Behavior Genetics, Vol. 35, No. 5, September 2005 ( 2005)
DOI: 10.1007/s10519-005-5357-7

Distinct Inﬂuences of Neonatal Epidermal Growth
Factor Challenge on Adult Neurobehavioral Traits
in Four Mouse Strains
Manavu Tohmi,1 Noriko Tsuda,1 Makoto Mizuno,1 Nobuyuki Takei,1Paul W. Frankland,2 and Hiroyuki Nawa1,3
Received 1 Jan. 2005 — Final 19 April 2005

Epidermal growth factor (EGF) receptor (ErbB1) signals regulate dopaminergic development
and function and are implicated in schizophrenia. We evaluated genetic eﬀects onneurobehavioral changes induced by neonatal EGF administration, using four mouse strains.
Subcutaneous EGF administration increased phosphorylation of brain ErbB1 in all strains,
although DBA/2 and C57BL/6 mice had lower basal phosphorylation. Neonatal EGF
treatment diﬀerentially inﬂuenced physical and behavioral/cognitive development, depending
on mouse strain. Prepulse inhibition was decreased in DBA/2 and C57BL/6 mice but not C3H/
He and ddY mice. Locomotor activity was accelerated in DBA/2 mice, but reduced in ddY
mice. EGF treatment enhanced fear-learning performance with a tone cue in DBA/2 mice, but
decreased performance with tone and context cues in C3H/He and ddY mice, respectively. The
strain-dependent behavioral sensitivity was correlated with basal ErbB1 phosphorylation.
Genetic components regulating brain ErbB1 signaling strongly inﬂuence the direction andstrength of behavioral responses stemming from the neonatal neurotrophic perturbation.
KEY WORDS: Animal behavior; cytokine; environment; genetic inﬂuence; neurotrophic factor; bodyweight; schizophrenia.

Nakagawa et al., 1998). These ErbB1 ligands enhanceneuronal survival and promote diﬀerentiation, particularly that of midbrain dopaminergic neurons(Alexi and Hefti, 1993; Casper et al., 1991; Ferrari
et al., 1991; Hanke et al., 2004). ErbB1 is expressedin many types of brain neurons, but is especiallyenriched in midbrain dopaminergic neurons(Adamson and Meek, 1984; Gomes-Pinilla et al.,
1988; Kaser et al., 1992; Seroogy et al., 1994). Consistent with ErbB1 distribution, in vivo experiments
demonstrate neurotrophic activity of ErbB1 receptor
ligands in midbrain dopaminergic neurons.
Intracerebroventricular infusion of EGF increases
the survival and function of dopaminergic neurons
in animal models of Parkinson’s disease (Hadjiconstantinou et al., 1991; Iwakura et al., 2005; Ventrella
et al., 1993). Gene-targeting and transgenic studies of
ErbB1 receptor and its ligands, conﬁrms their
biological signiﬁcance in prenatal and postnatal

INTRODUCTION
Epidermal growth factor (EGF) and EGF-related
peptide growth factors, such as transforming growth
factor alpha (TGFa), heparin-binding EGF-like factor (HB-EGF), amphiregulin, betacellulin, and epiregulin, are distributed in various brain regions andall bind to EGF receptors (ErbB1) (Gomez-Pinilla
et al., 1988; Kaser et al., 1992; Kornblum et al., 1997;

1

Division of Molecular Neurobiology, Brain Research Institute,
Niigata University, Asahimachi-dori 1-757, Niigata, 951-8585,
Japan.
2
Department of Neurobiology, Psychiatry, and Psychology, Brain
Research Institute, University of California, Los Angeles, CA
90095, USA.
3
To whom Correspondence should be addressed at Division of
Molecular Biology, Brain Research Institute, Niigata University,
Asahimachi-dori 1757, Niigata 951-8585, Japan. Tel: +81-25-2270613; Fax: +81-25-227-0815; e-mail: hnawa@bri.niigata-u.ac.jp6150001-8244/05/0900-0615/0  2005 Springer Science+Business Media, Inc.

Tohmi et al.

616
development of neocortical structures as well as indopamine physiology (Blum, 1998; Hilakivi-Clarke
et al., 1995; Sibilia et al., 1998).
EGF was ﬁrst described as Tooth-Lid Factor
because it enhances eyelid opening and tooth eruption in newborn mice. Using this activity as an assay,
Cohen (1962) puriﬁed this factor and nerve growth
factor to homogeneity from mouse salivary glandextracts. EGF is alternatively called urogastrone
because receptor-saturating concentrations of EGF
are often present in urine, blood, and amniotic ﬂuid
(Gregory and Preston, 1977). EGF concentrations inblood and amniotic ﬂuid vary among individuals(Peracchi et al., 2001; Varner et al., 1996). Although
EGF eﬃciently penetrates the immature blood–brain
barrier during early development (Futamura et al.,
2003; Pan and Kastin, 1999), the eﬀects of peripheral
EGF and other ErbB1 ligands on brain development
and behaviors remained unknown for many years.
We subsequently found that subcutaneously administered EGF acts on neonatal brain neurons to perturb dopaminergic development and alter animal
behaviors, such as sensory motor gating, social
interaction, exploratory motor activity and amphetamine sensitivity (Futamura et al., 2003; Mizuno
et al., 2004). These ﬁndings indicate that severe
abnormalities in EGF/ErbB1 signaling are induced insome human fetuses or neonates and might inﬂuence
their risk of schizophrenia.
Genetic background and rearing environment
mutually interact with each other and inﬂuence later
behavioral traits such as sensorimotor gating andlearning (Gendreau et al., 1997; Tong et al., 2001;
Waddell et al., 2004). Neurotrophic molecules
including ErbB1 ligands might mediate various
environmental signals and insults to the brain.
For example, the production of ErbB1 ligands, EGF
and HB-EGF, is regulated by neuronal activity,
inﬂammation, and brain injury (Isono et al., 2003;
Kawahara et al., 1999; Opanashuk et al., 1999;
Schaudies and Johnson, 1993; Tanaka et al., 1999).
Maternal separation also inﬂuences production of
TGFa in the neonatal brain (Romeo et al., 2004).
Thus, the injury-, stress- or inﬂammation-triggered
abnormal expression of ErbB1 ligands impairs normal brain development, potentially leading to dopamine-related cognitive/behavioral abnormalities suchas schizophrenia (Isono et al., 2003; Opanashuk
et al., 1999; Tanaka et al., 1999). Consistent with thishypothesis, abnormal ErbB signals are implicated inschizophrenia (Moldin and Gottesman, 1997; Nawa
et al., 2000; Petronis, 2004; Stefanson et al., 2002). A

