Habituation of exploratory behaviour in VPA rats: Animal model of autism

Abstract

Autism is a neurodevelopmental disorder with multifactorial aetiology, represented as impairment in social behaviour, communication and the occurrence of repetitive activities, which can be observed in the early life. The core features are frequently accompanied by other manifestations, including limited environmental exploration. The aim of the presented study, realised on an animal model of autism - VPA rats, i.e. animals prenatally affected with valproic acid on gestation day 12.5, was to investigate the habituation process of exploratory activity (manifested by a gradual decrease in the intensity of locomotor activity), which reflects the stage of the central nervous system. VPA rats were tested in open-field in three developmental periods - weaning (postnatal day 21 - PND 21), puberty (PND 42) and adulthood (PND 72). In each period of ontogenesis, the rapidity of habituation was evaluated by using the method of linear regression. Compared to controls, VPA rats showed a significant decrease in the intensity and an increase in the rapidity of exploratory activity habituation during puberty and adulthood. Our results indicate that the animal model of autism, i.e. VPA rats, showed disabilities in the development of the nervous system. These findings can help confirm not only the validity of this animal model of autism but can also help better understand neuronal changes in humans with autism.

Full text

interdisciplinary
Habituation of exploratory behaviour
in VPA rats: animal model of autism
Lucia OLEXOVÁ 1, Tomáš SENKO 1, Peter ŠTEFÁNIK 1, Alžbeta TALAROVIČOVÁ 2, Lucia KRŠKOVÁ 1
1 Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia
2 Neuroendocrinology Group, Faculty of Mathematics and Natural Sciences, University of Groningen, Groningen, The Netherlands
ITX060413A04 • Received: 03 December 2013 • Revised: 18 December 2013 • Accep ted: 28 Decembe r 2013
ABSTRACT
Autism is a neurodevelopmental disorder with multifactorial aetiology, represented as impairment in social behaviour, communication
and the occurrence of repetitive activities, which can be observed in the early life. The core features are frequently accompanied by
other manifestations, including limited environmental exploration. The aim of the presented study, realised on an animal model of
autism – VPA rats, i.e. animals prenatally affected with valproic acid on gestation day 12.5, was to investigate the habituation process
of exploratory activity (manifested by a gradual decrease in the intensity of locomotor activity), which reflects the stage of the central
nervous system. VPA rats were tested in open-field in three developmental periods – weaning (postnatal day 21 – PND 21), puberty
(PND 42) and adulthood (PND 72). In each period of ontogenesis, the rapidity of habituation was evaluated by using the method
of linear regression. Compared to controls, VPA rats showed a significant decrease in the intensity and an increase in the rapidity of
exploratory activity habituation during puberty and adulthood. Our results indicate that the animal model of autism, i.e. VPA rats,
showed disabilities in the development of the nervous system. These findings can help confirm not only the validity of this animal
model of autism but can also help better understand neuronal changes in humans with autism.
KEY WORDS: autism; animal model; VPA rats; exploratory behaviour; habituation
Correspondence address:
Lucia Olexová, PhD.
Department of Animal Physiology and Ethology,
Faculty of Natural Sciences, Comenius University in Bratislava
Mlynská dolina B-2, 842 15 Bratislava 4, Slovakia
TEL.: +421 2 60296 680 • FAX +421 2 60296 576
E-MAIL: olexoval@fns.uniba.sk
Psychiatric Association, 2000) and delayed motor devel-
opment (Ohta et. al., 1987; Landa & Garrett-Mayer, 2006;
Provost et al., 2007; Downey & Raport, 2012; Flanagan et
al., 2012). However studies on autism focused primarily
on social deficit. According to Kawa and Pisula (2010),
only the analysis of behaviour not directly linked with
social interaction may provide new important informa-
tion about autism.
Recently, some authors have explored links between
autism and limited environmental exploration. Children
with autism spent significantly less time by active explora-
tion than normal children (O’Neil & Happé, 2000; Pierce
& Courchesne, 2001).
