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Reproductive Toxicology 64 (2016) 116140

Contents lists available at ScienceDirect

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

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 117

changes and the behavior of a mouse or a rat is not the same as 1.1. Genetic animal models of autism: implication for human
the behavior of a child or adult. Indeed, a variety of neurobehav- studies (Tables 13)
ioral tests have been used to demonstrate in the animal models
a behavioral pattern that mimics the behavior of ASD children. Several human syndromes derived from a single gene mutation
Another approach is to create genetic models that are similar to increase the risk for ASD. The more common aberrations are Fragile
the human genetic changes observed sometimes in children with X syndrome, a mutation in FMR1 [10], Rett syndrome, a mutation
ASD. In these models, neurobehavioral tests often demonstrate an in MECP2 [11], tuberous sclerosis, mutations in TSC1 or TSC2 [12]
autistic like behavior as well as distinct changes in the brain that and Timothy syndrome, a mutation in CACNA1C [13]. Copy-number
resemble those sometimes found in children with ASD. variants that lead to inherited maternal 15q1113 duplication
Despite the heterogeneity of the clinical symptoms, animal resulting in Prader-Willi syndrome [14] and other duplications like
studies allow coherent investigations of the circuits, cells, and the NPHP1 gene [15] are also associated with autistic traits. The
pathophysiological processes affected in autism [5]. development in strategies for the identication of genetic variants
Basically, there are two types of animal models for ASD: environ- also led to the description of new syndromic forms of ASD and
mentally induced, by exposure of the pregnant animals to certain enabled the association between phenotype and genetic traits .
chemicals or infection/inammation and those induced by genetic The identication of such genetic variations with the devel-
manipulations. The rst are relevant mainly if it is shown that the opment of new strategies for genetic engineering facilitated the
same chemicals are also relevant for human; for example VPA. development of genetic animal models of ASD. Mice are the pre-
VPA was found to signicantly increase the rate of ASD among off- dominant animal model for ASD owing to their genetic tractability
spring of treated mothers [6,7] and was thereafter shown to induce and their ability to demonstrate analogs of behavioral decits
autism-like behavior in rats and mice [8]. Genetic animal models of associated with ASD [5]. Other animal models include rats [16],
ASD are also important especially when they mimic the ASD symp- zebrash [17], song birds [18] and the newly introduced macaque
toms that are present in distinct human genetic diseases such as monkeys [19]. Until now, only few of the current animal models
fragile X and tuberous sclerosis. Mouse models should therefore be were proven valid for evaluation of a known human aberration by
based on a known genetic cause of a disease, reect key aspects establishment of a measurable marker and an offer of a treatment
of the human symptoms and, if possible, respond to treatments option.
that are effective in the human disease [9]. However, new genetic
changes in animals that induce ASD like symptoms may also be 2. Animal models for genes of a syndromic disorder
of importance since they may push to seek similar changes in predisposing to autism (Table 1)
man.
ASD in human is manifested by a set of behavioral changes The animal models representing known human syndromes
that appear at early childhood. The most prominent are impaired derived from a gene mutation exemplify the difculties in estab-
social interaction, impaired language and motor behavior, includ- lishing a valid genetic trait.
ing stereotyped and repetitive motor movements and limited
interest in the surroundings. These behaviors can also be observed 2.0.1. Fragile X (FMR1)Table 1
and monitored in various animal models by using specic tests
that were developed for the measurement of such behavioral Children with Fragile X syndrome which is the most frequent
modications. Although it is valid to argue that animal behav- inherited cause of mental retardation have increased rates of ASD.
iors are different from those in men, the fact that they can be The Fragile Mental Retardation 1 locus (FMR1) resides in the X
specically demonstrated in the relevant models points to their chromosome and expansion of triplet repeats in the untranslated
similarity to the human behavior. As most genetic and non-genetic region of the FMR1 gene prevents synthesis of the FMR1 gene
models of ASD are in mice and rats, the more common and reli- product FMRP. FMRP is an RNA-binding protein that modulates
able behavioral tests were developed in these animals. In spite mRNA trafcking, dendritic maturation and synaptic plasticity.
of some limitations they are generally considered to represent Rodents, mostly mice knock out for the FMR1 gene, were shown to
adequately human behaviors. In the tables we name, wherever present autistic traits. Different traits depend on the background
relevant, the different behavioral tests used by the investigators strain. Some of the studies also found structural, biochemical and
to demonstrate autistic like behaviors in their models. It should physiological abnormalities including abnormal dendritic spine
be remembered, however, that not all tests used by the differ- morphology [20], elevated phosphorylation of translational control
ent investigators are unanimously accepted as representing ASD molecules and exaggerated protein synthesis in the hippocam-
like behaviors. Hence, this might be a weakness of some of the pus [21]. Additionally, enhanced metabotropic glutamate receptor
studies. (mGluR)-dependent long-term depression (LTD) recordings were
The purpose of the present review is to summarize the data described [21,22], implying reduction in the efcacy of neuronal
on more common experimental animal models of ASD either as a synapses following a stimulus. Treatment modalities by genetic
result of prenatal exposure (i.e. VPA, inammation) or those follow- engineering and medications are meant for prevention of exag-
ing genetic manipulations. Most models are in mice and rats. The gerated protein synthesis [21] rescue of synaptic tonic inhibitory
current review includes only investigations in which animals were currents [23] activation of translation to overcome the lack in Fmrp
evaluated by behavioral studies and the changes mimic ASD like [24] and genetic engineering for correction of the synaptic mGluR
behavior. The impacts of the genetic background or environmental signaling [22]. However, environmental stimulation was successful
insults on the phenotypic traits, anatomical, biochemical, physio- as well [20].
logical markers and treatment modalities are described whenever
applicable. The tests used, the neurobehavioral changes and mode 2.0.2. Rett syndrome and MECP2 mutations (Table 1)
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 Rett syndrome, an X linked disease that affects girls, is char-
describe the VPA induced models we also added the time of expo- acterized by neurodevelopmental delay, ASD and seizures. It is
sure and dose of VPA. 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, Impaired social interaction, Environmental stimulation The use of
background female T-maze and Fear hyperactivity and cognitive by additional non-lactating non-pharmacological
FVB.129P2-Fmr1tm1Cgr/J conditioning decits female improved impaired interventions for the
(FVB), male behavior and eliminated treatment of autism is
FVB.129P2Pde6bTyrc- the abnormalities in the benecial
ch/AntJ morphology of
hippocampal and
amygdala dendritic spines.
Guy et al. [27] Rett syndrome mice, silenced MECP2 gene Behavior chamber Repetitive and stereotypic Reduced LTP in the Rett syndrome is a
by a loss of lox-Stop behaviors, irregular hippocampus. The MECP2 neurodevelopmental
cassette breathing and abnormal activation under the rather than a
gait control of its own promoter neurodegenerative
and regulatory elements by disorder