single nucleotide polymorphism in the EGF gene that
positively regulates its transcription is associated withthe risk of schizophrenia in Finnish men (Anttila
et al., 2004). Abnormal expression of EGF and
ErbB1 receptors is found in the forebrain regions ofschizophrenic patients (Futamura et al., 2002).
Although abnormal EGF/ErbB1 signaling might
be associated with the risk of schizophrenia or contribute to its pathology, it remains to be determined
how genetic components can be incorporated into thishypothesis. In the present investigation, we used
EGF-treated mice as a schizophrenia model andevaluated the signiﬁcance of genotype in determining
behavioral responses to EGF at the adult stage. We
peripherally administered EGF to four different
mouse strains as newborns and compared the strengthand the direction of EGF-behavioral alterations ineach mouse strain. Mice were subjected mainly to the
behavioral tests that are often used to evaluate
schizophrenia models (Gendreau et al., 1997; Swerdlow
and Geyer, 1998). In addition, strain diﬀerence inbasal and EGF-triggered ErbB1 activation was correlated with strain-dependent behavioral changes.

MATERIALS AND METHODS
Animal Protocols
Four strains of neonatal mice, DBA/2 (DBA/2
Cr Slc; 4–6 per litter, 12 l total; SLC, Shizuoka,
Japan), ddY (8–15 per itter, 6 l total; SLC), C57BL/6(C57BL/6 Crj; 7–9 per litter, 7 l total; Nihon Charles
River, Yokohama, Japan) and C3H/He (C3H/He Cr
Slc; 4–6 per litter, 11 l total; SLC), were purchased
together with their dams. The number of pups per
dam was different among strains and not artiﬁciallyadjusted to other mouse strains. Recombinant human
EGF (0.875 lg/g; Higeta Syoyu, Chiba, Japan) was
administered subcutaneously to half of individual
litters of each mouse strain daily during postnataldays (PND) 2–10. The amount of EGF injected was
the minimum dose that reproducibly induces behavioral impairments in rats (Futamuta et al., 2003).
Control littermates of the other half received a saline
injection. After PND 20, mice were separated
according to gender and raised separately (3–5 mice
per cage; 20L · 30W · 12H cm). All micetreated with EGF or saline as neonates were housed
on a 12-h light-dark cycle with free access to food andwater. Physical indices such as body weight, eyelid
opening and tooth eruption were monitored daily(18:00–20:00 hour). All animal experiments wereInteraction between Brain ErbB1 and Genetic Backgrounds
authorized by the Animal Use and Care Committeeof Niigata University and performed during the nightcycle (20:00–2:00 hour). Animals (EGF-treated orsaline-treated mice, n = 12–17 per group for eachstrain) were assigned to two behavioral test batteries
to minimize the effects of the preceding behavioral
test; (a) locomotor test followed by prepulse inhibition (PPI) or (b) startle response followed by thecontextual conditioning task. Mice were subjected tobehavioral tests during PND 56–70 with 3–7-day
interval between tests. Both sexes were equally represented. Mice were habituated to experimentalrooms, at least, 2–3 hours before testing. Aftercompletion of the behavioral testing, mice were
weighed and dissected, and the wet tissue weight ofbrain, spleen and adrenal gland was measured.

617
Measurement of Acoustic Startle Response and PPIUsing an independent set of animals, EGF(0.875 lg/g) or saline was injected subcutaneously
into mouse pups (PND2) to examine the acute effecton ErbB1 activation in the brain. At this dose, thelevel of circulating EGF in the blood is greater than
10 ng/ml (ErbB1 receptor-saturating concentrations)
0.5–2 hour after administration in neonatal rats (datanot shown; Futamura et al., 2003). Thirty minutes
after acute EGF injection, brains were removed andhomogenized by sonication with 2· sample loading
buﬀer. Protein (50 lg/lane) was subjected to an 8–
12% gradient sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, and transferred to a nitrocellulose
membrane. The membrane was probed with
antibodies directed against phosphorylated ErbB1(1:1000; Cell Signaling, Beverly, MA) or against
ErbB1 (1:1000, Santa Cruz Biotech, Santa Cruz, CA),and subjected to chemiluminescence reaction(Amersham-Pharmacia, Tokyo, Japan).

The mice were placed in a plastic cylinder andﬁxed in an automated startle chamber (SR-Lab
Systems, San Diego, CA). After a 5-minute acclimation period with 70-dB-background noise (white
noise), an 80, 90, 100, 110 or 120-dB white noise
stimulus (40 millisecond duration) was given eighttimes to each mouse in the same pseudorandom orderat 15 second-intervals. Analysis for startle amplitudes
was based on the mean of the seven trials (ignoring
the ﬁrst trial) for each trial type.
Using a different set of mice, PPI responses
were measured with 120-dB acoustic stimuli combined with four different prepulse intensities. Eachmouse was placed in the startle chamber (SR-Lab)and initially acclimatized for 5 minute withbackground noise alone (70-dB white noise). The
mouse was then subjected to 48 startle trials, eachtrial consisting of one of six conditions: (i) a 40millisecond 120-dB noise burst presented alone (S),(ii–v) a 40-millisecond 120-dB noise burst following
prepulses by 100 millisecond (20-millisecond noise
burst) that were 3, 6, 9, or 12 dB above background noise (i.e., 73-, 76-, 79- or 82-dB prepulse,
respectively), or (vi) no stimulus (background noise
alone), which was used to measure baseline movement in the chamber. These six trial types (i–vi)
were each repeated eight times in a pseudorandom
order to give 48 trials. The inter-trial interval was
15 seconds. Each trial type was presented once
within a block of six trials and the order of 48 trial
presentations was ﬁxed for all mice. Analysis was
based on the mean of the seven trials for each trial
type. The percentage PPI of a startle response wascalculated as: 100 ) [(startle response on prepulsepulse stimulus trials ) no stimulus trials)/(pulsealone trials ) no stimulus trials)]) · 100) (Braff andGeyer, 1990; Swerdlow and Geyer, 1998).

