Reproductive Toxicology 64 (2016) 116140

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Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox

Genetic and non-genetic animal models for autism spectrum

disorders (ASD)
Zivanit Ergaz, Liza Weinstein-Fudim, Asher Ornoy
Laboratory of Teratology, Department of Medical Neurobiology Hebrew University Hadassah Medical School and Hadassah Hospital, Jerusalem, Israel

a r t i c l e i n f o a b s t r a c t

Article history: Autism spectrum disorder (ASD) is associated, in addition to complex genetic factors, with a variety of
Received 15 March 2016 prenatal, perinatal and postnatal etiologies. We discuss the known animal models, mostly in mice and
Received in revised form 18 April 2016 rats, of ASD that helps us to understand the etiology, pathogenesis and treatment of human ASD. We
Accepted 25 April 2016
describe only models where behavioral testing has shown autistic like behaviors. Some genetic models
Available online 30 April 2016
mimic known human syndromes like fragile X where ASD is part of the clinical picture, and others are
without dened human syndromes. Among the environmentally induced ASD models in rodents, the
Keywords:
most common model is the one induced by valproic acid (VPA) either prenatally or early postnatally. VPA
ASD
Mice
induces autism-like behaviors following single exposure during different phases of brain development,
Rats implying that the mechanism of action is via a general biological mechanism like epigenetic changes.
Genetic Maternal infection and inammation are also associated with ASD in man and animal models.
Environmental 2016 Elsevier Inc. All rights reserved.
VPA
Inammation

1. Introduction
Human ASD is a heterogeneous group of neurobehavioral prob-
lem with different recognized genetic and environmental origins.
Due to the complexity of these pathologies and the lack of a denite
known diagnostic marker, ASD is dened by phenotypic behav-
ioral traits. The DSM 5 denes ASD as a neurobehavioral disorder
manifested by persistent decits in social and communication
interaction, decits in developing, understanding and maintaining
relationships, as well as abnormal and xed interests and repetitive
behavior, with various degrees of severity [1,2]. Symptoms must be
present at early childhood and interfere with daily function. The eti-
ology is largely unknown, and seems to be the result of genetic and
environmental interaction [3].
In the last years environmental exposures, especially during
pregnancy, are increasingly being recognized as potential risk fac-
tors for ASD, and the possibility that the prenatal environment
affects fetal programming is a promising direction for research. In
addition, in many children with ASD a variety of gene mutations
and of changes in gene expression, most of them related to the
brain development and function were found. In addition, children
Corresponding author.
E-mail address: ornoy@cc.huji.ac.il (A. Ornoy).
with various well dened clinical entities (i.e. Fragile X, tuberous
sclerosis, Rett syndrome) exhibit ASD like behavior.
In addition to complex genetic susceptibility, as evidenced from
twin studies, epigenetic changes have also been proposed [2]. Other
mechanisms are: immune dysregulation that include abnormal lev-
els of cytokines and growth factors, fetal and maternal antibodies
to brain tissue, microglial activation, and others [4]. Additional
proposed mechanisms are increased oxidative stress, mitochon-
drial dysfunction, abnormalities in brain serotonin, abnormal white
matter connectivity and altered synapses resulting from genetic
changes such as changes in ERK pathway [4].
A variety of morphological and functional changes have been
demonstrated in the brain of children or adults with ASD. How-
ever, their presence is inconsistent and is generally not related to
the severity of the symptoms. Hence, in spite of the existence of var-
ious imaging and neurobehavioral tools for the diagnosis of ASD, its
diagnosis relies on clinical behavioral grounds.
Experimental animal models are of importance for the under-
standing of the etiology and pathogenesis of any human disease,
including ASD. However, these models are appropriate mainly
when the same diagnostic markers demonstrating resemblance to
the human situation are used. This is relatively easy whenever there
are distinct markers for a disease. For example, in animal models of
diabetes, one has to show similar metabolic derangements. How-
ever, it is more difcult to prove that an experimental animal is
the suitable model for ASD in man, since it is based on behavioral

http://dx.doi.org/10.1016/j.reprotox.2016.04.024
0890-6238/ 2016 Elsevier Inc. All rights reserved.
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
changes and the behavior of a mouse or a rat is not the same as
the behavior of a child or adult. Indeed, a variety of neurobehav-
ioral tests have been used to demonstrate in the animal models
a behavioral pattern that mimics the behavior of ASD children.
Another approach is to create genetic models that are similar to
the human genetic changes observed sometimes in children with
ASD. In these models, neurobehavioral tests often demonstrate an
autistic like behavior as well as distinct changes in the brain that
resemble those sometimes found in children with ASD.
Despite the heterogeneity of the clinical symptoms, animal
studies allow coherent investigations of the circuits, cells, and
pathophysiological processes affected in autism [5].
Basically, there are two types of animal models for ASD: environ-
mentally induced, by exposure of the pregnant animals to certain
chemicals or infection/inammation and those induced by genetic
manipulations. The rst are relevant mainly if it is shown that the
same chemicals are also relevant for human; for example VPA.
VPA was found to signicantly increase the rate of ASD among off-
spring of treated mothers [6,7] and was thereafter shown to induce
autism-like behavior in rats and mice [8]. Genetic animal models of
ASD are also important especially when they mimic the ASD symp-
toms that are present in distinct human genetic diseases such as
fragile X and tuberous sclerosis. Mouse models should therefore be
based on a known genetic cause of a disease, reect key aspects
of the human symptoms and, if possible, respond to treatments
that are effective in the human disease [9]. However, new genetic
changes in animals that induce ASD like symptoms may also be
of importance since they may push to seek similar changes in
man.
ASD in human is manifested by a set of behavioral changes
that appear at early childhood. The most prominent are impaired
social interaction, impaired language and motor behavior, includ-
ing stereotyped and repetitive motor movements and limited
interest in the surroundings. These behaviors can also be observed
and monitored in various animal models by using specic tests
that were developed for the measurement of such behavioral
modications. Although it is valid to argue that animal behav-
iors are different from those in men, the fact that they can be
specically demonstrated in the relevant models points to their
similarity to the human behavior. As most genetic and non-genetic
models of ASD are in mice and rats, the more common and reli-
able behavioral tests were developed in these animals. In spite
of some limitations they are generally considered to represent
adequately human behaviors. In the tables we name, wherever
relevant, the different behavioral tests used by the investigators
to demonstrate autistic like behaviors in their models. It should
be remembered, however, that not all tests used by the differ-
ent investigators are unanimously accepted as representing ASD
like behaviors. Hence, this might be a weakness of some of the
studies.
The purpose of the present review is to summarize the data
on more common experimental animal models of ASD either as a
result of prenatal exposure (i.e. VPA, inammation) or those follow-
ing genetic manipulations. Most models are in mice and rats. The
current review includes only investigations in which animals were
evaluated by behavioral studies and the changes mimic ASD like
behavior. The impacts of the genetic background or environmental
insults on the phenotypic traits, anatomical, biochemical, physio-
logical markers and treatment modalities are described whenever
applicable. The tests used, the neurobehavioral changes and mode
of treatment in each study are described in the tables of the genetic
models, as well as the specic gene defects. In the tables that
describe the VPA induced models we also added the time of expo-
sure and dose of VPA.
117
1.1. Genetic animal models of autism: implication for human
studies (Tables 13)
Several human syndromes derived from a single gene mutation
increase the risk for ASD. The more common aberrations are Fragile
X syndrome, a mutation in FMR1 [10], Rett syndrome, a mutation
in MECP2 [11], tuberous sclerosis, mutations in TSC1 or TSC2 [12]
and Timothy syndrome, a mutation in CACNA1C [13]. Copy-number
variants that lead to inherited maternal 15q1113 duplication
resulting in Prader-Willi syndrome [14] and other duplications like
the NPHP1 gene [15] are also associated with autistic traits. The
development in strategies for the identication of genetic variants
also led to the description of new syndromic forms of ASD and
enabled the association between phenotype and genetic traits .
The identication of such genetic variations with the devel-
opment of new strategies for genetic engineering facilitated the
development of genetic animal models of ASD. Mice are the pre-
dominant animal model for ASD owing to their genetic tractability
and their ability to demonstrate analogs of behavioral decits
associated with ASD [5]. Other animal models include rats [16],
zebrash [17], song birds [18] and the newly introduced macaque
monkeys [19]. Until now, only few of the current animal models
were proven valid for evaluation of a known human aberration by
establishment of a measurable marker and an offer of a treatment
option.
2. Animal models for genes of a syndromic disorder
predisposing to autism (Table 1)
The animal models representing known human syndromes
derived from a gene mutation exemplify the difculties in estab-
lishing a valid genetic trait.
2.0.1. Fragile X (FMR1)Table 1
Children with Fragile X syndrome which is the most frequent
inherited cause of mental retardation have increased rates of ASD.
The Fragile Mental Retardation 1 locus (FMR1) resides in the X
chromosome and expansion of triplet repeats in the untranslated
region of the FMR1 gene prevents synthesis of the FMR1 gene
product FMRP. FMRP is an RNA-binding protein that modulates
mRNA trafcking, dendritic maturation and synaptic plasticity.
Rodents, mostly mice knock out for the FMR1 gene, were shown to
present autistic traits. Different traits depend on the background
strain. Some of the studies also found structural, biochemical and
physiological abnormalities including abnormal dendritic spine
morphology [20], elevated phosphorylation of translational control
molecules and exaggerated protein synthesis in the hippocam-
pus [21]. Additionally, enhanced metabotropic glutamate receptor
(mGluR)-dependent long-term depression (LTD) recordings were
described [21,22], implying reduction in the efcacy of neuronal
synapses following a stimulus. Treatment modalities by genetic
engineering and medications are meant for prevention of exag-
gerated protein synthesis [21] rescue of synaptic tonic inhibitory
currents [23] activation of translation to overcome the lack in Fmrp
[24] and genetic engineering for correction of the synaptic mGluR
signaling [22]. However, environmental stimulation was successful
as well [20].
2.0.2. Rett syndrome and MECP2 mutations (Table 1)
Rett syndrome, an X linked disease that affects girls, is char-
acterized by neurodevelopmental delay, ASD and seizures. It is
caused by mutations in the gene encoding for the methyl-CpG bind-
ing protein 2 (MECP2) that binds to methylated-CpG dinucleotides
and inuences gene expression. MECP2 is expressed widely, but is
 118
Table 1
Animal models for genes of a syndromic disorder predisposing to autism among humans.