Moreover, animal models of autism, such as rats
prenatally exposed to valproic acid (VPA rats) (Schneider
& Przewłocki, 2005; Schneider et al., 2006; Kerr et al.,
2013), BALB/cByJ (Moy et al., 2008), Pax6 heterozygous
mutant (rSey
2
/+) rats (Umeda et al., 2010), and mice with
disruption in chromosome 7 (analogue of human locus
15q11-13) (Tamada et al., 2010), showed, in addition to
other ty pical autistic behavioural deficits, also a reduction
of exploratory behaviour.
The observed changes in exploratory behaviour are
strongly connected with alterations in particular brain
Introduction
Autism is a neurodevelopmental disorder with multifac-
torial aetiology characterised by severe deficits in social
reciprocity and communication and an occurrence of
repetitive behaviour (American Psychiatric Association,
2000). This disorder is usually diagnosed by three years
of age (Levy et al., 2009), but video analyses of infants
diagnosed later with autism have revealed disruptions in
development already by the first year of life (Teitelbaum et
al., 1998; Baranek, 1999; Osterling et al., 2002; Esposito,
2011). The core manifestations of autism are frequently
accompanied by other features, e.g. hyperactivity
(Canitano & Scandura, 2011), sleep disturbances, obses-
sive-compulsive behaviour (Amaral & Corbett, 2003),
aberrant sensitivity to sensory stimulation (Militerni et
al., 2000), anxiety (Groden et al., 1994; Gillott et al., 2001;
Tow b in et al., 2005), decrease of motivation (American
Interdiscip Toxicol. 2013; Vol. 6 (4): 222–227.
doi: 10.2478/ intox -2013-0 033
Published online in:
www.intertox.sav.sk & www.versita.com/it
Copyright © 2013 SETOX & IEPT, SASc.
This is an Open Acces s article distribu ted under the terms of the Cr eative Commons Attribu-
tion License (ht tp://creativecommons.org/li censes/by/2.0), which permits u nrestricted us e,
distribu tion, and reproductio n in any medium, provided the or iginal work is properly cited.
ORIGINAL AR TICLE

223
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Copyright © 2013 SETOX & Institute of E xperimental Pharmacol ogy and Toxicology, SASc.
areas. Changes in the number of Purkinje cells in cerebel-
lar vermal lobules have been observed in both VPA rats
(Rodier et al., 1996, 1997) and autistic patient (Pierce &
Courchesne, 2001). The decreased number of Purkinje
cells correlated with reduced environmental exploration
(Pierce & Courchesne, 2001). Alteration in structures
involved in regulation of fear processing can provide a
further explanation of reduced exploratory behaviour
and similar, changes have been observed in VPA rats
(Mark ram et al., 2008; Rinaldi et al., 2008; Sui & Chen,
2012) as well as in autistic patients (Schumann et al.,
2004; Bachevalier & Loveland 2006).
The aim of the presented study was to investigate the
habituation process of exploratory behaviour of VPA rats
(manifested by a gradual decrease in the intensity of loco-
motor activity in open-field). This process is known to
reflect a stage of the development of the central nervous
system (CNS) (Morokuma et al., 2004) and is changing
during development (Parsons et al., 1973; Chapillon &
Roullet, 1997; Lynn & Brown, 2009).
Habituation is defined as “the decrement in response
following repeated stimulation with the same stimu-
lus” and it is considered to be a basic form of learning
(Morokuma et al., 2004). In rodents, habituation is com-
monly evaluated as a quantitative decrease in exploratory
behaviour in response to continued or repeated exposure
to an unknown environment (Leussis & Bolivar, 2006).
Impaired habituation has been reported in individuals
with Down’s syndrome (Hepper & Shahidullah, 1992),
schizophrenia (Ludewig et al., 2003), mental retardation
(Gandhavadi & Melvin, 1985), depression (Michael et al.,
2004), epilepsy (Rogozea & Florea-Ciocoiu, 1982), and
Alzheimer’s disease (Vanini et al., 2010).
Impaired neural habituation has been reported in
people with autism (Kleinhans et al., 2009). Toddlers with
more severe feat ures of autism showed slower habit uat ion
to faces (Webb et al., 2010).
Information about changes of the VPA rat habitua-
tion process during ontogenesis can help to confirm not
only the validity of this animal model of autism but also
contribute to a better understanding of changes in CNS
functional development in humans with autism.