Analysis of Locomotor Activity

Contextual Conditioning

Exploratory motor activity was measured in anovel environment with the following test equipment
under dim light (27L · 27W · 20H cm, MED
Associates, St. Albans, VA). Mice were placed in an
automated activity monitor equipped with infrared
photosensors at 1.62-cm intervals. Horizontal activity
for every 5 minute was measured as beam crossings
during the initial 60 minute and analyzed by using
fully automated tracking system (Activity Monitor,
Med Associates).

The test paradigm of contextual conditioning was
based on a work by Frankland et al. (2004). Mice wereplaced in a shock chamber with a grid ﬂoor
(10L · 10W · 10H cm box; Obaraika Ltd. Tokyo,
Japan) for 2 minute to monitor baseline movement/
freezing and then exposed to 0.8-mA electric shocks (2second duration, six times for DBA/2, ddY, and C3H/
He, and three times for C57BL/6) all together with 30second tone cues (60 dB, 10 kHz). Preliminary tests
revealed that DBA/2, C3H/He, and ddY mice were

Immunoblot Analysis

618
relatively insensitive to three electric shocks and failed
to produce more than 50% freezing rates comparedwith C57BL/6 mice (data not shown). The number ofshocks was therefore increased for the DBA/2, C3H/
He and ddY mice. One day after conditioning, mice
were returned to the chamber. The time spent freezing,(no movements except those necessary for respiration)
was recorded and scored at 30-second intervals for
3 minute. After 3 hour, mice were moved to a diﬀerent
chamber with a ﬂat ﬂoor (10L· 10W · 10H cm box)
and the time spent freezing was recorded and scoredfor 3 minute before and after the tone cue. Freezing
behavior was automatically monitored by a video
camera during all sessions and analyzed by imaging
software (Obaraika Ltd.).

Tohmi et al.
neonatal brain through the immature blood–brain
barrier (Futamura et al., 2003; Kleshcheva, 1988). In
the present study, we examined whether there arestrain diﬀerences in ErbB1 activation by monitoring
ErbB1 expression and phosphorylation in the brain(Fig. 1a). In control mice, there was no signiﬁcantdiﬀerence in total ErbB1 levels among mouse strains.
A single subcutaneous injection of EGF increased
phosphorylation of ErbB1 in all four-mouse strains(F[1,24]=120, p<0.001) with signiﬁcant strain
differences in ErbB1 phosphorylation levels(F[3,24]=5.65, p=0.004) (Fig. 1b). There was amarginally non-signiﬁcant interaction between treatment and strain (F[3,24]=2.74, p=0.065). A post hoc
test indicated that basal ErbB1 phosphorylation levels in C3H/He and ddY mice were higher than those

Statistical Analysis
Behavioral scores were initially analyzed using
four-way analysis of variance (ANOVA) with genotypes (four strains), gender (two levels), and treatment(two levels) being between-subject factors and withtime (6 or 10 time points) or prepulse (four levels) being
within-subject factors. As the initial ANOVAs yieldedno signiﬁcant outcomes involving the gender variable,
the data of the two genders were combined for the ﬁnal
analyses. When there were signiﬁcant interactions between genotype and treatment for locomotor activity,and startle response, these behavioral scores wereanalyzed separately in each strain to evaluate treatment effects or at each treatment level (EGF or saline)
to examine the genotype effects. A Lavene test revealed
signiﬁcant differences in variance among strains oramong groups in almost all behavioral, physical andbiochemical data (data not shown). Accordingly a nonparametric analysis using a Student–Newman–Keuls
test was performed for post hoc multiple comparisonsto avoid type-2 errors. Only to evaluate EGF effects at
individual data points during repeated measurements
or in biochemical examination, a planned Student’s ttest with Bonferroni’s alpha compensation was usedalternatively. These statistical analyses were
performed using SPSS 11.0 for Windows (n values all
represent the number of animals used).

RESULTS
Strain Differences in ErbB1 Activation Triggered by
Peripheral Injection of EGF
We have previously reported that peripheral
administration of EGF activates ErbB1 in theFig. 1. Brain ErbB1 phosphorylation induced by subcutaneously
administered EGF in mouse pups. Relative magnitudes of ErbB1
phosphorylation in the brain were measured after EGF or saline
administration at postnatal day (PND) 2. Expression of ErbB1
levels was monitored in parallel. EGF solution (0.875 lg/g) orsaline was subcutaneously injected into neonatal DBA/2, C57BL/6,
C3H/He, and ddY mice (n=4 each). (a) Thirty minutes after
injection, the whole brain was dissected and protein extracts were
subjected to immunoblotting for the anti-ErbB1 antibody or antiphospho ErbB1 antibody. (b) The ordinate represents optical density of immunoreactive bands for phospholylated ErbB1 (arbitrary
units, mean ± SEM). *p<0.05, compared with saline-injected
control littermates for each strain and #p<0.05, compared withsaline-treated DBA/2 or C57BL/6 mice by Student’s t-test with
Bonferroni’s alpha compensation. Note; There was no difference in
ErbB1 levels between saline-treated and EGF-treated mice in all
strains (data not shown).

Interaction between Brain ErbB1 and Genetic Backgrounds

619

Table I. EGF Eﬀects on Physical Development of Four Mouse Strains
Control

EGF

Strain

Mean

s.e.