Reference Associated morbidities Type Behavioral tests Results of behavioral tests Other Conclusions
tests/markers/treatment

Zang et al. [42] Fragile X syndrome Mice, Fmr1 I304N knock-in Behavior chamber, PPI and Impaired social Altered synaptic plasticity Loss of RNA binding and
mutation by introducion of audiogenic seizures interactions, repetitive and in hippocampus, defective under expression of FMRP
the I304N mutation into stereotypic behaviors, KH2-mediated RNA caused the Fragile X
the endogenous mouse Lower level of anxiety, binding in neurons, and Syndrome.
Fmr1 locus by homologous hyperactivity and seizures decreased FMRP levels,
recombination., particularly in younger
background FVB and animals.
C57BL/6J
Olmos-Serrano Fragile X syndrome mice Fmr1 /-, background Behavior chamber, open Increased motor activity THIP, a GABA A receptor Enhancing tonic inhibition
et al. [23] congenic FVB eld and PPI and impaired response to agonist attenuated may be a putative target
auditory startle hyperactivity and PPI and for the treatment of fragile
not fear or startle response. X syndrome.
Pietropaolo et al. Fragile X syndrome mice Fmr1 /-,background Behavior chamber and Restricted interest and no C57BL/6 background was

Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

[43] FVB and C57BL/6 three chambered test intact level of sociability more suitable than FVB for
further research on
autistic-traits
Ronesi et al. [22] Fragile X syndrome mice, Fmr1-/-, background Open eld and audiogenic Reduced anxiety and Deletion of Homer1a Altered mGluR5-Homer
C57/Bl6J seizures normal locomotor activity restored mGluR5long scaffolds contributed to
Homer scaffolds and mGluR5 dysfunction and
corrected mGluR5 phenotypes
signaling, neocortical
circuit dysfunction and
behavior but not LTD.
Bhattacharya et al. Fragile X syndrome mice, Fmr1-/-, Behavior chamber, open impaired social Enhanced hippocampal Dysregulated protein
[21] eld, marble burying, interactions, repetitive LTD, elevated synthesis has a key role in
rotarod and Y-maze. behavior, restricted phosphorylation of fragile X syndrome and can
interest and hyperactivity translational molecules be cured by restoration of
and exaggerated protein normal translation.
synthesis in hippocampus.
Genetic deletion of SK1
restored translation and
phenotype
Udagawa et al. [24] Fragile X syndrome Mice, Fmr1-/y Cpeb/ Open eld, light chamber, Restricted interest, Lentivirus expressing Disruption of translational
T-maze and nest building decreased anxiety, shRNA that target CPEB homeostasis is causal for
enhanced learning and and/or the translational fragile X syndrome
poor nest building apparatus depleted CPEB in
the hippocampus and
rescued memory decits.
Hamilton et al. [16] Fragile X syndrome Rats, Fmr1 /- and Nlgn3 Elevated zero maze, Three Impaired social no Rat models for a syndromic
/- in the outbred chambered test, open eld, interactions, repetitive (FMRP) and non-syndromic
Sprague-Dawley PPI, olfaction and wood behavior, no decit on a (NLGN3) ASD was valid for
chew test fear and sensorimotor evaluation of autistic traits
gating
Han et al. [44] Fragile X syndrome mice, Open-eld, light and dark Enhanced PPI, Misregulation of Cyp2 Neurobehavioral
Cyp2+/,background box, auditory sensory hyperactivity and delayed function and its abnormalities were
C57BL/6J processing and hot plate sensory response mGluR-induced associated with
expression. misregulation of Cyp2
function and its
mGluR-induced
expression.
Table 1 (Continued)

Reference Associated morbidities Type Behavioral tests Results of behavioral tests Other Conclusions
tests/markers/treatment

Oddi et al. [20] Fragile X syndrome mice, Fmr1 /, Behavior chamber, USV,
background female
FVB.129P2-Fmr1tm1Cgr/J
(FVB), male
FVB.129P2Pde6bTyrc-
ch/AntJ
Guy et al. [27] Rett syndrome mice, silenced MECP2 gene
by a loss of lox-Stop
cassette
Impaired social interaction, Environmental stimulation
T-maze and Fear hyperactivity and cognitive
conditioning decits
Behavior chamber Repetitive and stereotypic
behaviors, irregular
breathing and abnormal
gait
The use of
by additional non-lactating
female improved impaired
behavior and eliminated
the abnormalities in the
morphology of
hippocampal and
amygdala dendritic spines.
Reduced LTP in the
hippocampus. The MECP2
activation under the
control of its own promoter
and regulatory elements by
non-pharmacological
interventions for the
treatment of autism is
benecial
Rett syndrome is a
neurodevelopmental
rather than a
neurodegenerative
disorder

Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

cassette deletion resulted
in phenotypic reversibility
Samaco et al. [25] Rett syndrome mice, MECP2+/ Elevated plus maze, light Reduced anxiety, restricted Breathing abnormalities Mutations associated with
backgrounds and dark box, open eld, interest, enhanced PPI, and reduced corticosterone behavioral changes caused
FVB/N 129S6/SvEv three-chambered test, reduced pain and normal levels in response to stress unique phenotypes specic
(FVB.129F1) olfaction test and hot plate olfaction to the background.
Sato et al. [31] Tuberous Sclerosis mice, Tsc1+/ and Behavioral chamber, tube Impaired social Whole-genome expression Rodent models of tuberous
+Tsc2 +/, background test, open eld, pole test, interactions, normal analysis showed sclerosis, which lack
C57BL/6J rotarod, hot plate, light and olfaction and intact motor uninhibited mTOR cortical tubers and epilepsy
dark box and sensory functions signaling. Rapamycin are appropriate for
reversed impaired social examining the causal role
behavior and was of mTOR signaling in ASD
associated with mTOR
inhibition
Normand et al. [45] Tuberous Sclerosis mice, thalamic Tsc1 Behavior chamber and Repetitive and stereotypic no Phenotype severity
deletion at E12.5 and E18.5 seizures behaviors and seizures depends on developmental
timing and degree of
genetic mosaicism.
Reith et al. [30] Tuberous Sclerosis mice, Tsc2+/ and Behavior chamber, open Impaired social Purkinje cell loss. Purkinje cell loss and/or
Tsc2f/;Cre, Purkinje cell eld, rotarod, marble interactions, repetitive Rapamycin prevented dysfunction may be the
specic Tsc2 deletion, burying, light and dark box, behavior and restricted social development decit link between tuberous
background 129 1/SvJ water maze and nest interest sclerosis and ASD
and C57BL/6J building,
Bader et al. [32] Timothy syndrome mice, TS2, missense Morris water maze, water Impaired social no Heterozygous TS2 mice
mutations in alternatively Y-maze, marble burying interactions, repetitive and that were allowed to keep
spliced exons that cause and USV stereotypic behaviors, an inverted neomycin
G406 replacement in the reduced USV and increased cassette (TS2-neo) survived
CaV1.2L-type calcium fear through adulthood and
channel displayed autism traits.
Giza et al. [34] Autism and mice, IB2/background Behavior chamber, Impaired social Reduced and delayed IB2 is essential for proper
PhelanMcDermid 129Svev elevated T-maze, olfaction interactions, restricted AMPA/kainate morphology and synaptic
syndrome and vision tests interest and cognitive and neurotransmission at IB2 transmission in cerebellum
motor decits /- cerebellar climbing
bre to Purkinje cell
synapses and disturbed IB2
proteins distribution in
brain.

119
 120
Speed et al. [33] Autism and SHANK3G/G mice Behavior chamber, open Restricted interest, decits Impaired hippocampal Alterations in synaptic
PhelanMcDermid eld, marble burying, light in learning, impaired excitatory transmission function and behavior in
syndrome and dark box, and tube test motor coordination, and plasticity and changes the SHANK3G/G mice
sensory decits, normal in baseline NMDA mimic ASD
social interactions and no receptor-mediated
repetitive behavior synaptic responses.
Allowing for deletion of the
mutant exon/neo-STOP
cassette and reversion to
wild-type SHANK3

Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

reversed SHANK3 levels.
Kwon et al. [35] Autism, increased cancer mice, PTEN mutant, Behavior chamber, Impaired social Macrocephaly, Abnormal activation of the
risk, deletion conned to olfaction test, nest interactions, restricted disorganized granular PI3 K/AKT pathway in
macrocephaly,seizures, discrete mature neuronal building, open eld, interest, sensory layer, enlarged dentate specic neuronal
Lhermitte-Duclos disease populations in the cerebral elevated plus maze, dark sensitivity, high anxiety, gyrus, increased axonal populations can underlie
and mental retardation cortex and hippocampus, and light box, rotarod and normal motor activity and growth, ectopic axonal macrocephaly and
background C57/BL6 Morris water maze fear conditioning and projections, and abnormal behavioral abnormalities.
seizures synapses. Increased
phosphorylation in the
PTEN-decient tissues.
activation of the
Akt/mTor/S6k pathway and
inactivation of Gsk3
Penagarikano et al. Cortical dysplasia focal mice, Cntnap2/, Morris water maze, T Impaired social Defects in the migration of CNTNAP2 has a role in
[46] epilepsy syndrome background C57BL/6J maze, Three chambered interactions, stereotypic cortical projection neurons brain development, its lack
test and USV behavior, sensory and a reduction in the may be alleviated with
sensitivity, increased number of GABAergic risperidone.
motor activity, impaired interneurons. Risperidone
nest-building, normal decreased the
olfaction and seizures. hyperactivity and reversed
impaired behavior.
Nakatari et al. [47] 15q11-13 duplication mice, patDp/+ Three-chambered test, Impaired social Alterations in the amount The 5-HT2cR increase due
Morris water maze, Barnes interactions, stereotypic of MBII52 RNA of the to increased MBII52
maze and USV behavior, restricted patDp/+ mice resulted in SnoRNA in the duplication
interest, high anxiety, fear, increased [Ca2 + ]I response region may play a role in
and depression. to 5-HT2cR signaling and autism and its ligand may
increased RNA editing be a potential treatment.
ratios in patDp/+ at the A
and B sites
Table 1 (Continued)
Reference Associated morbidities
Smith et al. [40] 15q11-13 maternal
duplication, autism
Piochon et al. [48] 15q11-13 duplication,
Autism
Shigemori et al. 15q11-13
[49] duplication
Autism
Zheng et al. [50] Rubinstein-Taybi
Syndrome
Meechan et al. [51] mice, LgDel transmitted
paternally, genomically
accurate model of
22q11DS, background
C57/Bl6N
Abbreviations: AMPA- -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, CPEBcytoplasmic polyadenylation element binding protein, Cyp2Cytoplasmic FMR1-interacting protein2, FMRPFragile X mental retardation
protein. IB2Islet Brain-2, LTDlong-term depression, MECP2methyl CpG binding protein 2, NMDAN-methyl-d-aspartate, PPIprepulse inhibition, SK1p70 ribosomal S6 kinase 1, USVultrasonic vocalization
Type
Mice, UBE3a 2 transgenic
Mice, patDp/+, human
15q11-13 duplication by
chromosome engineering
on mouse chromosome 7
mice, patDp+, paternal
duplication in mouse
chromosome 7
corresponding to human
chromosome 15q11-q13
mice, CBPCH1/CH1,
C57BL/6 129SvF1 hybrid
Autism, schizophrenia,
DiGeorge syndrome
Behavioral tests
Behavior chamber,
three-chambered test and
USV
DigiGait imaging system
and delayed eyeblink
conditioning,
open Field, marble burying
and USV
Behavior chamber, open
eld, elevated plus maze,
three-chambered test,
rotarod and hot plate test
LCD touchscreen apparatus
Results of behavioral tests
Impaired social
interactions, repetitive and
stereotypic behaviors
Impaired social
interactions, increased
activity and impaired
eyeblink conditioning
Impaired social
interactions, high anxiety,
fear in response to stress
and reduced USV
Impaired social
interactions, repetitive and receptordependent
stereotypic behaviors,
hyperactivity and reduced
anxiety
Restricted interest and
impaired cognitive
function.
Other
tests/markers/treatment
impaired glutamatergic
synaptic transmission,
reduced presynaptic
glutamate release via
reduced postsynaptic
excitability and phasic
excitatory synapse-like
stimuli
Saturated LTD in Purkinje
cell soma under baseline
condition preventing
subsequent LTD induction
and developmental
elimination of surplus
climbing bers.
Decreased Iba1, a
microglial activation
marker in the amygdala
was restored by maternal
minocycline treatment
during pregnancy and
lactation. Minocycline also
reduced anxiety-related
behaviors.
Disturbed NMDA
important for normal social
synaptic depression in
hippocampus
Altered inhibitory synaptic
organization, disrupted
parvalbumin
interneuron-dependent
inhibitory circuitry.
Conclusions
The autism traits depended
on UBE3a gene copy
number. Increased E3A
ubiquitin ligase gene
dosage results in reduced
excitatory synaptic
transmission.
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
Decits in synaptic
plasticity and pruning are
potential causes for motor
problems and abnormal
circuit development in
autism.
Perinatal microglia in the
basolateral amygdala may
play a pathogenic role in
the anxiety observed.
intact CH1 domain is
behavior, motor function,
and cognition
Disrupted parvalbumin
interneuron-dependent
inhibitory circuitry, is a key
feature of cortical
connectivity disorders that
arise during development,
including 22q11DS
121