Methods
Animals
Wistar rats (200–300 g, Institute of Experimental
Pharmacology and Toxicology, Dobra Voda, SR) were
hous ed in g roup s of t wo or t hree a nim als i n stan dard l ight
conditions (12:12; lights on at 6 a.m.) with food (Dos-2b
OVO, Dobra Voda, SR) and water ad libitum. After an
acclimatisation period of 7 days, male and female rats
were mated overnight and the presence of spermatozoa
in vaginal smear was considered the first day of gestation
(GD 1) (Schneider & Przewłocki, 2005; Schneider et al.,
2006). On GD 12.5 (in the middle of the light phase), half
of the females received a single intraperitoneal injection
of sodium valproate (VPA; Sigma, USA) dissolved in
saline (pH = 7.3; 250 mg/ml) in the dose of 600 mg/kg.
The other females served as a controls (C) and received
saline (pH = 7.3; 250 mg/ml) at the same time of gestation
(Schneider & Przewłocki, 2005; Schneider et al., 2006).
Females were housed in groups of two or three animals
until GD 20, later they were housed individually.
After delivery, valproate-treated (VPA) and control
(C) females were allowed to raise their offspring until
weaning on postnatal day (PND) 21. On PND 1, the litters
were culled to 8 animals per litter (4 males, 4 females).
The other animals of the litter were used for sample
preparation for fu rther analysis. After wea ning, the rats of
either sex were housed separately in groups of 4 animals
per cage.
The offspring of VPA (n=18; 9 males, 9 females) and
C (n=18; 9 males, 9 females) were tested in the open-field
test in three developmental periods: weaning (PND 21),
puberty (PND 42) and adulthood (PND 72).
Behavioural test
Spontaneous exploratory behaviour (locomotor activ-
ity) and its habituation were evaluated in an open-field
test. All animals (VPA and C) were tested in Conducta
(Experimetria Ltd., Hungary), a system used the continu-
ous recording of kinetic activity of laboratory rodents. The
testing chamber of this system consisted of a dark plastic
box (48×48×40 cm) with its floor divided into 25 squares
and built-in infrared beam lights (16 diodes at 16 mm
distance from each other in the three lines) for recording
the animals’ movement.
During the light phase (12:00–18:00 h), each animal
was put into the centre of the testing chamber and sub-
sequently tested for 20 minutes. The information about
locomotor activity (horizontal + vertical), expressed as
a number of beam breaks during horizontal and vertical
movement, were exported from Conducta system in four
five-minute intervals (the first interval: 0–5 min; the
second interval: 5–10 min, the third interval: 10–15 min
and the fourt h interval: 15–20 min). A gradual decrease of
locomotor activity during the test represented an uninter-
rupted habituation.
Statistical analysis
The habituation process was evaluated using the expo-
nential function Y(t) = Y
0
.e
–kt
(Y = the amount of locomo-
tor activity in the individual five minute interval, k = the
individual rate of habituation) was used as a model of the
habituation course of locomotor activity (Dubovický et
al., 1999).
The intensit y of locomotor activity (for the whole dura-
tion of the test) and k-value of habituation of VPA and C
animals were analysed using STATISTICA v 7.0 (StatSoft,
Inc., Tulsa, USA) and repeated measures analysis of vari-
ance (ANOVA) with the fixed effects of group, sex, and
age and their interaction. The effect of litter nested within
the group was included in the model. If the interaction
was significant, the dif ferences between individua l groups
were evaluated by Fisher LSD post hoc test. The results are
expressed as means ± SEM.

224
Lucia Olexová, Tomáš Senko, Peter Štefánik, Alžbeta Talarovičová, Lucia Kršková
Animal model of autism
ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)
Ethics statement
The methods and procedures of the present study were
approved by the local Ethical Committee of the Comenius
University in Bratislava, Slova k Republic and the Directive
of the European Parliament a nd of the Council on the pro-
tection of animals used for scientific purposes (2010/63/
EU) was followed. All efforts were made to minimise the
number of animals used and their suffering.