Rank

Mean

s.e

Rank

% Change

Eyelid open (PND)

DBA/2
C57BL/6
C3H/He
ddY

13.1
14.3
12.3
14.5

0.2
0.3
0.3
0.2

e
f
d
f

9.5
9.1
9.5
10.1

0.2
0.09
0.2
0.1

ab
a
ab
c

)27*
)36*
)23*
)30*

Tooth erupt (PND)

DBA/2
C57BL/6
C3H/He
ddY

10.0
11.1
10.2
9.4

0.2
0.2
0.2
0.3

d
e
d
cd

8.3
9.0
8.8
8.2

0.3
0.2
0.3
0.1

a
ab
ab
a

)17*
)19*
)13*
)13*

Body weight PND12 (g)DBA/2C57BL/6
C3H/He
ddY

4.8
4.7
5.9
5.9

0.2
0.3
0.3
0.09

ab
ab
c
c

4.0
4.4
5.7
5.3

0.2
0.3
0.1
0.2

a
a
c
bc

)16
)7
)4
)9

*Marks signiﬁcant diﬀerences between EGF-treated mice and saline-treated controls by Student–Newman–Keuls rank test (n=11–19 pergroup) as a Levene test revealed signiﬁcant diﬀerences in variance in all categories (p=0.002–0.024). The physical indices were indistinguishable between males and females at the early juvenile stage.

in the other strains, but the strain difference was notapparent following EGF treatment.

Effects of Neonatal EGF Treatment on PhysicalDevelopment of Mice
Repeated administration of EGF to mousenewborn pups accelerates eyelid opening and tooth
eruption (Cohen et al., 1962). To compare theperipheral eﬀects of EGF in the four mouse strains,
we administered EGF or saline subcutaneously into
mouse pup (PND 2) daily until PND 10 and monitored these physical indices at the end of the EGF
administration (Table I). Two-way ANOVA withstrain (4) · treatment (2) indicated that EGF treatment stimulated eyelid opening (F [1,102]=837,
p<0.001) and tooth eruption (F [1,107]=109,
p<0.001). There was a strain · treatment interaction
in eyelid opening (F [3,102]=13.2, p<0.001), but not
in tooth eruption. Although post hoc comparisonsrevealed signiﬁcant differences in EGF-triggered
eyelid opening among strains, the basal speedof lid opening in control mice (C3H/He>DBA/
2>ddY=C57BL/6) was negatively correlated withthe magnitude of EGF effects on eyelid opening (i.e.
accelerated days; C57BL/6>ddY>DBA/2>C3H/
He). Administration of EGF had similar effects on
tooth eruption in all strains, although there was abasal strain difference in tooth eruption of salinetreated mice.

We also monitored the immediate inﬂuence ofrepeated EGF administration on body weight(Table I). We compared mean body weight on
PND12, 2 day after completion of EGF treatment.
Two-way ANOVA indicated that EGF treatmentaﬀected body weight (F [1,93]=9.36, p=0.003 fortreatment). There was no strain · treatment interaction. Post hoc comparisons of body weights
among saline- and EGF-treated mice failed todetect a statistical difference in the effect of EGF ineach strain at this developmental stage. The
immediate effect contrasted with the delayed effectsof the treatment on body weight at the adult stage(see below).

Inﬂuences of Neonatal EGF Treatment on Exploratory
Motor Behavior in Adult Mice
The effect of EGF on locomotor activity wasassessed in a novel environment at the adult stage(PND56-62), more than 6 weeks following the ﬁnal
EGF injection. A multiple ANOVA with factors ofstrain (4) · gender (2) · treatment (2) · test duration(12, repeated) revealed a signiﬁcant main effect ofstrain (F [3,101]=16.0, p<0.001), but there was signiﬁcant interactions between strain and treatment(F [3,101]=2.76, p=0.046) and between gender andstrain (F [3,101]=3.21, p=0.026). Test duration had
a signiﬁcant overall effect (F [11,91]=44.6, p<0.001)
and an interaction with strain (F [33,279]=3.40,

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Tohmi et al.

Fig. 2. Exploratory locomotor activity of four mouse strains. Neonatal mice (DBA/2, C57BL/6, C3H/He, and ddY strains) were treated daily
with EGF (0.875 lg/g; closed circle) or saline (25 ll/g; open circle) (n=13–17 each group) from PND 2 to PND 10. Horizontal (the number of
1.7 cm-line crossing) activity in a novel open ﬁeld was measured every 5 minute in adult mice (PND56-62) and was plotted on the ordinate.
Asterisks indicate signiﬁcant relative diﬀerences in ANOVA (*p<0.05). Note: The changes in locomotor activity were indistinguishable
between males and females.

p<0.001) as well. Because of the signiﬁcantinteraction between treatment and strain and no interaction between gender and treatment, we pooled
male and female data to analyze the main effect of
EGF on locomotor activity separately in each strain
by using one-way repeated ANOVA (Fig. 2). Neonatal EGF treatment signiﬁcantly increased exploratory locomotor activity in the DBA/2 mice,compared to their saline-treated control littermates(F [1,24]=4.95, p=0.036). In contrast, EGF treatment suppressed locomotor activity in ddY mice(F [1,25]=5.89, p=0.023). There was no signiﬁcanteffect of EGF in C57BL/6 and C3H/He mice. Thus,
neonatal EGF treatment can either increase or decrease exploratory locomotor activity, depending
upon mouse genetic background.