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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

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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 Type Behavioral tests Results of behavioral tests Other Conclusions
tests/markers/treatment

Smith et al. [40] 15q11-13 maternal Mice, UBE3a 2 transgenic Behavior chamber, Impaired social impaired glutamatergic The autism traits depended
duplication, autism three-chambered test and interactions, repetitive and synaptic transmission, on UBE3a gene copy
USV stereotypic behaviors reduced presynaptic number. Increased E3A
glutamate release via ubiquitin ligase gene
reduced postsynaptic dosage results in reduced
excitability and phasic excitatory synaptic
excitatory synapse-like transmission.
stimuli

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


Piochon et al. [48] 15q11-13 duplication, Mice, patDp/+, human DigiGait imaging system Impaired social Saturated LTD in Purkinje Decits in synaptic
Autism 15q11-13 duplication by and delayed eyeblink interactions, increased cell soma under baseline plasticity and pruning are
chromosome engineering conditioning, activity and impaired condition preventing potential causes for motor
on mouse chromosome 7 eyeblink conditioning subsequent LTD induction problems and abnormal
and developmental circuit development in
elimination of surplus autism.
climbing bers.
Shigemori et al. 15q11-13 mice, patDp+, paternal open Field, marble burying Impaired social Decreased Iba1, a Perinatal microglia in the
[49] duplication duplication in mouse and USV interactions, high anxiety, microglial activation basolateral amygdala may
Autism chromosome 7 fear in response to stress marker in the amygdala play a pathogenic role in
corresponding to human and reduced USV was restored by maternal the anxiety observed.
chromosome 15q11-q13 minocycline treatment
during pregnancy and
lactation. Minocycline also
reduced anxiety-related
behaviors.
Zheng et al. [50] Rubinstein-Taybi mice, CBPCH1/CH1, Behavior chamber, open Impaired social Disturbed NMDA intact CH1 domain is
Syndrome C57BL/6 129SvF1 hybrid eld, elevated plus maze, interactions, repetitive and receptordependent important for normal social
three-chambered test, stereotypic behaviors, synaptic depression in behavior, motor function,
rotarod and hot plate test hyperactivity and reduced hippocampus and cognition
anxiety
Meechan et al. [51] mice, LgDel transmitted Autism, schizophrenia, LCD touchscreen apparatus Restricted interest and Altered inhibitory synaptic Disrupted parvalbumin
paternally, genomically DiGeorge syndrome impaired cognitive organization, disrupted interneuron-dependent
accurate model of function. parvalbumin inhibitory circuitry, is a key
22q11DS, background interneuron-dependent feature of cortical
C57/Bl6N inhibitory circuitry. connectivity disorders that
arise during development,
including 22q11DS

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

121
122
Table 2
Animal models for genes associated with phenotypic traits relevant to autism in rodents that do not cause recognized human diseases.

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

Jamain et al. [69] mice, loss-of-function Autism Elevated plus maze, open Impaired social Reduced brain volume Neuroligin-4 encodes the
mutation in the murine eld, rotarod, Morris water interactions, restricted synaptic cell adhesion
NLGN4 ortholog Nlgn4 maze, PPI, buried food, interest and reduced USV protein which takes part in
sucrose preference, USV upon contact with a female the regulation of the
and chemical seizures maturation and function of
synapses is associated with
autistic traits
Radyushkin et al. [70] mice, NL-3 /-, background Autism Elevated plus maze, open Impaired social Reduced brain volume A point mutations in
C57BL/6NCrl eld, hole board, rotarod, interactions, reduced USV, neuroligin-3 a homologue
Morris water maze, PPI, olfactory deciency and of neuroligin-4 results in a
USV, Buried food, Sucrose hyperactivity behavioral phenotype
preference and seizures related to olfactory and
communication deciency