Results
A statistical analysis of locomotor activity has revealed
a significant effect of the group (F
1,28
=19. 391; p<0.001),
litter (nested within the group) (F
4,28
=6.182; p<0.01),
age (F
2,56
=10.972 ; p<0.001), and interaction age*group
(F
2,56
=4.622; p<0.05). VPA rats showed decreased loco-
motor activity compared to the control group in puberty
(p<0.001) and in adulthood (p<0.001) (Figure 1). In wean-
ing, there were no significant differences between VPA
and C rats. Sex differences were not found at any stage of
ontogenesis. In the VPA group, there were no significant
differences in locomotor activity among the three ontoge-
netic stages. We observed changes in the developmental
period only in C animals. These animals showed an
increase of locomotor activity in puberty (p<0.001) and
adulthood (p<0.001), compared to the weaning period
(Figure 1).
Statistical analysis of habituation rate (k-value) has
revealed a significant effect of group (F
1,28
=8.702; p<0.01),
age (F
2,56
=25.5 03; p<0.001), and interaction age*group
(F
2,56
=7.605; p<0.01). Differences in habituation rate
during the weaning period were not significant between
VPA and C group (Figure 2). VPA rats showed a higher
rate of habituation in the open-field compared to C rats
in puberty (p<0.01) (Figure 3) and in adulthood (p<0.001)
(Figure 4). Similarly to locomotor activity, sex differences
were not found at any stage of ontogenesis.
0
100
200
300
400
500
600
700
800
900
1000
weaning puberty adulthood
locomotor activity
(number of complete-light suspension)
C
VPA
*** ***
+++
+++
locomotor activity
(number of complete-light suspension)
0
50
100
150
200
250
300
0-5 min 5-10 min 10-15 min 15-20 min
time interval
C
VPA
0.00
0.10
0.20
0.30
0.40
CVPA
k-value
locomotor activity
(number of complete-light suspension)
0
50
100
150
200
250
300
0-5 min 5-10 min 10-15 min 15-20 min
time interval
C
VPA
0.00
0.10
0.20
0.30
0.40
CVPA
k-value
**
Figure 1. Locomotor activity of control (C, n=18) and valproic
(VPA, n=18) rats in the open- eld test. Data are given as means
± SEM per 20 min. Asterisks indicate signi cant di erences
between C and VPA group (*** p<0.001), plus they indicate sig-
ni cant di erences between ontogenetic stages in the C group
(+++ p<0.001).
Figure 2. Habituation dynamics and rate (k-value) of control (C,
n=18) and valproic (VPA, n=18) rats during weaning. Data are
given as means ± SEM.
Figure 3. Habituation dynamics and rate (k-value) of control (C,
n=18) and valproic (VPA, n=18) rats in puberty. Data are given as
means ± SEM. Asterisks indicate signi cant di erences between
C and VPA groups (** p<0.01).
Significant changes in k-value were observed between
weaning and puberty (means ± SEM: weaning 0.01±0.03;
puberty 0.27±0.03; p<0.001), and between weaning and
adulthood (means ± SEM: weaning 0.01±0.03; adulthood
0.26±0.02; p<0.001) in VPA group as well as between
weaning and puberty (means ± SEM: weaning 0.07±0.02;
puberty 0.16±0.03; p<0.05) in C group.
Discussion
Exploratory behaviour is one of the basic forms of
behaviour and its prenatal disruption can reflect a CNS
developmental damage. Over the last 10 years, more

225
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Interdisciplinary Toxicology. 2013; Vol. 6(4): 222–227
Copyright © 2013 SETOX & Institute of E xperimental Pharmacol ogy and Toxicology, SASc.
links have been made between autism and limited envi-
ronmental exploration (O’Neil & Happé, 2000; Pierce &
Courchesne, 2001).
Our study performed on an animal model of autism
supports these findings. Prenatal exposure to VPA on
the 12.5
th
day of gestation had an effect on postnatal
exploratory behaviour and habituation of this activity in
rats during ontogeny.
