis revealed changes in synap-
tic structure and increased AMPA receptor expression in
the hippocampus of Arc/Arg3.1-deficient mice. These
findings suggest that Arc/Arg3.1 plays crucial roles not

K. Yamad a et al . / World Journal of Neuroscience 1 (2011) 1-8 5
0
1
2
3
R a ti o of i nte nsit y
WT-CA1
KO-CA1
WT-CA3
KO-CA3
*
*
GluR1
WT-CA1
KO-CA1
WT-CA3
KO-CA3
SYP
Figure 6. Ratio of fluorescence intensity. SYP: synaptophy sin,
GluR1: AMPA-type glutamate receptors, WT: wild-type mice,
KO: Arc/Arg3.1-deficient mice. Data represent mean + SEM. *:
p<0.01 (compared to wild-type mice).
only in LTM formation but also in th e memory retention
process. Although an AMPA potentiator did not lead to
recovery of the impaired memory in Arc/Arg3.1-defi-
cient mice (Supplemental information and Supplemental
Figure 2), this is the first study that used Arc/Arg3.1-
deficient mice to assess the effect of drugs on memory
impairment.
Arc/Arg3.1-deficient mice exhibited a lower percent-
age of freezing than did wild-type mice in the context
and cued-memory test, indicating that Arc/Arg3.1-de-
ficient mice were impaired in both contextual and cued
memory. Many studies have reported that contextual fear
reflects hippocampus-dependent memory function, and
cued fear reflects amygdala-dependent memory function
(for review, see LeDoux, [34]). Therefore, Arc/Arg3.1-
deficient mice may be impaired in both hippocampus-
and amygdala-dependent memory function. These re-
sults suggest that Arc/Arg3.1 may function in the amyg-
dala for auditory fear memory formation as well as in the
hippocampus for spatial memory formation. Arc/Arg3.1-
deficient mice did, however, show a significant decrease
in the freezing response to the new context without an
auditory cue in the cued test. This result indicates that
the freezing response in the cued test may reflect both
generalized contextual fear and cued fear. Therefore,
new experimental tasks or protocols should be devel-
oped to clarify the role of Arc/Arg3.1 in amygdala-de-
pendent memory processes.
In this study, we examined STM and LTM in Arc/
Arg3.1-deficient mice. Arc/Arg3.1-deficient mice exhib-
ited unimpaired memory performance 4 hr after condi-
tioning, indicating that their STM or early stage of LTM
was intact. In contrast, 24 hr after the conditioning trial,
the memory performance of Arc/Arg3.1-deficient mice
was greatly impaired. After 4 weeks, their memory was
almost completely absent, whereas that of wild-type
mice slightly but significantly increased. This differen-
tial retention process may be partly due to repeated ex-
posure to the conditioning context. Short exposure to
conditioned stimuli may enhance fear memory (reconso-
lidation: [33,35]). Decreased fear memory by repeated
exposure indicates that the reconsolidation process may
also be impaired in Arc/Arg3.1-deficient mice. Thus, we
assessed remote memory (ability to retrieve distant epi-
sodes/events) directly and confirmed complete impair-
ment of remote memory in Arc/Arg3.1-deficient mice.
Immunohistochemical analyses revealed increased
expression of AMPA-type glutamate receptors in hippo-
campal regions. This result is consistent with previous
studies reporting that Arc/Arg3.1 may be involved in
endocytosis of AMPA-type glutamate receptors and may
prompt the internalization of AMPA receptors [3-5]. Al-
though LTP, especially early-phase LTP, depends on
NMDA receptors ([36] for review), activation of AMPA
receptors may improve memory function in rats and
mice [37,38]. Therefore, this mutant mouse strain will be
useful for developing treatments including drugs for
memory impairment.
4. EXPERIMENTAL PROCEDURES
4.1. The Behavioral Laboratory Environment
and Housing Conditions of Mice
Mice were housed individually before transfer to the
behavioral laboratory. They were kept in the laboratory
during the behavioral analysis under a light/dark cycle of
12 hr/12 hr (lights on at 8:00). The laboratory was air
conditioned, and the temperature and humidity were
maintained at ~22˚C – 23˚C and 50% – 55%, respec-
tively. Food and water were freely available except dur-
ing experimentation unless otherwise indicated. We used
large tweezers with soft vinyl tips to handle the mice to
avoid potential differences in the handling technique of
the different researchers involved in the study.
Animal experiments in this study were conducted in
strict accordance with the guidelines of the Institute of
Physical and Chemical Research (RIKEN) and were
approved by the Animal Investigation Committee of the
Institute.