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


Umeda et al. [64] rats, rSey-/+, Pax6 Autism, absence or Behavior chamber, forced Impaired social Reduced 5-HT level in the The rSey2/+ rats likely have
heterozygous mutant hypoplasia of the anterior swim, electric shock interactions, repetitive and hippocampus. Clozapine some phenotypic
commissure, decreased sensitivity and USV stereotypic behaviors, was effective to improve components of autism
volumes of the corpus aggressiveness, impaired sensorimotor decits but
callosum, smaller brain fear-conditioned memory did not alter other
size, aniridia and various and reduced USV only in behavioral phenotypes
eye abnormalities. females.
Zaccaria et al. [71] mice, GAP43 (+/), Autism Three chambered test, Impaired social no Mice lacking one allele for
background C57BL/6J T-Maze, Morris water interactions, stereotypic the synaptic
129S3/SvImJ (B6129S3) maze, marble burying, food behavior, restricted growth-associated protein
preference and light and interest and high anxiety 43 can be used as an
dark box animal model for autism.
Zhao et al. [72] mice, glut3 (Slc2a3) /- no T-maze, Morris water Impaired social Compensatory increase in Neuronal glucose
background C57/BL6 maze, open-eld, rotarod, interactions, repetitive and neuronal MCT2 but not transporter (GLUT) isoform
radial arm maze, ultrasonic stereotype behaviors, endothelial/glial MCT1 3 deciency and increase
vocalization and olfactory abnormal spatial learning paralleling enhanced brain in lactate uptake in
sensitivity test and memory, with intact lactate uptake neurons is associated with
motor ability, reduced USV impaired neurobehavior
and seizures and seizures
Hiramoto et al. [73] mice, congenic Tbx1HT, Autism, social interaction Behavior chamber, Impaired social Tbx1 protein, Tbx1, a Tbx1 heterozygosity
background C57BL/6J decits and delayed elevated plus maze, and interactions, 22q11.2 gene was contributes to
development vocalization USV aggressiveness, reduced ubiquitously expressed 22q11.2-associated ASD
and language USV, impaired memory throughout the brain of presumably through its
and normal olfaction. C57BL/6J mice, and is expression in diverse brain
enriched in postnatally regions, some of which
proliferating cells include postnatally
generated neurons.
Horev et al. [74] mice, 16p11.2, either one Autism, Home Cage Scan Loss of 16p11.2: repetitive Brain volume size: The deletion and the
copy (df) allele], or with neurodevelopmental and and stereotypic behaviors, macrocephaly in deletion duplication affect behavior
three copies [heterozygous neuropsychiatric gain of 16p11.2: and microcephaly in and brain anatomy in
for a duplication (dp) syndromes hyperactivity and duplication. MRI show opposing ways, with
allele, background abnormal diurnal behavior changes in the posterior deletion mice exhibiting
C57BL/6N:129Sv 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, Motor decits, no SHANK1 is associated more
background C57BL/6 (B6) three-chambered test, mild-anxiety and with motor function and
and129SvJae (129Jae) olfactory habituation, open decreased cognitive memory and less with
eld, rotarod, Inverted abilities and memory. social decits.
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 Impaired social no Nlgn4/NLGN4X loss-of
background C57BL/6J. eld, hole board test, interactions, repetitive function mutation in
rotarod, PPI, buried food behavior, restricted mouse induce autistic
test, USV, sensor platform, interest and reduced USV traits
marble burying and nest
building
Kyzar et al. [75] mice, SERT+/ mice and Autism, schizophrenia, behavior chamber SERT+/ mice: Impaired no Decreased SERT and BDNF

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

Moy et al. [37] mice, C58/J, Grin1 /-, no Behavior chamber, open Impaired social Strain genome Repetitive phenotypes can
background 6 eld, marble burying, interactions, repetitive and comparisons for autism be used to distinguish ASD
heterozygous 129S6 and elevated plus-maze, nose stereotypic behaviors, candidate genes located mouse models
C57BL/, and mice, C58/J poke restricted interest and within regions divergent in
inbred strain 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


Shimamoto et al. [80] mice, Fabp3-/-, Fabp5-/- Autism, schizophrenia Behavior chamber, no Disturbances in
and Fabp7 background three-chambered test, social interactions and brain-expressed FABPs
mixed 129/B6 onto B6 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
Sungur et al. [81] mice, Shank1-/- and Autism and schizophrenia Behavior chamber, USV Impaired social no SHANK1 alterations is
Shank1+/, background interactions and reduced involved in acoustic
heterozygous C57BL/6J and USV communication resulting
129SvJae in social communication
decit.
Aller et al. [63] mice, over expressing grik4 Autism, schizophrenia and Open eld, Elevated plus Impaired social Altered information ow The model can be used for
in the forebrain 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
Li et al. [67] mice, (Prex1/) Autism Three chambered test, Impaired social Disturbed P-Rex1 signaling in CA1
background C57BL6 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
Mosienko et al. [59] mice, Tph2-/-, background Autism USV reduced USV Growth retardation Impaired ultrasonic
C57BL/6N communication is likely to
result in a decient
mother-infant interaction
and growth retardation

125
126
Sungur et al. [82] mice: Shank1-/-, Autism and schizophrenia Behavior chamber and USV Impaired social no SHANK1 alternation is
background strains: interactions, reduced USV involved in acoustic
C57BL/6J and 129SvJae. communication, resulting
in social communication
and interaction decits
Sztainberg et al. [28] mice, MECP2 duplication Autism, intellectual Three-chambered test, Impaired social Defects in long-term The neuroanatomy may