When compared to the C group, VPA rats showed
a lower level of locomotor component of exploratory
behaviour in puberty and adulthood, but the groups did
not differ at weaning. Lower exploratory behaviour of
VPA rats (compared to C group) was described in puberty
(Schneider & Przewłocki, 2005; Schneider et al., 2006;
Kerr et al., 2013) and also in adulthood (Schneider &
Przewłocki, 2005; Schneider et al., 2006), but informa-
tion about exploratory behaviour of these rats during the
weaning period are missing. Reduction in exploratory
behaviour in adulthood was documented in other animal
models of autism – Pax6 heterozygous mutant (rSey
2
/+)
rats with alter ations in serotonergic system (Umeda et al.,
2010), mic e with dis rup tion i n ch romo some 7 (an alo gue of
human locus 15q11-13) (Tamada et al., 2010), BALB/cByJ
mice (Moy et al., 2008).
Our findings that did not show any significant differ-
ences between the quantity of locomotor activity of VPA
and C groups in the weaning period support the research
of Ohta et al. (1987) realised on autistic individuals.
According to these authors, developmental motor delays,
only minimally different during infancy, may become
magnified with age. However, the human study of
Flanagan and colleagues (2012) documented the existence
of qualitative motor development differences in 6-month-
aged children. Therefore, further studies are needed
to reveal other potential qualitative motor as well as
behavioura l changes in the early development of VPA rats.
The intensity of exploratory behaviour and the loco-
motor part of this activity increased during adolescence
(Lynn & Brown, 2009). In our study, C animals showed a
significant increase of locomotor activity in puberty and
adulthood compared to the weaning period. But in the
VPA group, there were no significant differences between
ontogenetic stages and an increase in locomotor activity
during adolescence was absent. Changes in exploratory
behaviour can reflect developmental damage of the CNS.
One potential explanation of reduced exploratory behav-
iour in VPA rats may be reduced number of Purkinje cells
in cerebellar vermal lobules (Rodier et al., 1996, 1997).
Similarly, reduced cerebellar vermal lobules, which cor-
related with reduction of exploration, were observed by
Pierce and Courchesne (2001) in autistic children. The
second potential explanation could be changes in neural
structures involved in regulation of fear. This includes the
medial prefrontal cortex and amygdala. Abnormalities in
these structures were observed both in VPA rats (Markram
et al., 2008; Rinaldi et al., 2008; Sui & Chen, 2012) and
in autistic people (Schumann et al., 2004; Bachevalier &
Loveland 2006). According to Schneider and Przewłocki
(2005), decreased exploration in adult VPA rats may be
mediated rather by fear-related inhibition of exploratory
behaviour. In an autistic population, even minor changes
in the environment may induce confusion and distress,
while fear of a possible change can be a further source of
anxiety (Groden et al., 1994; Gillott et al., 2001).
However, reduction of exploration may be the result
of decreased motivation to explore a novel environment
(Schneider & Przewłocki, 2005), which is related with
changes in neural structures involved in regulation of
motivation. Decrease of motivation is one of the core
symptoms of autism defined by the American Psychiatric
Association (2000).
We did not observe significant changes in locomotor
activity between males and females, neither within indi-
vidual groups, nor in total. And yet it is well known that
females should be more active than males from pubert y
onwards (Lynn & Brown, 2009).
The quantitative changes in exploration of an unknown
environment (testing chamber) are associated with the
process of habituation. Habituation is classically defined
as the waning of a response, elicited by repeated exposure
to a novel stimulus not accompanied by any biologically
relevant consequence, either positive or negative (Leussis
& Bolivar, 2006). This process reflects the neurological
development of individuals and is changeable during
ontogenesis. Some studies indicate that younger animals
do not react as do older animals in exploratory situations
(Chapillon & Roullet, 1997).
Our VPA treated rats exhibited changes in decrease of
exploratory behaviour after exposure to a novel environ-
ment and habituated more rapidly than did C in puberty
and adulthood. Similarly as in exploratory behaviour, we
did not find any differences in the habituation process
during the weaning period.
locomotor activity
(number of complete-light suspension)
0
50
100
150
200
250
300
0-5 min 5-10 min 10-15 min 15-20 min
time interval
0.00
0.10
0.20
0.30
0.40
CVPA
k-value
***
C
VPA
Figure 4. Habituation dynamics and rate (k-value) of control
(C, n=18) and valproic (VPA, n=18) rats in adulthood. Data are
given as means ± SEM. Asterisks indicate signi cant di erences
between C and VPA groups (*** p<0.01).