Table 2
Animal models for genes associated with phenotypic traits relevant to autism in rodents that do not cause recognized human diseases.
Reference Type
Jamain et al. [69] mice, loss-of-function
mutation in the murine
NLGN4 ortholog Nlgn4
Radyushkin et al. [70] mice, NL-3 /-, background
C57BL/6NCrl
Umeda et al. [64] rats, rSey-/+, Pax6
heterozygous mutant
Zaccaria et al. [71] mice, GAP43 (+/),
background C57BL/6J
129S3/SvImJ (B6129S3)
Zhao et al. [72] mice, glut3 (Slc2a3) /-
background C57/BL6
Hiramoto et al. [73] mice, congenic Tbx1HT,
background C57BL/6J
Horev et al. [74] mice, 16p11.2, either one
copy (df) allele], or with
three copies [heterozygous
for a duplication (dp)
allele, background
C57BL/6N:129Sv
Associated morbidities
Autism
Autism
Autism, absence or
hypoplasia of the anterior
commissure, decreased
volumes of the corpus
callosum, smaller brain
size, aniridia and various
eye abnormalities.
Autism
no
Autism, social interaction
decits and delayed
development vocalization
and language
Autism,
neurodevelopmental and
neuropsychiatric
syndromes
Behavioral tests
Elevated plus maze, open
eld, rotarod, Morris water
maze, PPI, buried food,
sucrose preference, USV
and chemical seizures
Elevated plus maze, open
eld, hole board, rotarod,
Morris water maze, PPI,
USV, Buried food, Sucrose
preference and seizures
Behavior chamber, forced
swim, electric shock
sensitivity and USV
Three chambered test,
T-Maze, Morris water
maze, marble burying, food
preference and light and
dark box
T-maze, Morris water
maze, open-eld, rotarod,
radial arm maze, ultrasonic
vocalization and olfactory
sensitivity test
Behavior chamber,
elevated plus maze, and
USV
Home Cage Scan
Results of behavioral tests
Impaired social
interactions, restricted
interest and reduced USV
upon contact with a female
Impaired social
interactions, reduced USV,
olfactory deciency and
hyperactivity
Impaired social
interactions, repetitive and
stereotypic behaviors,
aggressiveness, impaired
fear-conditioned memory
and reduced USV only in
females.
Impaired social
interactions, stereotypic
behavior, restricted
interest and high anxiety
Impaired social
interactions, repetitive and
stereotype behaviors,
abnormal spatial learning
and memory, with intact
motor ability, reduced USV
and seizures
Impaired social
interactions,
aggressiveness, reduced
USV, impaired memory
and normal olfaction.
Loss of 16p11.2: repetitive
and stereotypic behaviors,
gain of 16p11.2:
hyperactivity and
abnormal diurnal behavior
122
Other Conclusions
tests/markers/treatment
Reduced brain volume Neuroligin-4 encodes the
synaptic cell adhesion
protein which takes part in
the regulation of the
maturation and function of
synapses is associated with
autistic traits
Reduced brain volume A point mutations in
neuroligin-3 a homologue
of neuroligin-4 results in a
behavioral phenotype
related to olfactory and
communication deciency
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
Reduced 5-HT level in the The rSey2/+ rats likely have
hippocampus. Clozapine some phenotypic
was effective to improve components of autism
sensorimotor decits but
did not alter other
behavioral phenotypes
no Mice lacking one allele for
the synaptic
growth-associated protein
43 can be used as an
animal model for autism.
Compensatory increase in Neuronal glucose
neuronal MCT2 but not transporter (GLUT) isoform
endothelial/glial MCT1 3 deciency and increase
paralleling enhanced brain in lactate uptake in
lactate uptake neurons is associated with
impaired neurobehavior
and seizures
Tbx1 protein, Tbx1, a Tbx1 heterozygosity
22q11.2 gene was contributes to
ubiquitously expressed 22q11.2-associated ASD
throughout the brain of presumably through its
C57BL/6J mice, and is expression in diverse brain
enriched in postnatally regions, some of which
proliferating cells include postnatally
generated neurons.
Brain volume size: The deletion and the
macrocephaly in deletion duplication affect behavior
and microcephaly in and brain anatomy in
duplication. MRI show opposing ways, with
changes in the posterior deletion mice exhibiting
region of the hypothalamus behaviors that resemble
sensorimotor decits in
rats with lateral
hypothalamic and
nigrostriatal lesions.
Table 2 (Continued)

Reference Type Associated morbidities Behavioral tests Results of behavioral tests Other Conclusions
tests/markers/treatment

Silverman et al. [54] mice, shank1 /-, Autism Behavior chamber,
background C57BL/6 (B6) three-chambered test,
and129SvJae (129Jae) olfactory habituation, open
eld, rotarod, Inverted
wire hang, elevated
plus-maze, light and dark
box, PPI and hot plate
El-Kordi et al. [56] mice, Nlgn4/, Autism Elevated Plus Maze, open
background C57BL/6J. eld, hole board test,
rotarod, PPI, buried food
test, USV, sensor platform,
marble burying and nest
building
Kyzar et al. [75] mice, SERT+/ mice and Autism, schizophrenia, behavior chamber
Motor decits,
mild-anxiety and
decreased cognitive
abilities and memory.
Impaired social
interactions, repetitive
behavior, restricted
interest and reduced USV
SERT+/ mice: Impaired
no SHANK1 is associated more
with motor function and
memory and less with
social decits.
no Nlgn4/NLGN4X loss-of
function mutation in
mouse induce autistic
traits
no Decreased SERT and BDNF

Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

BDNF+/, background depression, anxiety, social interactions and expression modulate
C57BL/6J attention decit and repetitive behavior. self-grooming (and its
hyperactivity disorder, BDNF+/ mice: decreased patterning) in opposite
Parkinsons disease and repetitive behavior, directions.
cognitive decit hypolocomotion and
increased turning behavior.
Veenstra-VanderWeele Setotonin receptor Ala56 Autism Behavior chamber, Impaired social Increased MAPKsensitive Altered serotonin
et al. [60] variant, background Slc6a4 three-chambered test, interactions, repetitive and basal phosphorylation, homeostasis can impact
elevated plus maze, Morris stereotypic behaviors, enhanced hippocampal risk for autistic traits
water maze, rotarod, reduced USV and sensory serotonin clearance rates
forced swim, and USV aversion and hyper-serotoninemia.
A decrease in the ring rate
under basal conditions
followed by serotonin1A
and serotonin2A receptor
hypersensitivity
Won et al. [66] mice, Shank2-/- Autism, intellectual Behavior chamber, Impaired social Impaired LTP induced by NMDAR function is an
disability three-chambered test, interactions, repetitive and high-frequency stimulation important mechanism
Morris water maze, open stereotypic behaviors, or the taburst stimulation underlying the
eld, elevated plus maze impaired spatial learning, reduced NMDA/AMPA development and rescue of
and USV impaired nesting behavior ratio. D-cycloserine,a ASD-like phenotypes
and hyperactivity partial agonist of NMDARs,
normalized NMDAR
function and improved
social interaction. CDPPB a
membrane-permeable
positive allosteric
modulator of mGluR5
improved social
interaction.
Grayton et al. [76] mice, Nrxn1a /- Autism, schizophrenia, Behavior chamber, open Impaired social no The Nrxn1a knock out mice
background C57BL/6J intellectual disability, eld, light and dark box, interactions, which represent the
developmental delay, Pitt elevated plus maze, Morris aggressiveness, reduced deletions within NRXN1
Hopkins-like Syndrome, water maze, nest building, no found in humans is a useful
epilepsy. three-chambered test, nest Impairments in memory model for autism.
building and buried food and no repetitive behavior
task.

123
 124
Greco et al. [77] mice, SynI/, SynII/ Autism, epilepsy Open eld, light and dark Impaired social no Missense mutations in the
and SynIII/ background box, tube test, buried food interactions, repetitive synapsin genes led to
C57BL/6J olfactory test and marble behavior and restricted autistic phenotype when
burying interest SynI or SynIII deletion
altered social behavior,
whereas SynII deletion also
impaired memory and
repetitive behavior
Becker et al. [62] mice, Oprm1-/- Autism Behavior chamber, Impaired social Increased number of Disrupted mu opioid
background 50% three-chambered test, interactions, symmetrical synapses in receptor signaling trigger
129SVPas50% C57BL l nest-building, Y-maze, aggressiveness, high the caudate putamen and an autistic syndrome,
marble burying and anxiety, motor clumsiness nucleus accumbens with maybe through blunted
penthylene and seizures. reduction in the social reward processes.
tetrazole-induced seizures. postsynaptic density.

Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

Changes in the expression
of genes coding for
adhesion and scaffold
proteins. VU0155041, a
positive mGluR4 allosteric
modulator improved
autistic-like symptoms.
Risperidone improved
autistic-like symptoms and
reduced striatal dopamine
levels
Jaramillo et al. [78] mice, neuroligin-3 R451C Autism, intellectual Three-chambered test, Impaired social no Different genetic
point mutant knockin, disability and Asperger elevated plus maze, open Interactions, Increased backgrounds of the
background 129S2/SvPasCr Syndrome eld and Morris water spatial learning, neuroligin-3 R451C point
maze. hyperactivity and low mutant knockin mouse
anxiety. model modied the
behavioral phenotype
through epistatic genetic
interactions.
Ju et al. [57] mice, Nlgn4/, Autism Behavior chamber, wire Reduced USV in females no The Nlgn4/ mice display
background C57BL/6J suspension and USV early manifestation of
communication decits
more pronounced in
immature females.
Mohn et al. [79] mice, APC conditional Autism, intellectual three-chambered test, Impaired social Increases in the frequency Loss of APC function in
knock-out predominantly disability Barnes maze, Y maze, interactions, repetitive of AMPA-mediated mEPSCs forebrain neurons leads to
to forebrain excitatory marble burying and behavior, restricted in APC Field potentials cognitive and autism-like
neurons during synaptic nonsocial olfaction test interest and decits in from CA1 neurons; phenotypes.
differentiation memory increased -Catenin,
hippocampal and striatal
neurons and Increased less
mature synaptic spines.
Table 2 (Continued)
Reference Type
Moy et al. [37] mice, C58/J, Grin1 /-, no
background 6
heterozygous 129S6 and
C57BL/, and mice, C58/J
inbred strain
Shimamoto et al. [80] mice, Fabp3-/-, Fabp5-/-
and Fabp7 background
mixed 129/B6 onto B6
Sungur et al. [81] mice, Shank1-/- and
Shank1+/, background
heterozygous C57BL/6J and
129SvJae
Aller et al. [63] mice, over expressing grik4
in the forebrain
Li et al. [67] mice, (Prex1/) Autism
background C57BL6
Mosienko et al. [59] mice, Tph2-/-, background
C57BL/6N
Associated morbidities Behavioral tests Results of behavioral tests Other Conclusions
tests/markers/treatment
Behavior chamber, open Impaired social Strain genome Repetitive phenotypes can
eld, marble burying, interactions, repetitive and comparisons for autism be used to distinguish ASD
elevated plus-maze, nose stereotypic behaviors, candidate genes located mouse models
poke restricted interest and within regions divergent in
hyperlocomotion C58/J identied the genes
Cntnap2 and Slc6a4, and
not Grin1, Nlgn1, Sapap3,
and Slitrk5. Amphetamines
increased number of
rearing movements
The Fabp3 /-: impaired
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
Autism, schizophrenia Behavior chamber, no Disturbances in
three-chambered test, social interactions and brain-expressed FABPs
open eld, Y maze, Morris restricted interest. The could represent an
water maze, elevated plus Fabp5 /-: no phenotypic underlying disease
maze, llght and dark box, changes. The Fabp7 /-: mechanism in
USV, forced-swim and PPI decreased PPI, schizophrenia and autism
hyperactivity and high
anxiety
Autism and schizophrenia Behavior chamber, USV Impaired social no SHANK1 alterations is
interactions and reduced involved in acoustic
USV communication resulting
in social communication
decit.
Autism, schizophrenia and Open eld, Elevated plus Impaired social Altered information ow The model can be used for
mental retardation maze, Y maze, Rotarod, interactions, repetitive and through the hippocampal specic treatment
Three-chambered test, stereotypic behaviors and trisynaptic circuit. targeting for abnormalities
forced swimming, light and restricted interest Tianeptine abolished signs in glutamatergic signaling
dark box and of anhedonia and
sucrose-intake improved behavioral
abnormalities. Fluoxetine
did not affect behavior
Three chambered test, Impaired social Disturbed P-Rex1 signaling in CA1
Morris maze and USV interactions, repetitive NMDARdependent LTD is critical for social
behavior, restricted synaptic depression in behavior and cognitive
interest and reduced USV hippocampus. d-serine a function
coagonist of the glycine
modulatory sites on the
NMDAR fully rescued LTD
in cerebellum
Autism USV reduced USV Growth retardation Impaired ultrasonic
communication is likely to
result in a decient
mother-infant interaction
and growth retardation
125