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


disability, motor open eld, elevated plus interactions, hypoactivity, hippocampal synaptic remain sufciently intact
dysfunction, anxiety, maze and rotarod high anxiety and abnormal plasticity. Correction of so that correction of the
epilepsy, recurrent motor function MECP2 levels by antisense molecular dysfunction can
respiratory tract infections oligonucleotide largely restore physiology
and early death reverses the behavioral,
molecular and
electrophysiological
decits
Tian et al. [68] mice, 16p11.2 df/+ Autism, language Contextual fear Impaired social Reduced basal protein Cognitive and
heterozygous null at the impairment, intellectual conditioning and inhibitory interactions and impaired synthesis in hippocampal neuropsychiatric
region of chromosome disability, anxiety, avoidance memory slices. The mGluR5 symptoms of 16p11.2
7qF3 that is syntenic to attention decit negative allosteric microdeletion disorder
human chromosome hyperactive disorder and modulator ameliorated the arise from altered synaptic
16p11.28, background epilepsy behavioral abnormalities signaling that can be
C57BL/6N reversed by chronic
treatment with a negative
allosteric modulator of
mGluR5
Whr et al. [83] Mice, parvalbumin /, Autism, schizophrenia and Behavior chamber, T-maze, Impaired social Altered inhibitory and Downregulation of
background C57BL/6J bipolar disorder. Water-maze, radial maze, interactions, repetitive and excitatory synaptic parvalbumin may result in
rotarod, open eld, light stereotypic behaviors, transmission; cortical disturbed synapse
and dark box, O-maze, reduced pain sensitivity hypertrophy and cerebellar structure/function
USV, PPI and hot-plate test and seizures hypoplasia phenotypes

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.
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140 127