Inﬂuence of Neonatal EGF Treatment on Acoustic
Startle Response
The effect of EGF on startle response was tested
with ﬁve intensities of white noise in four mousestrains at the adult stage (Fig. 3). A multiple ANOVA
with factors of strain (4) · gender (2) · treatment (2) ·sound intensity (5, repeated) revealed signiﬁcant maineﬀects of EGF (F [1,91]=10.7, p=0.002) and strain(F [3,91]=65.6,
p<0.001),but
notgender(F [1,91]=0.43, p=0.51). There were interactions
between sound intensity and strain (F [12,270]=8.54,
p>0.001) and between sound intensity and treatment(F [4,88]=5.87, p<0.001). Because of signiﬁcantsound intensity · treatment interaction and no
gender · treatment interaction, EGF effects wereInteraction between Brain ErbB1 and Genetic Backgrounds

621

Fig. 3. Comparison of acoustic startle reactions among mouse strains. Neonatal mice (DBA/2, C57BL/6, C3H/He, and ddY strains) were
given EGF (closed circle) or saline (open circle) (n=10–17 for each group) during the early postnatal period. At the adult stage (PND56–62),
relative amplitudes of the startle response to 80-, 90-, 100-, 110- and 120-dB white noise were measured. Data represent mean ± SEM(arbitrary units). *p<0.05, a single startle level compared with saline-injected control littermates by Student’s t-test with Bonferroni’s alphacompensation. A Student–Newman–Keuls test was applied to detect minimum sensitivity of startle in each strain, comparing with thebackground startle at 80-dB noise (+p<0.05), or to compare basal startle amplitudes at 120-dB among strains; C3H/He>C57BL/
6=ddY>DBA/2. Note: The changes in startle amplitude were indistinguishable between males and females. A Levene test revealed signiﬁcant differences in variance at 120-dB level among groups (F[7,99]=3.65, p=0.002).

examined in each strain at 120-dB pulses, poolingmale and female data. Startle amplitudes were signiﬁcantly elevated in DBA/2, C3H/He and C57BL/6
mice but not ddY mice. A Student–Newman–Keuls
test revealed signiﬁcant stain difference in basal sensitivity of startle responses: A minimum response was
detected with the 100-dB pulse in C3H/He and
C57BL/6 mice while that in DBA/2 and ddY mice
was with the 110-dB pulse. These results indicatedthat the magnitudes or threshold levels of soundstartle were not associated with EGF responsivenessof individual mouse strains. Thus, EGF treatment
differentially affected startle responses, depending on
mouse strain, but irrespective of basal startle sensitivity or amplitudes.

Inﬂuence of EGF on PPI in Four Mouse Strains
We measured PPI in all mouse strains at theadult stage (Fig. 4). A multiple ANOVA with factorsof strain (4) · gender (2) · treatment (2) · prepulse
intensity (4, repeated) yielded signiﬁcant maineﬀects for treatment (F [1,97]=12.5, p<0.001),
strain (F [3,97]=12.0, p<0.001), prepulse intensity(F [3,97]=69.8, p<0.001) but not for gender. There
was a marginally non-signiﬁcant interaction between
strain and treatment (F [3,97]=2.20, p=0.093). There
was no other signiﬁcant two-way, three-way, or fourway interaction. Accordingly, PPI scores of males
and females were combined and overall PPI levels
were compared in each strain and at each treatment

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Tohmi et al.

Fig. 4. Strain-speciﬁc eﬀect of neonatal EGF administration on PPI. PPI was measured with 73-, 76-, 79- and 82-dB prepulse in mice treatedneonatally with EGF (closed bar) and control (open bar) littermates of four mouse strains (DBA/2, C57BL/6, C3H/He, and ddY, n=12–17
for each group). Data represent mean ± SEM (% inhibition of main pulse responses). *p<0.05, a single PPI level compared with salineinjected control littermates by planned Student’s t-test with Bonferroni’s alpha compensation. A Student–Newman–Keuls test was applied tocompare basal PPI levels among strains; ddY=DBA/2=C57BL/6>C3H/He Note: The changes in PPI were indistinguishable between males
and females. A Levene test revealed signiﬁcant diﬀerences in variance at all prepulse levels (F[7,105]=3.38–4.53, p<0.003).

level. The neonatal EGF treatment signiﬁcantly
decreased PPI levels in DBA/2 (F [1,24]=12.4,
p=0.002) and C57BL/6 mice (F [1,24]=9.80,
p=0.005), but not in C3H/He or ddY mice. Comparisons of baseline PPI among strains revealed that
only C3H/He mice displayed the lower PPI (seeFig. 4 legend). Although both control ddY and DBA/
2 had the similar basal PPI level, DBA/2 mice reactedwith EGF to reduce PPI levels and ddY mice did not
respond to EGF, retaining the higher basal PPI level.
Strength of the EGF eﬀects on PPI diﬀered amongmouse stains and appeared not to correlate withbaseline PPI levels.

Effect of EGF on Contextual and Auditory
Conditioning in Four Strains of Mice
The effect of EGF on learning was examinedat the adult stage by measuring freezing behavior
after training, in which an electric shock was coupled with a context plus a tone. Because of thedifference in shock sensitivity strains, a factorial
ANOVA was not applied to detect strain differences and a strain-treatment interaction. Freezing
levels were compared separately in each strain as
well as in each testing session with ANOVA[treatment · gender · test duration (repeated, 6
points over 3 minute)]. In DBA/2 mice, ANOVAInteraction between Brain ErbB1 and Genetic Backgrounds

623

Fig. 5. Tone-dependent and context-dependent learning in EGF-treated and untreated mice. EGF- and saline-treated mice of each strain(DBA/2, C57BL/6, C3H/He, and ddY; n=12–16 each) were subjected to shock-paired contextual conditioning with a tone cue at PND 63–70
and, after 1 day, their learning performance was measured with the contextual cue and the tone cue. Mean percentage (±SEM) of freezingtime of saline-treated (open circle) and EGF-treated mice (closed circle) was scored every 30 seconds during conditioning as well as during
testing periods (3 minute). Striped arrows and closed arrows indicate the timing of the context cue given and that of the tone cue given during
test sessions, respectively. Note. The number of electric shocks was reduced in C57BL/6 mice to adjust their freezing levels to those of theother mouse strains (see Materials and methods).

detected a signiﬁcant EGF effect on freezing time
coupled with the tone cue (F [1,27]=18.5,
p<0.001), but not with context. In contrast, theeffect of EGF was reversed in C3H/He mice
(Fig. 5). Neonatal EGF treatment impaired tonedependent learning (F [1,26]=5.02, p=0.034), but
not context-dependent learning in C3H/He mice.
EGF treatment in C57BL/6 mice did not alterlearning performance in either paradigm. Neonatally EGF-treated ddY mice exhibited lower learning ability in the context-dependent test
(F [1,21]=11.0, p=0.003) but not in the tonedependent test. In any of mouse strains, there wereno signiﬁcant differences in freezing rate duringconditioning between EGF- and saline-treated animals, suggesting no inﬂuence of neonatal EGF
treatment on shock sensitivity. There was no gendereffect or no gender · treatment interaction in theabove learning performance of any strain, either.
Thus, shock-triggered learning ability in EGFtreated mice was differentially altered, depending
upon mouse strain and learning paradigm.