Sungur et al. [82] mice: Shank1-/-,
background strains:
C57BL/6J and 129SvJae.
Sztainberg et al. [28] mice, MECP2 duplication
Tian et al. [68] mice, 16p11.2 df/+
heterozygous null at the
region of chromosome
7qF3 that is syntenic to
human chromosome
16p11.28, background
C57BL/6N
Whr et al. [83] Mice, parvalbumin /, Autism, schizophrenia and
background C57BL/6J
Abbreviations: PPIprepulse inhibition, USVultrasonic vocalization, SERTserotonin transporter, BDNFBrain-derived neurotrophic factor, MAPKp38- Mitogen-activated protein kinases, LTPlong-term potentiation,
NMDAN-methyl-d-aspartate. AMPA--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. APCadenomatous polyposis coli protein, mEPSCminiature excitatory postsynaptic currents.
Autism and schizophrenia
Autism, intellectual
disability, motor
dysfunction, anxiety,
epilepsy, recurrent
respiratory tract infections
and early death
Autism, language
impairment, intellectual
disability, anxiety,
attention decit
hyperactive disorder and
epilepsy
Behavior chamber, T-maze,
bipolar disorder.
Behavior chamber and USV Impaired social
interactions, reduced USV
Three-chambered test, Impaired social
open eld, elevated plus interactions, hypoactivity,
maze and rotarod high anxiety and abnormal
motor function
Contextual fear Impaired social
conditioning and inhibitory interactions and impaired
avoidance memory
Impaired social
Water-maze, radial maze, interactions, repetitive and
rotarod, open eld, light stereotypic behaviors,
and dark box, O-maze, reduced pain sensitivity
USV, PPI and hot-plate test and seizures
126
no SHANK1 alternation is
involved in acoustic
communication, resulting
in social communication
and interaction decits
Defects in long-term The neuroanatomy may
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
hippocampal synaptic remain sufciently intact
plasticity. Correction of so that correction of the
MECP2 levels by antisense molecular dysfunction can
oligonucleotide largely restore physiology
reverses the behavioral,
molecular and
electrophysiological
decits
Reduced basal protein Cognitive and
synthesis in hippocampal neuropsychiatric
slices. The mGluR5 symptoms of 16p11.2
negative allosteric microdeletion disorder
modulator ameliorated the arise from altered synaptic
behavioral abnormalities signaling that can be
reversed by chronic
treatment with a negative
allosteric modulator of
mGluR5
Altered inhibitory and Downregulation of
excitatory synaptic parvalbumin may result in
transmission; cortical disturbed synapse
hypertrophy and cerebellar structure/function
hypoplasia phenotypes
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
most abundant in neurons of the mature nervous system. Knock
out of the Mecp2 in male animals leads to a total loss of function
without the potential confounding effects of variable X chromo-
some inactivation (according to the Lyon hypothesis of random
chromosome X inactivation). In females, however, the animals
have a short life span. The female Mecp2 +/ model evaluated by
Samaco et al. [25] showed autistic traits that varied according to the
background strains, suggesting that the more physiologic heterozy-
gote strain can be used for future studies. Mecp2 is important for
normal GABAergic neuronal function as shown among mice lack-
ing Mecp2 from -amino-butyric-acid-(GABA)-ergic neurons that
showed reduced levels of glutamic acid decarboxylase (GAD65/67)
mRNA and decreased miniature inhibitory post-synaptic currents
(mIPSC) in the cerebral cortex [26]. Phenotypic reversal by acti-
vation of Mecp2 expression was rectied by delayed restoration
of that gene, implying that correction of the molecular dysfunc-
tion underlying the disorder can restore physiology, most probably
since Rett syndrome is a neurodevelopmental rather than a neu-
rodegenerative disorder [27].
MECP2 duplication syndrome is characterized by autism, intel-
lectual disability, motor dysfunction, anxiety, epilepsy, recurrent
respiratory tract infections and early death. Given the absence
of neurodegeneration in MECP2 duplication syndrome Sztainberg
et al. showed that correction of MECP2 levels by either tamoxifen
or human-specic antisense oligonucleotides induced a phenotypic
rescue [28].
2.0.3. Tuberous sclerosis TSC1 or TSC2 (Table 1)
Tuberous sclerosis complex is an autosomal dominant disor-
der caused by mutations in either the TSC1 or TSC2 gene that
is associated with cerebral cortical tubers and may be compli-
cated by astrocytomas. Besides intellectual disability and often
seizures of the type of infantile spasm, there is increased rate of
ASD. The protein products of TSC1, hamartin and TSC2, tuberin,
inhibit Ras homologue enriched in the brain (Rheb) that activates
the mammalian target of rapamycin (mTOR) complex 1 (mTORC1).
Heterozygous defects in either TSC gene prevents the Rheb inhibi-
tion and allows for excessive mTOR activation, which induce cell
growth and proliferation. Since homozygous mutants do not sur-
vive the embryonic period, heterozygote strains were developed,
in which most mouse strains have no cortical tubers, do not suffer
from seizures but show autistic traits. To produce the brain anoma-
lies other animal models were generated by targeting Tsc1 or Tsc2
homozygous deletion to specic cell types as neurons, astrocytes
or radial glial cells [29]. The Tsc2f/; cre mice exhibit progressive
Purkinje cell degeneration. Since loss of Purkinje cells was reported
in patients with ASD, this model was offered for future studies
[30]. The reversal of the autistic traits of the/Tsc2+/ by Rapamycin
indicate the role of mammalian target of rapamycin signaling in
decient social behavior in mouse models of tuberous sclerosis
complex [31]. (Table 1).
2.0.4. Timothy syndrome CACNA1C (Table 1)
Timothy syndrome (TS) is a rare dysmorphic disease associ-
ated with autism. It is a single nucleotide mutation in the gene
encoding the pore-forming subunits of an L-type calcium channel
(CaV1.2). The sporadic glycine-to-arginine mutation is located at
position 406 in exon 8A [Splawskis terminology). Both variants
of Timothy Syndrome (the milder TS1 and the more severe TS2)
arise from missense mutations in alternatively spliced exons that
cause the same G406R replacement in the CaV1.2 L-type calcium
channel. Since knock out mice, both homozygote and heterozygote,
did not survive to weaning, Bader et al. [32] used heterozygous
mice that were allowed to keep an inverted neomycin cassette in
127
exon 8A (Ts2-neo) that resulted in lowered expression of the G406R
L-type channel via transcriptional interference, blunting deleteri-
ous effects of the mutation. The mice survived and still exhibited
impaired social interaction and vocalization.
2.0.5. PhelanMcDermid syndrome, SHANK3 (Table 1)
Deletion of the human SHANK3 gene near the terminus of chro-
mosome 22q is associated with PhelanMcDermid syndrome and
autism. Attempts to delete SHANK3 in mice by targeting regions
of the gene failed, since none succeeded in deleting all isoforms
due to the presence of multiple promoters within the gene. Speed
et al. [33] used a frameshift mutation by inserting a single gua-
nine nucleotide into exon 21 (Shank3G) that caused a premature
STOP codon and loss of major higher molecular weight Shank3
isoforms at the synapse. Giza et al. [34] showed that deletion of
the Mapk8ip2 gene (also termed IB2 or JIP2) situated 70 kbp from
Shank3 is nearly always co-deleted. They found that mice knock out
for IB2 had reduced AMPA and enhances NMDA receptor-mediated
glutamatergic transmission in the cerebellum, disturbances in the
morphology of Purkinje cell dendritic arbors and autistic traits.
They hypothesized that IB2 mutation has a role in the Chr22qter-
associated cognitive disorders.
2.0.6. PTEN mutations (Table 1)
Phosphatase and tensin homolog on chromosome ten (PTEN) is
a tumor suppressor gene that has been reported in autistic indi-
viduals with macrocephaly, especially the Hippocampus Cowdon
syndrome and the Lhermitte-Duclos disease. Pten or Pten null mice
die during embryogenesis. Conditional loss of Pten can have dif-
fering consequences depending on the cell type or its state of
differentiation. Known et al. [35] employed a Neuron-specic eno-
lase (Nse) promoter-driven cre transgenic mouse line, in which cre
activity is conned to mature neuronal populations in the cere-
bral cortex and hippocampus. This resulted in a mutant mouse that
developed macrocephaly due to cre specic neuronal hypertrophy,
and showed autistic traits.
2.0.7. Cortical dysplasia focal epilepsy, CNTNAP2 (Table 1)
A recessive nonsense mutation in the Contactin associated
protein-like 2 (CNTNAP2) gene was shown to cause a syndromic
form of ASD, cortical dysplasia and focal epilepsy syndrome (CDFE).
This is a rare disorder resulting in epileptic seizures, language
regression, intellectual disability, hyperactivity and ASD. Neuronal
migration defects were found in about half of the patients. The CNT-
NAP2 variant that increases risk for the language endophenotype in
autism was shown to lead to abnormal functional brain connectiv-
ity in human subjects. Knock out mice for the mutation showed,
beside migration abnormalities, reduced number of interneurons
and abnormal neuronal network activity as well as autistic traits.
The abnormal behavior was reversed with risperidone treatment
[36]. The importance of this gene was also found in the inbred strain
C58/J when strain genome comparisons identied autism candi-
date genes, including Cntnap2, located within regions divergent in
C58/J [37].
2.0.8. 15q11-13 duplication maternal/paternal (Table 1)
Mutations in 15q11-13 are associated with either duplication
or gene deletion. Prader-Willi syndrome results from the loss of
imprinted genomic material within the paternal 15q11.2-13 locus
and deletions, unbalanced translocations, or uniparental maternal
disomy. The loss of maternal genomic material at the 15q11.2-
13 locus results in Angelman syndrome since normally, only the
128 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
maternal copy of the UBE3A gene is active in the brain [38].
Region duplications are the most frequent genetic abnormality in
autismand the penetrance rate in individuals with a maternally
derived duplication is >85% [39].
Maternally inherited 15q11-13 duplications and triplications
are among the most common genomic copy number variants iden-
tied in patients with autism. The dosage of an imprinted gene
or genes within the duplicated region underlies the autism risk in
these patients. E3 ubiquitin-protein ligase, UBE3A is the only gene
within the 15q11-13 duplicated segment consistently expressed
solely from the maternal allele in mature neurons, and inactivating
mutations or deletions of UBE3A cause Angelman syndrome. Smith
et al. [40] showed also in mice that the autistic traits were depend-
ent on the Ube3a gene dosage. Mice with paternal duplication of
the loci showed autistic traits in association with synaptic plastic-
ity decits in the developing and adult cerebellum and long-term
depression (LTD) at parallel bre-Purkinje cell synapses [41].
2.1. Genes associated with ASD in mice that do not cause
recognized human diseases (Tables 2 and 3)
Besides the well-recognized human syndromes derived from
gene mutations, some genetic variations were described in
increased susceptibility among patients with ASD. Genetic ani-
mal models include natural models, induced models (Table 2) and
inbred strains that naturally display ASD-related behaviors, such
as the inbred strains BTBR T+ tf/J, BALB/cByJ, A/J, and 129S1/SvImJ
[5]. (Table 3). The list of the available mouse models related to
ASD can be found in the SFARI gene database (https://gene.sfari.
org). The information in the site includes the nature of the tar-
geting construct, the background strain and a thorough summary
of the phenotypic features of the mice that are most relevant to
autism. ASD candidate genes are divided to those for which a rig-
orous statistical comparison between cases and controls has been
performed in a series of independent studies, yielding genome-
wide statistical signicance of ASD association (categories 1 and
2) and Genes with suggestive or minimal evidence of ASD associ-
ation for which only small/unreplicated genetic studies have been
performed (categories 3 and 4).
2.1.1. Neural pathways evaluated by animal models (Tables 13)
The different genetic animal models often demonstrate abnor-
mal neural and synaptic pathways which are difcult to study in
human ASD. Hence, in that respect these models reveal valuable
information that might have direct reection on the pathogenesis
of human ASD. The following are the more common pathways that
were studied:
2.1.2. The synapse: excitatory/inhibitory neurotransmission
Studies from Animal models show that the primary decit
occurs early in neural development and in the development of
synaptic function resulting in abnormal development in the super-
cial cortical layers and in the cerebellum. Continuous synapse
formation occurs throughout development, with synaptogenesis
dominating early in development, followed by synapse pruning
which lasts through adolescence. Intact glutamate signaling is crit-
ical for sensory processing and cognitive function. Calcium inux
results in release of glutamate and its concentration is regulated
in the synaptic cleft by the neurons and neighboring astro-
cytes. The spines, the postsynaptic dendritic protrusions contain
scaffolding proteins, receptors, and signaling molecules. Gluta-
mate works through ionotropic glutamate receptors (iGluRs), and
glutamate-activated G proteincoupled receptors (mGluRs). iGluRs
are subdivided into N-methyl-d-aspartate (NMDA), -amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate
(KA) receptors [52]. Animal studies support synapse dysfunction
as a core pathological feature of ASD. Models of dysmorphic syn-
dromes associated with ASD like Fmr1 knockout mice [53] show
disturbed synaptic plasticity and exhibit impaired mGluR LTD
[22]. Models for syndromic (Phelan-McDermid syndrome) [34]
and non-syndromic ASD shows abnormalities in the Shank family
of postsynaptic scaffolding proteins [54]. Shank2 knock out mice
were found to have a reduced NMDA/AMPA ratio [55]. The trans-
synaptic cell adhesion complex of pre and postsynaptic neurexins
are encoded by four genes (Nlgn14) and interact intracellularly
with Shank protein. Nlgn4 knockout mice exhibit ASD-relevant
behavioral abnormalities, including decits in social interaction
[56,57].
2.1.3. mTOR pathway
Mechanistic target of rapamycin (mTOR) is a component of pro-
tein complexes; mTORC1 lies downstream from proteins encoded
by genes associated with ASD: TSC1 and or TSC2 in Tuberous
sclerosis and phosphatase and tensin homolog (PTEN) in Lhermitte-
Duclos disease and ASD. TSC1 and TSC2 form a heterodimer that
negatively regulates mTOR signaling and PTEN is an upstream reg-
ulator of TSC1/TSC2-mTOR. Deletion of PTEN conned to discrete
mature neuronal populations in the cerebral cortex and hippocam-
pus mature neurons results in dendritic and axonal overgrowth,
increased synapse number, neuronal hypertrophy, and macro-
cephaly [35]. Mouse models decient in Tsc1 or Tsc2, which lie
downstream of Pten in regulating mTORC1 activity, show autistic
traits. Rapamycin reversed disturbed social behavior in Knock out
mice of either Tsc1 or Tsc2 [31] and prevented the pathological and
behavioral decits among mice with selective cerebellar Purkinje
cells Tsc2 knock out [30].
2.1.4. Serotonin
Serotonin (5-hydroxytryptamine) signaling mediates neuro-
genesis, cell migration and survival, synaptogenesis, and synaptic
plasticity. Elevated levels of serotonin in blood have been reported
for up to 45% of ASD subjects [5,58]. Serotonin knock out mouse
pups displayed ultrasonic communication impairment during early
development that may contribute to decient mother-infant inter-
action, presumably contributing to growth retardation phenotype
[59]. Serotonin is accumulated in blood almost exclusively in
platelets, via the serotonin transporter. Linkage studies impli-
cated the 17q11.2 region containing the serotonin transporter gene
SLC6A4 to be associated with ASD. Conversion of a single amino acid,
Gly56 to Ala56 in the transport gene resulted in autistic pheno-
type in conjunction with increased p38- Mitogen-activated protein
kinases (MAPK)sensitive basal phosphorylation. It also resulted
in enhanced hippocampal serotonin clearance rates and hyper-
serotonemia. Additionally, a decrease in the ring rate under basal
conditions was followed by serotonin1A and serotonin2A receptor
hypersensitivity [60].
2.2. The future: treatment modalities (Tables 13)
The development and evaluation of treatments that are effec-
tive in human diseases is one of the main goals of animal models.
The lack of objective clinical endpoints or biomarkers to appraise
treatment modalities complicates the development and evaluation
of medications. Since ASD is a developmental disorder, diagnosis
at earlier ages will allow treatment during development while the
neurons in the brain are still organizing, possibly before irreversible
abnormal changes have already occurred. However, using genetic
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140 129