226
Lucia Olexová, Tomáš Senko, Peter Štefánik, Alžbeta Talarovičová, Lucia Kršková
Animal model of autism
ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)
In ontogenesis, VPA rats, comparably to C, showed an
increased habituation rate during adolescence. Younger
animals, in both cases, showed a lower rate of habituation,
which corresponds to literature information that young
animals habituate more slowly than do their older coun-
terparts (Parsons et al., 1973; Chapillon & Roullet, 1997).
Information ab out the habituation process in VPA rats have
so far been absent, but in autistic patients a slower neural
habituation i n the amygdala and h ippocampus wa s reported
after pre sentation of images of faces – visua l social stimulu s
(Kleinhans et al., 2009; Webb et al., 2010). In our observa-
tions, the rate of habituation, as a reaction to an unknown
environmental stimulus was increased in VPA rats.
The habituation process can be affected by a variety of
factors including arousal level, attention, learning, mem-
ory, and fear of novelty influencing exploratory behaviour
and thus also habituation (Leussis & Bolivar, 2006).
We hypothesise that differences in the rate of habitu-
ation may be related with differences in stimulus charac-
ters and with different motivational value of this stimulus.
However, the understanding of changes occurring in
habituation requires further studies, aimed especially
on discovering neural mechanisms participating in the
regulation of this process in VPA rats.
VPA rats show many features characteristic for the
autistic population, but in our opinion these findings call
for further research. In our article we mentioned only
behavioural changes in VPA rats but we will continue in
deciphering neural mechanism associated with regula-
tion of the specific types of behaviour observed in these
animals.
The questions why behavioural changes were dis-
covered in puberty and adulthood and why we did not
observe intersex differences in contrast to other authors,
remain still open.
Conclusion
Our behavioural study supports the findings of devel-
opmental changes in the nervous system of VPA rats.
These changes are not only demonstrating as changes in
locomotor activity but also as so far unpublished changes
in the process of habituation. We believe that these find-
in gs w ill cont rib ute t o the v ali dit y of th e mod el to improv e
research of autism.
Acknowledgments
This work was supported by grants VEGA 1/0686/12
and VEGA 2/0107/12. The authors would like to thank
Dr. Monika Okuliarová for helpful comments and
suggestions.
Conflict of interest statement
The authors declared no potential conflict of interest with
respect to the research, authorship, and/or publication of
this study.
REFERENCES
Amaral DG, Corbett BA. (2003). The amygdala, autism and anxiety. Novartis
Found Symp 251: 177–187.
American Psychiatric Association. (2000). Diagnostic and statistical manual of
mental disorders DMS-IV-TR, 4
th
edition (text revision). American Psychiatric
Association, Washington, DC.
Bachevalier J, Loveland KA. (2006). The orbitofrontal-amygdala circuit and
self-regulation of social-emotional behavior in autism. Neurosci Biobehav
Rev 30: 97–117.
Baranek GT. (1999). Autism during infancy: a retrospective video analysis of
sensory-motor and social behaviors at 9–12 months of age. J Autism Dev
Disord 29: 213–224.
Canitano R, Scandurra V. (2011). Psychopharmacology in autism: an update.
Prog Neuropsychopharmacol Biol Psychiatry 35: 18–28.
Downey R, Rapport MJ. (2012). Motor activity in children with autism: a re-
view of current literature. Pediatr Phys Th er 24: 2–20.
Dubovický M, Škultétyová I, Ježová D. (1999). Neonatal stress alters habitua-
tion of exploratory behavior in adult male but not female rats. Pharmacol
Biochem Behav 64: 681–686.
Esposito G. (2011). Early postures and gait development in Autism Spectrum
Disorders (ASD). Neurosci Res 71: Suppl. e101.
Flanagan JE, Landa R, Bhat A, Bauman M. (2012). Head lag in infants at risk for
autism: a preliminary study. Am J Occup Ther 66: 577–585.
Gandhavadi B, Melvin JL. (1985). Electrical blink re ex habituation in men-
tally retarded adults. J Mental De c Res 29: 49–54.