Impact of EGF on other Physical Indices at the AdultStage
We also monitored EGF effects on body weight
and tissue weight of brain, spleen and adrenal gland
after completion of the behavioral testing. A multiple
ANOVA of body weight with main factors of strain(4) · gender (2) · treatment (2) revealed that EGF
treatment (F [1,115]=7.07, p=0.009), mouse strain (F[3,115]=97.9, p<0.001), and gender (F [1,115]=13.4,
p<0.001) had signiﬁcant effects on body size without
any interaction between main factors or among thefactors. EGF effects on body weight were further
analyzed in each gender by Student–Newman–Keuls
test (Table II). In male adult mice, there was a signiﬁcant increase in body weight detected in ddY mice.
Female C57BL/6 and C3H/He mice exhibited
increasing trends in averaged body weight, which
failed to reach a signiﬁcance range. The overall
increasing trend in body weight of the adult EGFtreated mice is contrasted with the overall decreasing
trend during postnatal EGF treatment as shown in
Table I.

Tohmi et al.

624
Table II. Delayed Eﬀects of Neonatal EGF Treatment on Body Weight at the Adult Stage
Control

EGF

Strain

Mean

s.e.

Rank

Mean

s.e.

Rank

% Change

Male body weight (g)DBA/2
C57BL/6
C3H/He
ddY

21.1
23.7
23.2
33.8

0.4
0.2
0.7
1.6

a
a
a
b

22.6
24.5
22.8
38.1

0.7
0.4
0.7
1.4

a
a
a
c

+7.1
+3.3
)1.8
+12.7*

Female body weight (g)DBA/2
C57BL/6
C3H/He
ddY

19.3
21.9
16.2
31.4

1.0
0.8
1.0
2.0

ab
ab
ab
c

20.2
25.4
19.4
33.5

0.8
2.2
1.4
2.2

ab
b
ab
c

+4.6
+16.0
+19.8
+6.7

Adult mice, which were treated with EGF or saline as neonates, were weighed on PND 70–84 (n = 5–13 per group for males and n = 4–11
per group for females). *Marks a signiﬁcant diﬀerence between EGF-treated mice and saline-treated controls by Student–Newman–Keuls test.
A Levene test revealed signiﬁcant diﬀerences in variance among groups (p<0.001).

ANOVA revealed that EGF treatment(F [1,131]=19.8, p<0.001) and mouse strain(F [3,131]=72.1, p<0.001) had signiﬁcant effects on
brain size without any interaction between main
factors or among the factors (Table III). There was asigniﬁcant decrease in brain size of the ddY mice, but
not in that of the other strains. It is noteworthy that
the EGF-treated ddY mice were smaller in brain size
but larger in body size at the adult stage. There wasno signiﬁcant eﬀect of EGF treatment on tissue sizeof spleen or adrenal gland and no factorial interaction between EGF treatment and strain or between
treatment and gender, although there were strain andgender diﬀerences in their sizes (data not shown).

DISCUSSION
Our previous study demonstrated that peripherally administered EGF in neonatal Sprague–
Dawley rats efﬁciently penetrates the brain–blood
barrier and acts on immature neurons in the developing brain (Futamura et al., 2003). The immediateeﬀects of EGF injections include the upregulation oftyrosine hydroxylase and dopamine turnover in the
striatum. There are, however, no detectable alterations in cell proliferation and brain structures. Although these neurochemical inﬂuences disappear
until adulthood, neonatal EGF treatment conversely
induces several abnormalities in various behavioral

Table III. Inﬂuence of Neonatal EGF Treatment on Tissue Sizes at the Adult Stage
Control

EGF

Strain

Mean

s.e.

Rank

Mean

s.e.

Rank

% Change

Brain weight (mg)DBA/2
C57BL/6
C3H/He
ddY

370
427
431
461

5.2
2.8
7.5
8.5

a
bc
bc
d

354
411
410
436

3.6
8.2
4.8
5.1

a
b
b
c

)4.3
)3.7
)4.9
)5.4*

Spleen size (mg)DBA/2
C57BL/6
C3H/He
ddY

95
88
72
148

7.9
8.7
2.7
17

a
a
a
b

102
84
71
147

6.3
5.3
3.7
17

a
a
a
b

+7.3
)4.5
)1.4
)0.7

Adrenal gland size (mg)DBA/2
C57BL/6
C3H/He
ddY

0.1
0.1
0.1
0.5

a
a
a
b

0.2
0.1
0.1
0.5

a
a
a
b

0
)5.2
)5.9
+6.6

1.8
1.9
1.7
3.0

1.8
1.8
1.6
3.2

Adult mice, which were treated with EGF or saline as neonates were weighed and dissected on PND 70–84 (n=15–22 per group). Wet tissue
weight of brain, spleen and adrenal gland was measured. *Marks a signiﬁcant diﬀerence between EGF-treated mice and saline-treatedcontrols by Student–Newman–Keuls test. A Levene test revealed signiﬁcant diﬀerences in variance in spleen (p=0.013) and adrenal sizes(p<0.001).