Table 3
Inbred strains of rodents expressing phenotypic traits relevant to autism.

Reference Type Behavioral tests Results/outcome Other Conclusions

tests/markers/treatment

McFarlane et al. [84] mice, BTBR T1tf/J Behavior chamber and Impaired social Polymorphisms for the The BTBR inbred strain
open eld interactions and gene encoding is suitable as an animal
repetitive behavior kynurenine model for autism
3-hydroxylase, a
glutamate antagonist
with neuroprotective
actions
Yang et al. [85] mice, BTBR T + tf/J Arena chamber and Impaired juvenile no The repetitive behavior
three chambered test social interactions, despite cross-fostering
repetitive behavior and favors a genetic
restricted interest not background for ASD.
altered by foster
mother
Ryan et al. [86] mice, C58/J mice, Angled screen, Impaired social no The C58/J mice show
background C57BL/6J plexiglas chamber, interactions, repetitive autistic traits in both
beaker box and and stereotypic adolescence and
three-chambered test behaviors and adulthood.
restricted interest
Zhao et al. [72] mice, glut3 (Slc2a3) /- T-maze, Morris water Impaired social Compensatory increase Neuronal glucose
background C57/BL6 maze, open-eld, interaction, repetitive in neuronal MCT2 but transporter isoform 3
rotarod, radial arm and stereotype not endothelial/glial deciency and increase
maze, USV and behaviors, abnormal MCT1 paralleling in lactate uptake in
olfactory sensitivity spatial learning and enhanced brain lactate neurons is associated
test memory, with intact uptake with impaired
motor ability, reduced neurobehavior and
USV and seizures seizures
Hamilton et al. [87] mice, transgenic line Open-eld, swim test, Impaired social Lower brain weight The Fam46 gene family
generated by three chambered test, interactions, repetitive and Fam46 gene may be linked to
microinjection of a USV, olfactory and stereotypic upregulated in cortex. autism.
tyrosinase minigene detection, audiogenic behaviors, restricted
into 1-cell stage FVB seizures interest, male
embryos aggressiveness and
reduced USV
Jacome et al. [88] mice, inbred Balb/c Three chambers test Impaired social no The domains of
interactions, repetitive sociability, repetitive
and stereotypic and stereotyped
behaviors behaviors are
independent of each
other
Pearson et al. [89] mice, BTBR T + tf/J Behavior chamber Impaired social no The BTBR inbred strain
background C57Bl/6J interactions, is suitable as an animal
Stereotyped and model for autism
repetitive behavior.
Silverman et al. [65] mice, BTBR T + tf/J Three-chambered test Impaired social GRN-529 a selective mGluR5 intervention
and open eld interactions, repetitive negative allosteric are effective in ASD
behavior and restricted modulator of the
interest mGluR5 receptor
improved sociability
and reduced repetitive
and stereotyped
behaviors
Zhang-James etal. [90] rats, inbreds: Elevated plus maze, Impaired social no The small genetic
WKY/NCrl and open eld, three interactions, restricted difference between
WKY/Nhsd compared chambered test and interest, reduced USV, WKY/NHsd and
to spontaneous USV high anxiety WKY/NCrl rats may be
hypertensive and useful for identifying
Sprague Dawley genetic mechanisms

Abbreviations: MCTmonocarboxylate transporter, USVultrasonic vocalization.