Gillott A, Furniss F, Walter A. (2001). Anxiety in high-funct ioning children with
autism. Autism 5: 277–286.
Groden J, Cantela J, Prince S, Berryman J. (1994). The impact of stress and
anxiety on individuals with autism and developmental disabilities, in Be-
havioural issues in autism (Schopler E, Mesibov GB eds) pp. 177–195, Plenum
Press, New York.
Hepper PG, Shahidullah S (1992). Habituation in normal and Down’s syn-
drome fetuses. Q J Exp Psychol B 44: 305–317.
Chapillon P, Roullet P. (1997). Habituation and memorization of spatial ob-
jects’ con gurations in mice from weaning to adulthood. Behav Process 39:
249–256.
Kawa R, Pisula E. (2010). Locomotor activity, object exploration and space
preference in children with autism and Down syndrome. Acta Neurobiol
Exp 70: 131–140.
Kerr DM, Downey L, Conboy M, Finn DP, Roche M. (2013). Alterations in the
endocannabinoid system in the rat valproic acid model of autism. Behav
Brain Res 249: 124–132.
Kleinhans NM, Johnson LC, Richards T, Mahurin R, Greenson J, Dawson G, Ay-
lward E. (2009). Reduced neural habituation in the amygdala and social im-
pairments in autism spectrum disorders. Am J Psychiatry 166: 467–475.
Landa R, Garrett-Mayer E. (2006). Development in infants with autism spec-
trum disorders: a prospective study. J Child Psychol Psychiatry 47: 629–638.
Leussis MP, Bolivar VJ. (2006). Habituation in rodents: a review of behavior,
neurobiology, and genetics. Neurosci Biobehav Rev 30: 1045–1064.
Levy SE, Mandell DS, Schultz RT. (2009). Autism. Lancet 374: 1627–1638.
Ludewig K, Geyer MA, Vollenweider FX. (2003). De cits in prepulse inhibition
and habituation in never-medicated,  rst-episode schizophrenia. Biol Psy-
chiatry 54: 121–128 .
Lynn DA, Brown GR. (2009). The ontogeny of exploratory behavior in male
and female adolescent rats (Rattus norvegicus). Dev Psychobiol 51: 513–
520.
Markram K, Rinaldi T, La Mendola D, Sandi C, Markram H. (2008). Abnormal
fear conditioning and amygdala processing in an animal model of autism.
Neuropsychopharmacology 33: 901–912.
Michael N, Ostermann J, Sörös P, Schwindt W, P eiderer B. (2004). Altered
habituation in the auditory cortex in a subgroup of depressed patients by
functional magnetic resonance imaging. Neuropsychobiology 49: 5–9.
Militerni R, Bravaccio C, Falco C, Puglisi-Allegra S, Pascuci T, Fico C. (2000).
Pain reactivity in children with autistic disorder. J Headache Pain 1: 53–56.
Morok uma S, Fukush ima K, Kawa i N, Tomonaga M , Satoh S, Nakano H . (2004).
Fetal habituation correlates with functional brain development. Behav
Brain Res 153: 459–563.

227
Also available online on PubMed Central
Interdisciplinary Toxicology. 2013; Vol. 6(4): 222–227
Copyright © 2013 SETOX & Institute of E xperimental Pharmacol ogy and Toxicology, SASc.
Moy SS, Nadler JJ, Poe MD, Nonneman RJ, Young NB, Koller BH, Crawley JN,
Duncan GE, Bod sh JW. (2008). Development of a mouse test for rep etitive,
restricted behaviors: relevance to autism. Behav Brain Res 18 8: 178–194.
O’Neil DK, Happé FGE. (2000). Noticing and commenting on what’s new: dif-
ferences and similarities among 22–month–old typically developing chil-
dren, children with Down syndrome and children with autism. Dev Sci 3:
457–478.
Ohta M, Nagai Y, Hara H, Sasaki M. (1987). Parental perception of behavioral
symptoms in Japanese autistic children. J Autism Dev Disord 17: 549–563.
Osterling JA, Dawson G, Munson JA. (2002). Early recognition of 1-year-old
infants with autism spectrum disorder versus mental retardation. Dev Psy-
chopathol 14: 239–251.