Interaction between Brain ErbB1 and Genetic Backgrounds

625
traits. Endogenous expression of ErbB1 receptor
ligands (EGF, TGFa, HB-EGF, amphiregulin etc.) is
induced by neuronal activity, inﬂammation, andinjury (Isono et al., 2003; Opanashuk et al., 1999;
Tanaka et al., 1999). Rearing conditions also aﬀect
the production of TGFa in the neonatal brain (Romeo et al., 2004). Therefore, genetic background of
individual animals might inﬂuence physiologic andpathologic consequences of brain ErbB1 activation
during brain development.

and pharmacological tests for locomotion, PPI,
antipsychotic sensitivity and methamphetamine sensitization (Futamuta et al., 2003; Mizuno et al.,2004), which involve the dopaminergic system(Gendreau et al., 1997; Swerdlow and Geyer, 1998;
Zocchi et al., 1998). Thus, this animal appears to be
useful as an animal model for schizophrenia (Nawaand Futamura, 2003). The present study demonstrates that neonatal EGF treatment produces similar
behavioral alterations in mice, but EGF inﬂuences on
individual behaviors varied considerably depending
on mouse strain (Table IV).

Strain-speciﬁc Behavioral Responses and Peripheral
Reactions to EGF

Irregular Behavioral Responses of Mouse Strains andBrain ErbB1 Signaling

In our experiments, neonatal EGF treatment
facilitated eyelid opening and tooth eruption, conﬁrming the biological activity, as described previously(Cohen, 1962). There was no correlation, however,between the acceleration of these physical indices andthe adult behavioral changes induced by EGF. BothPPI responders (DBA/2 and C57BL/6 mice) and PPI
non-responders (C3H/He and ddY mice) had thesame degree of accelerated eyelid opening and tooth
eruption. The strength of EGF eﬀects on body weightor brain weight at the adult stage was most marked in
ddY mice and, therefore, was not consistent with thaton behavioral traits, either. These results suggest thatstrain-dependent behavioral responses to EGF are
independent of the peripheral actions of EGF. In
contrast, the behavioral responses appeared to becorrelated with basal phosphorylation levels of brain
ErbB1 in individual mouse strains. Although there
was no signiﬁcant diﬀerence in total ErbB1 levels,
basal phosphorylation levels were lower in PPI
responders and higher in PPI non-responders. After
treatment with receptor-saturating EGF, however,ErbB1 phosphorylation was approximately equivalent in all four strains. Therefore, the strain diﬀerence
in basal ErbB1 phosphorylation might reﬂect

Neonatal EGF treatment had opposite effects on
exploratory locomotor activity in DBA/2 and ddY
mice (Table IV). Similar strain diﬀerences following
neonatal EGF treatment were observed in the PPI
test and learning tasks. EGF treatment enhanced
auditory learning performance in DBA/2 mice,
whereas it depressed contextual and auditory learning
in ddY and CH3/He mice, respectively. Neonatal
EGF treatment reduced PPI levels in DBA/2 and
C57BL/6, but not in C3H/He and ddY mice. Basedon the behavioral inﬂuence of EGF, we classiﬁed themouse strains into two categories: PPI responders
and PPI non-responders. The PPI responders include
DBA/2 and C57BL/6 mice, which show a decrease in
PPI and/or improved learning following neonatal
EGF challenge. The PPI non-responders include
C3H/He and ddY mice, which exhibit decreased
learning and no signiﬁcant response to EGF in PPI.
These behavioral variations among mouse strains are
not correlated with physical responses to EGF. The
present ﬁndings highlight the biological signiﬁcanceof genetic background in determining the direction
and strength of EGF inﬂuences on neurobehavioral

Table IV. Summary of Behavioral Eﬀects of Neonatal EGF Treatment in Four Mouse Strains
Context and auditory fear
learning
Mouse strains
DBA/2
C57BL/6
C3H/HeddY

Exploratory locomotion

Startle response

PPI

Context

Tone cue

UP
=
=
Down

UP
UP
UP
=

Down
Down
=
=

=
=
=
Down

UP
=
Down
=

=not statistically signiﬁcant. All behavioral data were obtained at the adult stage (PND56-75).