engineering, Guy et al. showed in an animal model of Rett syndrome Enriched environment improved behavioral and neuronal
a phenotypic reversal following activation of Mecp2 expression abnormalities in adult Fmr1 knock out mice as well [61] supporting
[27]. Acute depletion of the cytoplasmic polyadenylation element the paradigm of benecial effects of environmental stimulation.
binding protein (CPEB), an activator of translation in the hippocam-
pus of Fmr1 /y mice rescued working memory decits as well [24].
2.2.1. Psychoactive drugs
The phenotype reversibility raises the possibility that neurological
defects seen in human disorders are not irrevocable.
Risperidone is an atypical antipsychotic approved for symp-
Currently, since there is no effective pharmacotherapy for the
tomatic treatment of ASDs. Risperidone acts at dopamine 2 and
core symptoms of ASD, neuropsychiatric drugs are used to allevi-
serotonin 2a receptors. Risperidone alleviated behavioral decits in
ate severe behavioral symptoms. The aim of animal models is to
Mu Opioid Receptor Null Mice less efciently than positive mGluR4
develop new approaches based on the pathophysiology of ASD.
Allosteric Modulator. A chronic moderate dose of risperidone
reduced striatal dopamine levels [62]. Risperidone decreased activ-
130 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
ity level, improved nest-building and reversed increased grooming
behavior and perseveration in a Cntnap2/ cortical dysplasia focal
epilepsy syndrome [46].
Tianeptine, an antidepressant structurally similar to tricyclic
antidepressants, but not Fluoxetine that increases synaptic sero-
tonin, abolished signs of anhedonia and depression in mice
overexpressing Grik4, a gene coding for a glutamate receptor sub-
unit of the kainate type in the forebrain [63]. Clonazepine, an
anti-psychotic drug that targets serotonin and dopamine recep-
tors recovered the defects in sensorimotor gating function, but
not in fear-conditioned memory among Pax6 heterozygous mutant
rats that had decreased brain and serum serotonin levels. Muta-
tions in the Pax6 gene were described in both syndromic and
non-syndromic ASD patients [64].
2.2.2. Behavioral therapy
environmental stimulation by housing pups from birth until
weaning with the mother and an additional non-lactating female
rescued the behavioral decits displayed in adulthood by Fmr1-
knock out mice including hyperactivity, reduced social interactions,
and cognitive decits. Abnormalities in the morphology of hip-
pocampal and amygdala dendritic spines, improved as well.
The manipulation affected motherinfant interaction, resulting in
enhanced physical contact with the pups, by the nursing and
non-nursing postures [20]. Enriched environment cages improved
behavioral and neuronal abnormalities in adult Fmr1 knock out
mice as well [61] supporting the paradigm of environmental stim-
ulation.
2.2.3. Synaptic modulation
GABAergic transmission mostly in the amygdala, striatum or
cerebral cortex is central to fragile X behavioral abnormalities.
Enhancing GABAergic transmission by the GABA A receptor agonist
THIP (gaboxadol) corrected attenuated hyperactivity and reduced
prepulse inhibition (evaluated by the Auditory Startle Testing)
of the Fmr1 knock out mouse [23]. The metabotropic gluta-
mate receptor subtype 5 (mGluR5), which modulates excitatory
neurotransmission and mediates signaling and protein synthesis
is increased in fragile X knockout mice. The selective negative
allosteric modulator of the mGluR5 receptor GRN-529 reduced
repetitive behaviors and spontaneous stereotyped jumping and
improved sociability [65]. D-cycloserine, a partial agonist at the
glycine-binding site of NMDARs, recovered the NMDA/AMPA ratio
and improved social interaction in a Shank2/ knock out mouse
model [66]. D-serine, a co-agonist of the glycine modulatory
sites on the NMDAR fully rescued the NMDAR-dependent LTD
impairment and signicantly improved social recognition at P-
Rex1 knock out mice in the CA1 region of the hippocampus
[67]. Based on VU0155041, a positive mGluR4 Allosteric Modula-
tor improved autistic-like symptoms and normalized immediate
early gene expression induced by social exposure in the brain of
Mu Opioid Receptor Null Mice [62]. Treatment with a negative
allosteric modulator of mGluR5 2-chloro-4-((2,5-dimethyl-1-(4-
(triuoromethoxy)phenyl)-1H-imidazol-4-yl) ethynyl) pyridine
(CTEP) (NAM), reversed the cognitive decit in a mouse model for
human chr16p11.2 microdeletion [68].
Rapamycin reversed impaired social behavior [30] and was asso-
ciated with inhibition of mTOR signaling in a mouse model of
Tuberous sclerosis [31].
2.2.4. Microglial modulator
Since several studies have demonstrated microglial distur-
bances in different brain regions of individuals with ASD,
Minocycline, a microglial modulator was used in a model of pater-
nal duplication (patDp/+) corresponding to human chromosome
15q11-q13. Perinatal treatment with minocycline restored the Iba1
expression and reduced the anxiety related behavior of patDp+ mice
[49].
2.2.5. Antisense oligonucleotides
Antisense oligonucleotides (ASO) are small, modied nucleic
acids that can selectively hybridize with messenger RNA tran-
scribed from a target gene and silence it. ASOs designed to bind
several regions of the human MECP2 precursor mRNA (pre-mRNA)
so as to reduce the levels of both alternatively spliced MECP2
isoforms, MECP2-e1 and MECP2-e2, were successfully used in
Mecp2-overexpressing mouse model and corrected MECP2 levels
in lymphoblastoid cells, abolished abnormal EEG discharges and
eliminated behavioral phenotype and electrographic seizure spikes
[28].
2.2.6. Summary
Genetic animal models are helpful to elucidate mechanisms
related to the genetic etiology and the pathogenesis of ASD. In
recent years these models are also used to evaluate the possible
success of different treatment modalities; this is of special impor-
tance as it may close a gap in our understanding of the effects of
therapy and help in the search for new and effective ways for the
treatment of ASD. The fact that many different genetic modica-
tions result in a similar phenotype, point to the complexity of the
gene environment interaction in the etiology of ASD.
3. Animal models for ASD induced by chemicals
3.1. Exposure to VPA (Tables 46)
VPA is a frequently used antiepileptic drug, also being used for
treatment of bipolar disorders, migrain. headaches and neuropathic
pain. VPA is well known for its teratogenic effects, including neural
tube defects, cardiovascular anomalies, limb anomalies, cranio-
facial abnormalities and neurodevelopmental delay [91,92]. Its
main mechanisms of action are the inhibition of histone deacety-
lase, being an epigenetic modulator and the ability to increase
oxidative stress. Prospective and retrospective human studies have
demonstrated that prenatal exposure to VPA, especially during the
rst trimester of pregnancy, is also associated with reduced cogni-
tive function and high risk for ASD among the offspring [6,7,9396].
Supporting data to the human studies have been seen in VPA rodent
models. Exposure of mice and rats to VPA can lead to behav-
ioral abnormalities that are similar to those observed in autistic
patients, including decreased social interactions, increased repet-
itive or stereotypic behaviors, lower sensitivity to pain, impaired
sensorimotor gating or eye blink conditioning, increased anxiety,
reduced exploratory behavior and abnormally high and longer
lasting fear memories [9799]. Moreover, Along with the behav-
ioral phenotype, VPA-exposed rodents present neuroanatomical
and cellular changes similar to human ASD including reduced num-
ber of motor cells in cranial nerve motor nuclei in the brain stem,
decreased cerebellar volume and reduced number of Purkinje cells
in cerebellar vermal lobules, as well as enhanced synaptic plastic-
ity of the prefrontal cortex. Signicant changes in gene expression
following prenatal VPA exposure have also been reported [100].
Rodier et al. [101] were the rst to develop an animal model of
ASD by exposing rats to VPA in utero. They found that injection of
350 mg/Kg of VPA to pregnant rats on embryonic day 11.512.5, the
stage of neural tube closure and brain stem nuclei formation, pro-
duced damage to the motor cranial nerve nuclei and reduced the
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
number of their neurons. Since then, and especially within the last
decade, the number of studies using the prenatal or early postna-
tal VPA exposure animal model is increasing, demonstrating many
of the structural and behavioral features that can be observed in
ASD patients [8,92,97,98,102,103]. Prenatal administration mimics
VPA exposure during different stages of human pregnancy, while
postnatal models investigate the effect of VPA exposure during the
third trimester of pregnancy and after, in the rst several months
after birth.
3.2. Prenatal rodent models of VPAinduced ASD (Tables 45)
3.2.1. Exposure time points
To mimic human intra uterine exposure to VPA, several time
points for VPA exposure in rodents have been suggested. In most
studies animals were exposed during organogenesis, a develop-
mental period during which exposure to teratogens may cause
specic abnormalities according to the embryonic developmental
stage at time of exposure, occurring in mice and rats during embry-
onic days 815. Therefore, the most common time points for VPA
exposure are days 9 [104106], 12 [107,108] and 12.5 [99,109112]
in rats, and 12.5 [113116] and 13 [102,117119] in mice, around
the time of neural tube formation that complete at approximately
gestational days 10.5-11 in these animals.
The most sensitive time for ASD induction by VPA was assessed
by Kataoka et al. [113] and Kim et al. [108] who compared behav-
ioral phenotypes to different time points of prenatal exposure.
Kataoka examined exposure to VPA on E days 9, 12.5 and 14.5 in
mice, while Kim compared between exposure on E days 7, 9.5, 12
and 15 in rats. E day 12.5 for mice and 12 for rats were found to be
the most vulnerable to VPA exposure.
3.2.2. Doses
The VPA doses ranged between 300 and 800 mg/kg, while most
of the studies used the amount of 600 mg/kg. In clinical populations
the dose of VPA administered is 2003600 mg/day, equivalent to
355 mg/kg for an average female that weight 65 kg [111], while
adverse effects were common in association with plasma lev-
els above 100 micrograms/ml [120]. VPA doses administered to
rodents are approximately 1020 times higher than the human
therapeutic dose, and results in a peak level of 900 mg/ml in mater-
nal plasma in less than 1 h [121]. Only few studies addressed these
differences and explained them by species differences in the phar-
macokinetics of VPA, human higher bioavailability of VPA [111,122]
and rapid elimination of VPA in rodents, with enhanced reduction
in the average plasma concentrations [123]. Furthermore, most of
the studies used a single time exposure to VPA while in human it
is generally an accumulated effect .
Lucchina et al. [114] compared between prenatal exposure to
400 or 600 mg/kg VPA. They found that mice prenatally exposed to
maternal doses of 600 mg/kg VPA had signicantly impaired social
interactions and increased anxiety behavior while mice prenatally
exposed to 400 mg/kg had much lower behavioral effect. Most of
the investigators injected a single dose of VPA dissolved in nor-
mal saline; several studies administered the VPA orally by using an
infant feeding tube [104,105] or mix of peanut butter [124].
3.2.3. Behavioral phenotype
Studies in rodents demonstrated behaviors analogous to those
observed clinically, including the core symptoms of human ASD:
decits in social interaction and communication and presence of
restricted, repetitive behavior. Schneider and Przewlocki [69] eval-
uated the behavioral aberrations of prenatally VPA exposed rats
using a large battery of tests in order to validate the VPA animal
model of autism. They found lower sensitivity to pain, locomo-
tor and repetitive/stereotypic-like hyperactivity, increased anxiety,
131
and decreased number of social behaviors, in addition to delayed
maturation and motor development. Following these ndings,
many other researchers found this model to be reliable and demon-
strated autistic like behaviors in VPA offspring using different
behavioral tools. Decrease in social interaction and social prefer-
ence was demonstrated in prenatally VPA-treated offspring using
different tools including the 3 chamber test [108,124], open eld
test [99,113,117] and play behavior measurements [125]. Decits
in social communication was demonstrated by ultrasonic vocaliza-
tions [117], nest seeking response [124] or olfactory habituation.
Increased repetitive, stereotyped behaviors were demonstrated by
open eld [99], self-grooming, marble burying [118] or Y maze tests
[109]. Additional behavioral impairments as found in subsets of
ASD children were also detected in rodents, including enhanced
anxiety by elevated plus maze, lower sensitivity to pain and learn-
ing and memory disabilities by radial maze and open eld test. VPA
offspring demonstrated faster eye-blink conditioning, consistent
with studies in autistic children [107]. Taken together, these stud-
ies demonstrate a relatively good similarity between the behavioral
phenotype observed in ASD individuals and in prenatally VPA
exposed rodents strengthening the validity of the different studies
on VPAinduced ASD. However, we should remember that some
of the tests measure behaviors that are not necessarily typical to
the behavior of children with ASD, and they therefore should be
considered questionable. Moreover, since there are obvious varia-
tions in the behavior of different offspring in the same litter, these
variations were not considered in most studies .
3.2.4. Anatomical and cellular changes in the brain
Prenatal exposure to VPA was associated with anatomical and
morphological alterations similar to those observed in the brains
of ASD patients, such as reduced size of cerebellar hemispheres,
diminished number of cerebellar Purkinje cells and cranial neu-
rons [103], enhanced synaptic plasticity of the prefrontal cortex and
amygdala, alterations in monoamines levels and amino acid neu-
rotransmission [104,106] and lower cortical expression of BDNF
[124]. Immunological alterations such as decreased splenocytes
proliferative in response to concanavalin A have also been reported
in this model [126].
3.3. Postnatal rodent models (Table 6)
Most studies that used postnatal VPA models, exposed mice
and rats to VPA on postnatal day 14. This time point has been
chosen because it is a sensitive period during which cerebellar
organization and neuronal proliferation are essentially complete,
but neuronal differentiation, myelination, synaptogenesis and glia
genesis are continuing in the cerebellum, striatum and hippocam-
pus. Wagner et al. [102] examined male and female mice exposed
prenatally to VPA or males exposed on postnatal day 14 (P14).
They found that in utero exposure to VPA resulted in develop-
mental retardation, with delayed appearance in the maturation
of surface and mid-air righting as well as negative geotaxis, grip
strength, motor activity and water maze performances. Postnatal
VPA administration caused similar retardation in the maturation
of negative geotaxis and water maze performance. Regression of
acquired skills (mid-air righting) was also demonstrated following
postnatal VPA exposure. They concluded that a single postna-
tal exposure to VPA also has long-lasting effects mimicking ASD.
Moreover, this study has shown that critical cerebellar-mediated
behaviors of mid-air righting and negative geotaxis mature rst
appear on P14 in mice. Following these ndings, Yochum et al. [127]
examined BALB/c mice (male and female) exposed to single VPA
injection on post-natal day 14. They found decreased ano-genital
snifng, crawl-under/over behavior and allogrooming in pairs of
VPA-treated mice compared to controls. They also demonstrated by
132 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

Table 4
Studies on prenatally treated mice: time of exposure and dose, outcome and conclusions from each study.