Parsons PJ, Fagan T, Spear NE. (1973). Short-term retention of habituation in
the rat: a developmental study from infancy to old age. J Comp Physiol Ps y-
chol 84: 545–553.
Pierce K, Courchesne E. (2001). Evidence for a cerebellar role in reduced ex-
ploration and stereotyped behavior in autism. Biol Psychiatry 49: 655–664.
Provost B, Lopez BR, Heimerl S. (2007). A comparison of motor delays in
young children: autism spectrum disorder, developmental delay, and de-
velopmental concerns. J Autism Dev Disord 37: 321–328.
Rinaldi T, Perrodin C, Markram H. (2008). Hyper-connectivity and hyper-plas-
ticity in the medial prefrontal cortex in the valproic acid animal model of
autism. Front N eural Circu its 2: 4.
Rodier PM, Ingram JL, Tisdale B, Nelson S, Romano J. (1996). Embryological
origin for autism: developmental anomalies of the cranial nerve motor nu-
clei. J Comp Neurol 370: 247–261.
Rodier PM, Ingram JL, Tisdale B, Croog VJ. (1997). Linking etiologies in hu-
mans and animal models: studies of autism. Reprod Toxicol 11: 417–422.
Rogozea R, Florea-Ciocoiu, V. (1982). Habituation of the orienting reaction in
patients with post-meningoencephalitic epilepsy. Electroencephalogr Clin
Neurophysiol 53: 115 –118 .
Schneider T, Przewłocki R. (2005). Behavioural alterations in rats prenatally
exposed to valproic acid: animal model of autism. Neuropsychopharmacol-
ogy 30: 80–89.
Schneider T, Turczak J, Przewłocki R. (2006). Environmental enrichment re-
verses behavioral alterations in rats prenatally exposed to valproic acid: is-
sues for a therapeutic approach in autism. Neuropsychopharmacology 31:
36–46.
Schumann CM, Hamstra J, Goodlin-Jones BL, Lotspeich LJ, Kwon H, Buono-
core MH, Lammers CR, Reiss AL, Amaral DG. (2004). The amygdala is en-
larged in children but not a dolescents with autism; the hippocampus is en -
larged at all ages. J N eurosci 24: 6392–64 01.
Sui L, Chen M. (2012). Prenatal exposure to valproic acid enhances synaptic
plasticity in the medial prefrontal cortex and fear memories. Brain Res Bull
87: 556–563.
Tamada K, Tomonaga S, Hatanaka F, Nakai N, Takao K, Miyakawa T, Nakatani
J, Takumi T. (2010). Decreased exploratory activit y in a mouse model of 15q
duplication syndrome; implications for disturbance of serotonin signaling.
PLoS One 5: e15126.
Teitelbaum P, Teitelbaum O, Nye J, Fryman J, Maurer RG. (1998). Movement
analysis in infancy may be useful for early diagnosis of autism. Proc Natl
Acad Sci U S A. 95: 13 982–13 987.
Towbin KE, Pradella A, Gorrindo T, Pine DS, Leibenluft E. (2005). Autism spec-
trum traits in children with mood and anxiety disorders. J Child Adolesc Psy-
chopharmacol 15: 452–464.
Ume da T, Takashi ma N, Nak agawa R , Maek awa M, Ik egami S , Yoshikaw a T, Ko-
bayashi K, Okanoya K, Inokuchi K, Osumi N. (2010). Evaluation of Pax6 mu-
tant rat as a model for autism. PLoS One 5: e15500.
Vannin i P, Sulli van C, Sch ultz A, Pu tcha D, Vito lo O, Rentz D, Frey M, Be cker AJ,
Johnson KA, Sperling RA. (2010). Increased amyloid deposition is related
to failure of habituation of the default network but preserved repetition
suppression in the hippocampus during successful repetition encoding in
cognitively normal older adult s. Alzheimers Dement 6: 103 –104.
Webb SJ, Jones EJ, Merkle K, Namkung, J, Toth K, Greenson J, Murias M, Daw-
son G. (2010). Toddlers with elevated autism symptoms show slowed ha-
bituation to faces . Child Neuropsychol 16: 255–278.