most abundant in neurons of the mature nervous system. Knock exon 8A (Ts2-neo) that resulted in lowered expression of the G406R
out of the Mecp2 in male animals leads to a total loss of function L-type channel via transcriptional interference, blunting deleteri-
without the potential confounding effects of variable X chromo- ous effects of the mutation. The mice survived and still exhibited
some inactivation (according to the Lyon hypothesis of random impaired social interaction and vocalization.
chromosome X inactivation). In females, however, the animals
have a short life span. The female Mecp2 +/ model evaluated by 2.0.5. PhelanMcDermid syndrome, SHANK3 (Table 1)
Samaco et al. [25] showed autistic traits that varied according to the
background strains, suggesting that the more physiologic heterozy- Deletion of the human SHANK3 gene near the terminus of chro-
gote strain can be used for future studies. Mecp2 is important for mosome 22q is associated with PhelanMcDermid syndrome and
normal GABAergic neuronal function as shown among mice lack- autism. Attempts to delete SHANK3 in mice by targeting regions
ing Mecp2 from -amino-butyric-acid-(GABA)-ergic neurons that of the gene failed, since none succeeded in deleting all isoforms
showed reduced levels of glutamic acid decarboxylase (GAD65/67) due to the presence of multiple promoters within the gene. Speed
mRNA and decreased miniature inhibitory post-synaptic currents et al. [33] used a frameshift mutation by inserting a single gua-
(mIPSC) in the cerebral cortex [26]. Phenotypic reversal by acti- nine nucleotide into exon 21 (Shank3G) that caused a premature
vation of Mecp2 expression was rectied by delayed restoration STOP codon and loss of major higher molecular weight Shank3
of that gene, implying that correction of the molecular dysfunc- isoforms at the synapse. Giza et al. [34] showed that deletion of
tion underlying the disorder can restore physiology, most probably the Mapk8ip2 gene (also termed IB2 or JIP2) situated 70 kbp from
since Rett syndrome is a neurodevelopmental rather than a neu- Shank3 is nearly always co-deleted. They found that mice knock out
rodegenerative disorder [27]. for IB2 had reduced AMPA and enhances NMDA receptor-mediated
MECP2 duplication syndrome is characterized by autism, intel- glutamatergic transmission in the cerebellum, disturbances in the
lectual disability, motor dysfunction, anxiety, epilepsy, recurrent morphology of Purkinje cell dendritic arbors and autistic traits.
respiratory tract infections and early death. Given the absence They hypothesized that IB2 mutation has a role in the Chr22qter-
of neurodegeneration in MECP2 duplication syndrome Sztainberg associated cognitive disorders.
et al. showed that correction of MECP2 levels by either tamoxifen
or human-specic antisense oligonucleotides induced a phenotypic 2.0.6. PTEN mutations (Table 1)
rescue [28].
Phosphatase and tensin homolog on chromosome ten (PTEN) is
2.0.3. Tuberous sclerosis TSC1 or TSC2 (Table 1) a tumor suppressor gene that has been reported in autistic indi-
viduals with macrocephaly, especially the Hippocampus Cowdon
Tuberous sclerosis complex is an autosomal dominant disor- syndrome and the Lhermitte-Duclos disease. Pten or Pten null mice
der caused by mutations in either the TSC1 or TSC2 gene that die during embryogenesis. Conditional loss of Pten can have dif-
is associated with cerebral cortical tubers and may be compli- fering consequences depending on the cell type or its state of
cated by astrocytomas. Besides intellectual disability and often differentiation. Known et al. [35] employed a Neuron-specic eno-
seizures of the type of infantile spasm, there is increased rate of lase (Nse) promoter-driven cre transgenic mouse line, in which cre
ASD. The protein products of TSC1, hamartin and TSC2, tuberin, activity is conned to mature neuronal populations in the cere-
inhibit Ras homologue enriched in the brain (Rheb) that activates bral cortex and hippocampus. This resulted in a mutant mouse that
the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). developed macrocephaly due to cre specic neuronal hypertrophy,
Heterozygous defects in either TSC gene prevents the Rheb inhibi- and showed autistic traits.
tion and allows for excessive mTOR activation, which induce cell
growth and proliferation. Since homozygous mutants do not sur- 2.0.7. Cortical dysplasia focal epilepsy, CNTNAP2 (Table 1)
vive the embryonic period, heterozygote strains were developed,
in which most mouse strains have no cortical tubers, do not suffer A recessive nonsense mutation in the Contactin associated