Reference Exposure window and Behavioral tests Results/outcome Other Conclusions

dose tests/markers/treatment

Wagner et al. [102] E day 13, 600 mg/kg Surface righting, Impaired mid-air no Developmental decits are
(Subcutaneous mid-air righting, righting, shorter in an ontogenic fashion
injection) hanging wire grip latency to fall from a that parallels the clinical
strength, negative suspended wire, signs of autism
geotaxis, water maze, impaired motor
passive avoidance, activity and water
motor activity maze performance
Gandal et al. [117] E day 13, 600 mg/kg Neonatal vocalizations, Reduced social Auditory evoked Abnormal auditory evoked
(Subcutaneous developmental interactions and potentials were potentials were in
injection) milestones, prepulse ultrasonic recorded correlation with autistic
inhibition of startle, vocalizations, like behaviors
locomotor testing, increased repetitive
open eld testing, self-grooming, and
social testing, rotarod, decits in prepulse
adult vocalizations and inhibition
olfactory testing
Roulett et al. [124] E day 11, 800 mg/kg Nest-seeking response, Delayed nest seeking Expression of BDNF Alterations in
(orally, food) social behavior using a response, decit in and NMDA receptor plasticity-related genes
modied 3-chamber sociability, impaired subunits NR2A and may contribute to the
apparatus, locomotor olfactory NR2B- behavioral phenotype in
activity discrimination, no plasticity-related genes prenatally exposed mice
difference in locomotor
activity
Mehta et al. [118] E day 13, 600 mg/kg Self-Grooming, open Increased repetitive Treatment by 2- mGluR5-antagonists may
(Subcutaneous eld and locomotor and anxiety-like methyl-6-phenylethyl- have therapeutic efcacy
injection) testing, Marble burying behaviors pyrididine, an for core symptoms of
assay mGluR5-receptor autism
antagonist
Kataoka et al. [113] E day 9, 12.5 or 14.5 Open-eld, elevated Social interaction Nissl staining, BrdU VPA induces behavioral
500 mg/kg(Subcutaneous plus-maze, social decits, anxiety-like neuronal birth dating decits and decreases
injection) interaction and water behavior and memory and morphometric neocortical neuron density.
maze tests decits in male mice quantitation
Kim et al. [130] E day 10, 300 mg/kg Open eld locomotor Social decits: AChE activity in the Subchronic dysregulation
(Subcutaneous test, three chambers signicantly lower prefrontal cortex. of AChE system may serve
injection) social interaction test, social approach index Treatment with as an effective
elevated plus maze, and social preference Donepezil pharmacological
marble burying test, approach index, therapeutic target against
self-grooming and reduced cognitive autistic behaviors
digging test, novel exibility, greater
object recognition test, locomotor activity;
nest building test. upregulated AChE
expression in the
neural progenitors
Lucchina et al. [114] E day 12.5 400 or Social interaction test, VPA600 mice spent less Peripheral Prenatal VPA results in
600 mg/kg(Subcutaneous evaluated plus maze, time snifng the inammatory stimulus autism-related behaviors
injection) open eld, novel object stimulus in the social challenge and long-lasting alterations
recognition test, tail interaction test. No in peripheral and brain
suspension differences between inammatory responses
VPA and control mice
in the tail suspension
or forced swimming
tests
de Theije et al. [134] E day 11, 600 mg/kg Social behavior test Reduced social Measure of serotonin Males had autism-related
(Subcutaneous interaction in males; (5-HT) in brain and behaviors and
injection) disturbed serotonergic intestinal tissue inammatory conditions in
system in the brain and the small intestines.
intestinal tract
Kang et al. [131] E day 13.5 Three chamber test, Rescue of impaired Administration of Suppression of NMDAR
600 mg/kg(Subcutaneous self-grooming, social interaction, memantine, an NMDAR function in VPA mice
injection) jumping, and digging, enhanced repetitive antagonist rescues repetitive behavior
forty eight-hour self-grooming and as well as social decits
movements jumping, no
measurement impairment in social
novelty recognition, no
alterations in
self-grooming,
horizontal locomotion
or rearing movement
Al-Amin et al. [132] E day 12.5 600 mg/kg Three-chamber social Decits in pain Increase in oxidative VPA exposure leads to
(Subcutaneous interaction device; sensation, increased stress markers: SOD, increased oxidative stress.
injection) locomotor activity and locomotor activity, MDA, APOP, GSH, CAT Treatment with
anxiety tested by open higher anxiety level, and NO level; astaxanthin signicantly
eld test, nociception lower exploratory treatment with the improved the behavioral
by hot-plate test. activity, reduced antioxidant astaxantin disorder and reduced the
sociability and social oxidative stress in brain
novelty. and liver
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140 133

Table 4 (Continued)

Reference Exposure window and Behavioral tests Results/outcome Other Conclusions

dose tests/markers/treatment

Cheaha et al. [119] E day 13, Local eld potentials Decreased numbers of Local eld potentials Hippocampal and olfactory
600 mg/kg(Subcutaneous ultrasonic vocalization (LFP) acquisition bulb LFP might be
injection) calls, sociability and surrogate biomarkers of
increased repetitive VPA mouse model.
self-grooming
behavior,
Hara et al. [115] E day 12.5 500 mg/kg Social interaction test, Reduced duration of Morphological analysis Treatment with stimulants
(Subcutaneous novel object snifng, decrease in of dendritic spines. improved the behavioral
injection) recognition test the time exploring the Treatment with decits and suppressed the
novel object, methylphenidate or reduction in spine density
recognition memory atomoxetine in male mice via a
impairment (males) prefrontal dopaminergic
system-dependent
mechanism
Hara et al. [116] E day 12.5 Locomotor activity Decrease in the Measurement of ASD symptoms in male
500 mg/kg(Subcutaneous locomotor activity in dopamine, mice may be related to
injection) male mice noradrenaline, hypofunction of
serotonin and their dopaminergic system in
metabolites levels in the prefrontal cortex
the prefrontal cortex
and striatum

TUNEL stain that VPA treatment enhanced cell death in the cerebel-
lum and hippocampus. Earlier postnatal VPA exposure described by
Wang et al. [128] demonstrated anxious, depressive, and impaired
social behaviors in addition to changes in neural cell proliferation
and differentiation in male rats exposed to VPA on post-natal day
7, that is equivalent to a fetus at 3640 weeks gestation.
Taken together, these studies demonstrate that postnatal VPA
exposure results in anxiety-related and depressive behaviors and
fewer social interactions along with diminished number of Purkinje
cells and enhanced cell death in the cerebellum and hippocampus
[102,127,129]. It should be emphasized, however, that many of the
behavioral changes described in these studies are not necessarily
typical of ASD like behaviors .
3.4. Gender-specic differences
ASD is 45 times more common in male children than in females.
However, in children born to women treated during pregnancy
with VPA there seems to be no gender difference in the rate of
ASD [96]. Most animal studies, which used prenatal VPA exposure,
did not report any differences between genders in ASD behavioral
phenotype [104,106,107,109,117,118,124,125,130133]. Several
investigators used only males to eliminate the possible sex differ-
ence [99,105,110,112,114,115,119]. Wagner et al. [102] compared
between males and females and found no difference in the behav-
ioral assessment. However, Kataoka et al. [113] and de Theije
et al. [134] reported a decrease in social interaction in male
VPAexposed offspring compared to females. In contrast, Farve
et al. [111] demonstrated more signicant olfactory decit in
female offspring of VPA rats compared to males.
Similar data can be seen in the postnatal studies as in sev-
eral of them, only males were used for the behavioral tests
[102,127,128], others did not mention any gender-specic differ-
ences in the behavioral tests [135,136]. Two studies have shown
ndings that are consistent with the male preponderance of ASD;
Oguchi-Katayama et al. [129] reported enhanced anxiety in post-
natal exposed males but not in females. Reynolds et al. [137]
found Gender-specic differences in sensory processing. While
VPA exposed males had signicantly diminished auditory startle
responses and altered sensory motor gating during both the juve-
nile (P day 23) and the adolescence (P day 45) stages, female rats
have shown similar results only during adolescence. They sug-
gested that this may indicate differences in the timing of symptom
manifestation between males and females, as observed in human
studies [138]. The lack of gender differences in the VPA induced
ASD can be explained by the fact that the specic effects of VPA on
the brain during different stages of brain development are appar-
ently not related to the genetic differences between genders and in
that case gender differences play only a minimal role.
3.5. Summary
We should remember that rodents are not man, and the
observed neurobehavioral decits, although mimicking human
ASD, may not be exactly the same. Moreover, these studies also
suffer from the disadvantage that no study seems to describe the
results in relation to the individual behavioral changes in each one
of the prenatally or postnatally exposed animals. From our studies
on early postnatal VPAinduced ASD like symptoms in ICR albino
mice (Ornoy et al., personal communication), we know that there
are signicant variations in the behavioral abnormalities among the
animals of the same litter and between males and females. Hence,
in spite of the difculties, it is important to try and assess all bio-
chemical, morphological and molecular changes in relation to the
type and extent of the individual neurobehavioral decits. The fact
that prenatal exposure to VPA at different phases of brain develop-
ment, lead to a similar phenotype of ASD like behavior, implies that
there might be a general mechanism of action of VPA not related
to the developmental stages of the brain. Indeed, many of the envi-
ronmental factors associated with ASD in pregnant women would
increase the rate of ASD with no correlation to the specic time
of exposure in pregnancy [4]. Hence, these agents might produce
their damage through a different mechanism, perhaps by epige-
netic changes.
3.6. Exposure to thalidomide
Thalidomide caused multiple birth defects such as limb
reduction defects, ocular and cardiovascular anomalies. Today,
thalidomide is still used in the treatment of leprosy and mul-
tiple myeloma [139]. Strmland et al. [140] have reported an
increased number of children with ASD following prenatal expo-
sure to thalidomide. Very few Animal studies have examined the
use of thalidomide exposure as a model of autism. Narita et al. [141]
exposed rats to 500 mg/kg of thalidomide on embryonic day E2, E4,
E7, E9, and E11. They also exposed rats to 500 mg/kg of VPA on E day
134
Table 5
Studies on prenatally VPA treated rats: time of exposure and dose, outcome and conclusions from each study.The table shoud start from the left with the reference and and
with the conclusion.
Reference
Schneider et al. [99]
Stanton et al. [107]
Tsujino et al. [104]
Markam et al. [109]
Dufour-Rainfray et al. [106]
Narita et al. [105]
Kim et al. [108]
Bringas et al. [110]
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
Exposure window and Behavioral tests Results/outcome Other Conclusion
dose tests/markers/treatment
E day 12.5 600 mg/kg Negative geotaxis, Lower sensitivity to no Similarities between
(Subcutaneous swimming pain; higher sensitivity the observed pattern of
injection) performance, olfactory to nonpainful stimuli; behavioral alterations
discrimination, diminished acoustic in VPA rats and
nociception and tactile prepulse inhibition; features of disturbed
threshold, locomotor and behavior in autistic
sensorimotor repetitive/stereotypic- patients.
gatingprepulse like hyperactivity;
inhibition, locomotor, lower exploratory
repetitive/stereotypic- activity; decreased
like, and exploratory number of social
activity, social behavior behaviors and
increased latency;
delayed maturation;
lower body weight,
delayed motor
development;
attenuated integration
of coordinated
reexes; delayed
nest-seeking response
mediated by olfactory
system.
E day 12, 600 mg/kg Acquisition and Faster eye blink no Faster eye blink is
(Subcutaneous reversal of conditioning. similar to the condition
injection) discriminative eye in autistic children
blink
E day 9800 mg/kg Open-eld test, Hyperactivity in the Serotonin counting in Higher serotonin levels
(water) circadian activity novel environment; a rat brains in the prefrontal cortex
(locomotor activity and higher mean count of might be responsible
feeding under a feeding during the light for the irregular
reversed 12-h cycle) phase and lower sleep/awake rhythm in
during the dark phase autism
E day 12.5 500 mg/kg Social Interaction, Impaired social Electrophysiol-ogical Enhanced
(Subcutaneous repetitive behavior, interactions, increased recording from the overgeneralized and
injection) nociception, startle repetitive behaviors amygdala extinction-resistant
response and prepulse and less sensitivity to conditioned fear
inhibition, anxiety, fear pain, more anxiety and memories may be
conditioning, Morris abnormal high and caused by the
water maze longer lasting fear hyperactivity and
memories hyperplasticity in the
lateral amygdala,
which may be due to
impairments in the
inhibitory system of
the amygdala
E day 9 600 mg/kg Sociability test Lower tendency to Serotonin Behavioral alterations
(Subcataneous initiate social measurements in the could be a consequence
injection) interactions and hyper cerebellum, prefrontal of decreased serotonin
locomotor activity in cortex and levels in the
juvenile male rats hippocampus. hippocampus
E day 9800 mg/kg (in Eight-arm radial maze Impaired achievement no VPA might alter
water) learning assay, open of learning; no behavior in a manner
eld and social signicant differences that is, in part,
interaction test in social interaction consistent with human
autism
E days 7, 9.5, 12 and Sociability and social E12 rats showed no E12 is the critical
15; 400 mg/kg preference test signicantly impaired period in rats when
(Subcutaneous social interactions and VPA exposure has
injection) vulnerability to seizure prominent effects for
inducing the altered
social behavior similar
to human autistic
behavior
E day 12.5 500 mg/kg Exploratory behavior Hyper-locomotion and Morphological VPA altered behavioral
(Subcutaneous and locomotor activity impaired exploratory examination of phenotype is
injection) in a novel environment behavior neuronal accompanied by
rearrangement in neuronal
various parts of the morphological
brain rearrangement in the
hippocampus,
prefrontal cortex and
basolateral amygdala
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140 135