4.2. Classical Fear Conditioning
4.2.1 Contextual and Cued Test
Twenty mice (wild type, n = 10; Arc/Arg3.1 deficient, n
= 10; 9 weeks of age) were used. This test consisted of

K. Yamad a et al . / World Journal of Neuroscience 1 (2011) 1-8 6
three parts: a conditioning trial, a contex t test trial, and a
cued test trial. Fear conditioning was carried out on a
clear plastic chamber equipped with a stainless-steel grid
floor (34  26  30 [H] cm). The luminance of the floor
was 225 Lux in the conditioning and context test trial
and 0–1 Lux in the cued test trial. A CCD camera was
installed on the ceiling of the chamber and was con-
nected to a video monitor and a Windows PC. The grid
floor was wired to a shock generator. White noise (65 dB)
was supplied from a loudspeaker as an auditory cue
(conditioned stimulus, CS). The conditioning trial con-
sisted of a 2-min exploration period followed by two
CS–unconditioned sti mulus (US) pairings sep arated by 1
min each. A US (foot-shock: 0.5 mA, 2 sec) was admin-
istered at the end of the 30-sec CS period. A context test
was performed in the same conditioning chamber for 3
min in the absence of the white noise 24 hr after the
conditioning trial. In addition, a cued test was performed
in an alternative context with distinct cues: the test
chamber was different from the conditioning chamber in
brightness (dark environment, almost 0–1 Lux), color
(white), floor structure (no grid), and shape (triangular).
The cued test was conducted 24 hr after the contextual
test was finished, and consisted of a 2-min exploration
period (no CS) to evaluate the nonspecific contextual
fear followed by a 2-min CS period (no foot shock) to
evaluate the acquired cued fear.
4.2.2. STM and LTM Test
A second set of mice (wild type, n = 10; Arc/Arg3.1 de-
ficient, n = 10; 9 weeks of age) was used for these tests.
The conditioning trial was the same as in the above con-
textual and cued test (see 4.2.1). After 4 hr, an STM test
was conducted, and LTM tests were conducted 24 hr, 1
week, and 4 weeks later. STM and LTM tests were the
same as the context test of 4.2.1. In this test, the cued
test was not conducted to exclude any sensitization ef-
fect caused by the auditory cue.
4.2.3. Remote Memory Test
A third set of mice (wild type, n = 10; Arc/Arg3.1 defi-
cient, n = 10; 9 weeks of age) was used. The condition-
ing trial was the same as in the above STM and LTM test
(see 4.2.2). In this test, a context test was conducted 4
weeks after the conditioning trial.
Both the contextual and cued tests and the STM and
LTM tests were replicated five times to confirm their
reproducibility. The rate of the freezing response (im-
mobility excluding respiration and heartbeat) of mice
was measured as an index of fear memory. Data were
collected and analyzed with Image J FZ2 (O’Hara, To-
kyo, Japan; Image J XX is modified software based on
the public domain Image J progr am developed at the U.S.
National Institutes of Health and is available at
http://rsb.info.nih.gov/ij).
4.3. Immunohistochemical Analysis
Brains (n = 4 male mice per group) were fixed with me-
thacarn fixative (methanol/chloroform/acetic acid, 60:30:
10 [v/v]), and paraffin-embedded sections were prepared
[39]. Mouse monoclonal anti-neurofilament (NF-M,
sc-20013; Santa Cruz Biotechnology, CA, USA; 1:1000;
a marker for axons), rabbit polyclonal anti- micro-
tubule-associated protein 1A (MAP1A, sc-25728; Santa
Cruz Biotechnology; 1:1000; a marker for dendrites),
mouse monoclonal anti-synaptophysin (SYP, sc-17750;
Santa Cruz Biotechnology; 1:1000; a pre- synaptic mar-
ker), rabbit polyclonal anti-homer (HOM, sc-15321;
Santa Cruz Biotechnology; 1:500; a post- synaptic
marker), and anti-GluR1(GluR1, sc-28779; Santa Cruz
Biotechnology; 1:500) were used for immunohisto-
chemistry. Sections were pretreated with HistoVT-One
(Na- calai Tesque, Japan) and incubated with primary
antibodies. Signals were visualized with Alexa
568-conjugated anti-mouse IgG and Alexa
488-conjugated anti-rabbit IgG (Molecular Probes, OR).
Images were obtained with an FV-300 confocal la-
ser-scanning microscope (Olympus, Japan).
For semi-quantitative analysis of image, the ratio of
fluorescence intensity was calculated and compared
Arc/Arg3.1-deficient mice to wild-type by using IMAGE
J program (http://rsb.info.nih.gov/ij/index.html. National
Institute of Health, Bethesda), after adjusting back-
ground noise (n = 4 images per mouse).
5. ACKNOWLEDGEMENTS
We thank Dr. Yoshitake Sano for his comments and suggestions re-
garding this study. We also thank Dr. Mariko Katayama for her assis-
tance in maintaining the Arc/Arg3.1-deficient mouse line.
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Abbreviations
Arc/Arg3.1: activity-regulated cytoskeleton-associated
protein; LTP: long-term potentiation; LTM: long-term
memory; STM: short-term memory; NF-M: neuro-
filament-M; MAP1A: microtubule-associated protein
1A; SYP: synaptophysin; HOM: homer; DG: dentate
gyrus.