626
variations in endogenous EGF levels among mousestrains. In agreement, C57BL/6 mice are reported to
produce the lowest level of EGF in the salivary gland
among many mouse strains (Tom-Moy and Barka,
1981). EGF/TGFa/ErbB1 signals have crucial rolesin the development of dopaminergic neurons andneural stem cell proliferation (Alexi and Hefti, 1993;
Blum, 1998; Casper et al., 1991; Ferrari et al., 1991;
Hanke et al., 2004; Sibilia et al., 1998). There are
genetic diﬀerences among mouse strains in basal
tyrosine hydroxylase activity and the number of
dopamine neurons in the midbrain area (Baker et al.,1980). Thus, the strength of the ErbB1 phosphorylation before or after neonatal EGF treatment inﬂuences ErbB1-dependent dopaminergic diﬀerentiationor maturation, leading to strain-speciﬁc behavioral
alterations.
Interaction between EGF-triggered Behavioral
Changes and Basal Traits
Many behavioral traits, such as sensorimotor
gating, cognitive performance, and learning ability,
are under apparent genetic control (Crawley et al.,
1997; Marks et al., 1989; Nguyen et al., 2000;
Wehner et al., 1997). Inbred mouse strains diﬀer inbasal acoustic startle response and in PPI levels;
Among the various mouse strains, DBA/2 mice are
relatively insensitive in sound-startle reactions andC3H/He mice have a lower PPI level, which is consistent with the present results (Bullock et al., 1997;
Crawley et al., 1997; Dulawa and Geyer, 1996; Markset al., 1989). The baseline startle amplitudes of PPI
non-responder mice, ddY, were similar to those of the
PPI responder mice, C57BL/6. Control ddY, C57BL/
6, and DBA/2 mice all had similar basal PPI levels,
but only ddY mice failed to alter PPI levels in response to neonatal EGF challenge. Thus, there wasno correlation between baseline startle amplitudes
and EGF eﬀects on PPI or between baseline PPIlevels and the EGF eﬀects.
Neonatal EGF treatment in DBA/2 mice
improved performance in learning with an auditorycue. In contrast, EGF treatment led to the opposite
behavioral changes in learning of other strains: PPI
non-responders of C3H/He and ddY mice displayed
reduced performance of learning with tone and visualcues in response to EGF, respectively. Althoughhigher auditory sensitivity of EGF-treated DBA/2
mice contributed to their better tone-dependent
learning, (Nguyen et al., 2000; Wehner et al., 1997), it
is diﬃcult to correlate the strength and direction ofTohmi et al.
the EGF-triggered changes in learning of C3H/He
and ddY mice with higher or lower sensory sensitivityof these animals. Learning involves multiple brain
processes including primary sensation, sensory
modality, and integration, which are supported by
diﬀerent brain regions. For example, visual fear
learning is known to depend hippocampal mechanism while auditory fear learning involves amygdala
processing (Anagnostaras et al., 2000; Phillips andLeDoux. 1992; Sacchetti et al., 1999). Thus, one ofthe potential explanations is that the strain-dependent or area-speciﬁc sensitivity to EGF might diﬀer
among strains and contribute to the above straindiﬀerence in EGF responses in learning. The fact thatthe cell proliferation activity of EGF is signiﬁcantly
diﬀerent among mouse strains supports this hypothesis (Parzefall et al., 2002). Alternatively, it is possiblethat EGF permeability into these brain regions might
diﬀer considerably among mouse strains, although
this assumption also remains to be tested.
Implication of Genetic Background in Schizophrenia
Abnormal brain development as well as dopaminergic dysfunction is implicated in the etiology or
pathology of schizophrenia (Murray and Lewis, 1987;
Weinberger, 1987). Among many neurodevelopmental regulators, ErbB1 ligands (EGF, HB-EGF,
TGFa, etc.) have strong trophic activity on midbrain
dopaminergic neurons (Hadjiconstantinou et al.,
1991; Iwakura et al., 2005; Seroogy et al., 1994;
Ventrella et al., 1993). Our previous postmortem
study on schizophrenic patients indicates that EGF
content is speciﬁcally reduced and ErbB1 receptor
levels are conversely upregulated in the striatum(Futamura et al., 2002). Based on the above hypothesis, as well as the fact that schizophrenic patients
carry abnormal EGF/ErbB1 signals (Futamura et al.,2002), the present animal model was initially established with EGF (Futamura et al., 2003). This animal
has several characteristics that ﬁt a schizophrenia
model; (1) Behavioral abnormalities, such as reduced
sensorimotor gating and accelerated rearing behaviors, develop at post-pubertal stages and persist forlife. (2) Most of these abnormalities can be ameliorated by typical or atypical antipsychotic drugs. (3)There is a reduction in brain weight with no apparent
sign of gliosis or neurodegeneration. Accordingly, theEGF-treated rat presumably serves as a good model
for schizophrenia. EGF-treated mice exhibited or did
not exhibit impairments in sensorimotor gating
and context learning, depending on mouse genetic

Interaction between Brain ErbB1 and Genetic Backgrounds
backgrounds. Although patients with schizophrenia
display similar sets of cognitive and learning impairments such as sensorimotor gating and context
learning, there are large inter-individual diﬀerences inpatients’ cognitive/learning performance (Braﬀ et al.,
1992; Hsieh et al., 2004; Kumari et al., 2004; Sullivan
et al., 1997). Thus, the present mouse study indicatesthat the inter-individual variation in pathophysiologic indices of schizophrenic patients might also involve genetic diﬀerences in ErbB1/EGF signaling orother neurotrophic sensitivity.
Previous extensive studies on schizophrenia
indicate the importance of both environmental andgenetic inﬂuences on schizophrenia etiology. Environmental factors include maternal viral infection
and birth complications that increase the risk forschizophrenia or impair animal behaviors (Cannon
et al., 2002; Lipska et al., 1998; Narayan et al., 1983;
O’Callaghan et al., 1991, 1992; Salvatore et al., 1997;
Shi et al., 2003). Both brain insults and infections are
known to induce a variety of neurotrophic cytokines
including EGF (Isono et al., 2003; Kawahara et al.,
1999; Opanashuk et al., 1999; Schaudies and Johnson,
1993; Tanaka et al., 1999). It is noteworthy that EGF
in the amniotic ﬂuids is markedly increased toreceptor-stimulating levels in some pregnant women(Hofmann and Abramowicz, 1990; Varner et al.,1996). Thus, it is possible that neurotrophic cytokines, such as EGF, mediate the above environmental
insults or stress in human embryos and neonates toinﬂuence the future risk of schizophrenia (Nawaet al., 2000; Petronis, 2004).
A gene(s) is another factor that markedly inﬂuences the risk of schizophrenia as well as the behavioralfeatures of its models (Lipska and Weinberger, 1995;
Moldin and Gottesman, 1997; Pletnikov et al., 2002).
In particular, the present results illustrate the strengthand complexity of the interaction between genetic(mouse strain) and environmental (neonatal EGF
challenge) factors in behavioral development,
although many questions regarding their interactions
remain unanswered. Genetic background inﬂuences
whether a particular set of behavioral changes occur,
but it also determines the direction of the behavioral
change following exposure to EGF. We assume that, if
abnormal EGF/ErbB1 activity occurs in human fetuses or neonates with diﬀerent genetic backgrounds,
the resulting behavioral traits will be diverse and possibly distinct in pathophysiology. The present results
indicate that the biological interaction between genetic
background and neurotrophic conditions during

627
development determines later psychobehavioral traits
in animals, possibly including human beings.
ACKNOWLEDGMENTS
This work was supported by a grant-in-aid for
Creative Scientiﬁc Research, the Targeted Research
Grant for Brain Sciences, and a grant-in-aid from the
Ministry of Welfare and Labor, Japan. We thank Dr.
A. Silva for many kind instructions of behavioral
analyses and Ms. E. Higuchi for technical assistances.

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Edited by Jeanne Wehner