Table 5 (Continued)

Reference Exposure window and Behavioral tests Results/outcome Other Conclusion

dose tests/markers/treatment

Favre et al. [111] E day 12.5 500 or, Olfactory VPA 500 had olfactory Body and brain mass VPA at 500 mg/kg on
600 mg/kg discrimination decits measurement E12.5 is sufcient to
(Subcutaneous behavioral test induce a core
injection) autism-like symptoms
Gao et al. [112] E day 12.5 600 mg/kg Morris water maze Males had poorer Histology and DHA prevent cognitive
(Subcutaneous learning ability than enzymatic activity impairment in male
injection) controls measurement. offspring of
Treatment with VPA-treated rats and
docosahexaenoic acid may play a
(DHA) neuroprotective role in
hippocampal neuronal
cell and ameliorates
dysfunctions in
learning and memory
in this rat model
Olde Loohuis et al. [125] E day 12.5 495 mg/kg Social play behavior, Signicantly lower Genome-wide The changes in miR and
(Subcutaneous elevated plus maze, motor performance transcriptomics mRNA expression
injection) prepulse inhibition, and coordination in expression in the proles of the rat
open eld tests swimming amygdala amygdala most likely
performance on P12; underlay many of its
impaired social play anatomical and
behavior. functional alterations
that have also been
observed in autistic
individuals
Frisch et al. [133] 470 and 720 mg/kg (in Negative Geotaxis, Memory impairments MRI analysis of brain Long-term valproate in
drinking water, during rotarod performance, and delayed motor volumetric changes utero affects offspring
the entire pregnancy) water maze test learning in offspring cognitive capabilities
from dams receiving and probably depends
high doses of VPA; on the total drug load
medium dosages led to differentially affecting
improved water maze cerebral development
performance during adolescence

9. They found that thalidomide and VPA increase serotonin in the
hippocampus, increase dopamine in the frontal cortex, and cause
hyperserotonemia when given on E9. In a later study by Narita et al.
[105] they examined the behavioral phenotype of rats exposed to
VPA or thalidomide on E day 9. They reported impaired achieve-
ment of learning in both VPA and thalidomide exposed pups. They
did not nd other behavioral effects typical of ASD. Hence, it seems
that thalidomide is not an appropriate model for ASD in rats that
are generally insensitive to the teratogenic effects of that teratogen.
3.7. Exposure to misoprostol
Shultz et al. [148] reported that exposure to PPA consistently
impaired social behavior measured as distance apart, proximity,
and play behavior in rats.
MacFabe et al. [147] demonstrated that rats treated with PPA
displayed restricted behavioral interest to a specic object among
a group of objects, impaired social behavior, and impaired rever-
sal in a T-maze task compared to controls, in addition to reactive
astrogliosis and activated microglia in the brain.
These results provide evidence of occurrence of ASD-like behav-
iors in the PPA rodent model. However, the relevance to the human
situation is unknown.

Misoprostol is a prostaglandin analog drug commonly pre-

scribed for the prevention and treatment of gastric ulcers [142],
There is evidence that prenatal exposure to high doses of miso-
prostol during the rst or second trimesters of pregnancy can lead
to the occurrence of Mbius Syndrome, a disorder characterized
by uni- or bilateral eye-face palsy due to damage to cranial nerve
nuclei, associated with muscle or skeletal malformations and a
high prevalence of autistic- like behavioral symptoms [4,143,144].
Koenig et al. [145] were the only ones who examined the effect
of Misoprostol on autistic behavioral phenotype in mice using a
battery of behavioral tests. They found no signicant neurodevel-
opmental or behavioral effects of injection with 0.4, 4 or 40 g/kg
misoprostol on postnatal day 7 in mice.
3.8. Exposure to PPA
Propionic acid (PPA) is a short chain fatty acid, a metabolic
end-product of enteric bacteria in the gut, and a common food
preservative. A number of studies indicated that PPA can cause
autistic like behaviors and a neuro-inammatory response in rats
[146149].
4. Animal models of infection and inammation in relation
to ASD
Large population studies found increased rate of ASD in the
offspring of women with fever and infection in pregnancy, espe-
cially when maternal infection was more severe and necessitated
hospitalization. This association is apparently related to maternal
inammatory process. Maternal immune activation may play a role
in neuro-developmental perturbation [4].
Animal models in which early viral infection results in behav-
ioral changes later in life include the inuenza virus model in
pregnant mice and the Borna disease virus model in newborn Lewis
rats. Respiratory infection with human inuenza virus in pregnant
mice at mid-gestation resulted in behavioral abnormalities in some
of the offspring, mostly decits in social interaction and prepulse
inhibition (PPI) and in histological abnormalities in the hippocam-
pus and cortex of the neonatal offspring [150]. Neonatal Borna
virus infection resulted in decreased play activity among pups and
a stereotypic behavior with decrease in the exploratory activity
at adulthood. Immunohistochemistry was positive for the pres-
ence of Borna virus throughout the brain. Pathological ndings

Table 6
Studies on postnatally VPA treated mice and rats: time of exposure and dose, tests used, outcome and conclusions from each study.
Reference
Wagner et al. [102]
Yochum et al. [127]
Pragnya et al. [135]
Oguchi- Katayama et al. [129]
Wang et al. [128]
Chomiak et al. [136]
Reynolds et al. [137]
Type and gender
BALB/c male mice
BALB/c male mice
BALB/c mice (males
and females)
Wistar Hannover/Rcc
rats (males and
females)
Sprague-Dawley male
rats
Sprague-Dawley rats
(males and females)
Long-Evans rats (males
and females)
Exposure window and Behavioral tests
dose
P day 14, 200 or Mid-air Righting,
400 mg/kg hanging wire, negative
(Subcutaneous geotaxis, balance
injection) beam, water maze,
passive avoidance,
motor activity and
self-injury behavior
P day 14, 400 mg/kg Social interactions and
(Subcutaneous play behavior (open
injection) eld) behavior and allogrooming,
P day 14, 400 mg/kg Negative geotaxis,
(Subcutaneous nociception, locomotor
injection) activity, Rota rod test,
Moris water maze,
elevated plus maze
P day 14, Elevated plus maze
400 mg/kg(Subcutaneous
injection)
P day 7200 mg/kg Elevated plus maze,
(Subcutaneous open eld test, forced anxiety-like behaviors at
injection) swimming test and
social interaction test
from P6 for maximum Social (play) behavior,
2 weeks 150 mg/kg/day cued sensorimotor task
Daily Subcutaneous Auditory and tactile
injection from P day startle, auditory
612 150 mg/kg sensory gating, motor auditory sensory gating,
testing including
sunower seed eating speed and performance
task and a vermicelli
handling task
Results/outcome
Retardation in the
maturation of negative
geotaxis and water maze
performance. Regression of
acquired skills (mid-air
righting)
Decreased ano-genital
snifng, crawl-under/over
no signicant difference for
nonsocial behavior such as
self-grooming, decrease in
motor activity; enhanced
cell death in the
cerebellum and
hippocampus
Impaired performance on
negative geotaxis, elevated
plus maze and Morris
water maze tests.
Decreased motor
co-ordination, thermal
nociception and social
behavior increased
locomotor activity,
increase oxidative stress
Impaired performance on
elevated plus maze in
males
Depressive and
the late postnatal ages;
impaired social interaction
Reduced social play
behavior
Under responsiveness to
auditory stimuli, decits in
impairments in motor
Other
tests/markers/treatment
no
Histopathological analysis,
TUNEL assay for apoptosis
Malondialdehyde
concentration, glutathione
and total nitrite levels, and
serotonin determination;
pepirine treatment
microarray assay analysis
of changes in gene
expression in prole in the
amygdala
Studies of differential
expression of genes
associated with neural cell
differentiation mainly in
the hippocampus
Cortical thickness
measurements
no
136
Conclusion
Postnatal VPA exposure
cause developmental
decits in an ontogenic
fashion that parallels the
clinical signs of autism
VPA- enhanced cell death
in the cerebellum and
hippocampus may be
responsible for ASD like
behavior
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
Postnatal VPA exposure
causes behavioral decit,
increased oxidative stress,
increased serotonin levels
in the hippocampus, and
diminished number of
Purkinje cells in the
cerebellum. Treatment
with pepirine can
signicantly improve these
outcomes.
The gene expression
changes in the amygdala
might be an important
cause for impaired social
behavior and enhanced
anxiety rather than
expression changes of
particular genes
VPA disrupts neuronal
differentiation program
related to anxious,
depressive, and impaired
social behavior and
involved in anxiety
disorder typical to autism
VPA exposure can lead to
synchronous
developmental alterations
reminiscent of ASD
Postnatal valproate
treatment elicits sensory
and motor features often
seen in individuals with
ASD.
 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140
included complete degeneration of the dentate gyrus, thinning of
the neocortex and loss of Purkinje cells in the cerebellum [151].
The implication for human disease is controversial since there is no
strong experimental evidence that Borna virus is a human pathogen
[152]. Most animal models for the impact of maternal infection on
the offspring evaluate the inammatory process and not specic
viruses. Revising most models of maternal inammation and its
effect on the offspring behavior and neurodevelopment is beyond
the scope of this review. Hence, we will summarize only some
studies that touch on this subject.
Maternal Inammation is triggered by injection of immunogens
during pregnancy and the offspring are evaluated. In a literature
review Boksa [153] found that different models demonstrated that
maternal prenatal immune activation was associated with changes
in various interleukins in the fetal brain, mostly including: IL1, IL6
and TNF- , when their levels were associated with the time of
injection of the immunogen to the pregnant animal and the time
of the offspring evaluation. Morphological changes were described,
mostly in the hippocampus and cerebral cortex. However, changes
were noted in other areas as well. The more common immunogens
used are lipopolysaccharides [154] and Polyriboinosinic: polyri-
bocytidylic acid (poly I:C) system, a double-stranded RNA, which
mimics viral infection [155]. The injection of immunogens trig-
gered tissue necrosis in the placenta, hypoperfusion of the fetus
and reduced neural progenitor cell mitotic index in the develop-
ing cortex [154]. Ohkawara et al. suggested that immune activation
induced by poly I:C may cause ASD in the offspring since they found
decrease in hippocampal serotonin levels in the murine adult off-
spring which may cause behavioral and cognitive abnormalities
[155]. The higher rate of ASD among male mice was found following
injected with LPS or Poly I:C during mid-gestation when stereo-
typed, repetitive behavior was found in male but not in female
offspring [156].
Transparency document
The Transparency document associated with this article can be
found in the online version.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.reprotox.2016.
04.024.
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