from seizures but show autistic traits. To produce the brain anoma- protein-like 2 (CNTNAP2) gene was shown to cause a syndromic
lies other animal models were generated by targeting Tsc1 or Tsc2 form of ASD, cortical dysplasia and focal epilepsy syndrome (CDFE).
homozygous deletion to specic cell types as neurons, astrocytes This is a rare disorder resulting in epileptic seizures, language
or radial glial cells [29]. The Tsc2f/; cre mice exhibit progressive regression, intellectual disability, hyperactivity and ASD. Neuronal
Purkinje cell degeneration. Since loss of Purkinje cells was reported migration defects were found in about half of the patients. The CNT-
in patients with ASD, this model was offered for future studies NAP2 variant that increases risk for the language endophenotype in
[30]. The reversal of the autistic traits of the/Tsc2+/ by Rapamycin autism was shown to lead to abnormal functional brain connectiv-
indicate the role of mammalian target of rapamycin signaling in ity in human subjects. Knock out mice for the mutation showed,
decient social behavior in mouse models of tuberous sclerosis beside migration abnormalities, reduced number of interneurons
complex [31]. (Table 1). and abnormal neuronal network activity as well as autistic traits.
The abnormal behavior was reversed with risperidone treatment
2.0.4. Timothy syndrome CACNA1C (Table 1) [36]. The importance of this gene was also found in the inbred strain
C58/J when strain genome comparisons identied autism candi-
Timothy syndrome (TS) is a rare dysmorphic disease associ- date genes, including Cntnap2, located within regions divergent in
ated with autism. It is a single nucleotide mutation in the gene C58/J [37].
encoding the pore-forming subunits of an L-type calcium channel
(CaV1.2). The sporadic glycine-to-arginine mutation is located at 2.0.8. 15q11-13 duplication maternal/paternal (Table 1)
position 406 in exon 8A [Splawskis terminology). Both variants
of Timothy Syndrome (the milder TS1 and the more severe TS2) Mutations in 15q11-13 are associated with either duplication
arise from missense mutations in alternatively spliced exons that or gene deletion. Prader-Willi syndrome results from the loss of
cause the same G406R replacement in the CaV1.2 L-type calcium imprinted genomic material within the paternal 15q11.2-13 locus
channel. Since knock out mice, both homozygote and heterozygote, and deletions, unbalanced translocations, or uniparental maternal
did not survive to weaning, Bader et al. [32] used heterozygous disomy. The loss of maternal genomic material at the 15q11.2-
mice that were allowed to keep an inverted neomycin cassette in 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]. are subdivided into N-methyl-d-aspartate (NMDA), -amino-3-
Region duplications are the most frequent genetic abnormality in hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate
autismand the penetrance rate in individuals with a maternally (KA) receptors [52]. Animal studies support synapse dysfunction
derived duplication is >85% [39]. as a core pathological feature of ASD. Models of dysmorphic syn-
Maternally inherited 15q11-13 duplications and triplications dromes associated with ASD like Fmr1 knockout mice [53] show
are among the most common genomic copy number variants iden- disturbed synaptic plasticity and exhibit impaired mGluR LTD
tied in patients with autism. The dosage of an imprinted gene [22]. Models for syndromic (Phelan-McDermid syndrome) [34]
or genes within the duplicated region underlies the autism risk in and non-syndromic ASD shows abnormalities in the Shank family
these patients. E3 ubiquitin-protein ligase, UBE3A is the only gene of postsynaptic scaffolding proteins [54]. Shank2 knock out mice
within the 15q11-13 duplicated segment consistently expressed were found to have a reduced NMDA/AMPA ratio [55]. The trans-
solely from the maternal allele in mature neurons, and inactivating synaptic cell adhesion complex of pre and postsynaptic neurexins
mutations or deletions of UBE3A cause Angelman syndrome. Smith are encoded by four genes (Nlgn14) and interact intracellularly
et al. [40] showed also in mice that the autistic traits were depend- with Shank protein. Nlgn4 knockout mice exhibit ASD-relevant
ent on the Ube3a gene dosage. Mice with paternal duplication of behavioral abnormalities, including decits in social interaction
the loci showed autistic traits in association with synaptic plastic- [56,57].
ity decits in the developing and adult cerebellum and long-term
depression (LTD) at parallel bre-Purkinje cell synapses [41]. 2.1.3. mTOR pathway

Mechanistic target of rapamycin (mTOR) is a component of pro-


2.1. Genes associated with ASD in mice that do not cause
tein complexes; mTORC1 lies downstream from proteins encoded
recognized human diseases (Tables 2 and 3)
by genes associated with ASD: TSC1 and or TSC2 in Tuberous
Besides the well-recognized human syndromes derived from sclerosis and phosphatase and tensin homolog (PTEN) in Lhermitte-
gene mutations, some genetic variations were described in Duclos disease and ASD. TSC1 and TSC2 form a heterodimer that
increased susceptibility among patients with ASD. Genetic ani- negatively regulates mTOR signaling and PTEN is an upstream reg-
mal models include natural models, induced models (Table 2) and ulator of TSC1/TSC2-mTOR. Deletion of PTEN conned to discrete
inbred strains that naturally display ASD-related behaviors, such mature neuronal populations in the cerebral cortex and hippocam-
as the inbred strains BTBR T+ tf/J, BALB/cByJ, A/J, and 129S1/SvImJ pus mature neurons results in dendritic and axonal overgrowth,
[5]. (Table 3). The list of the available mouse models related to increased synapse number, neuronal hypertrophy, and macro-
ASD can be found in the SFARI gene database (https://gene.sfari. cephaly [35]. Mouse models decient in Tsc1 or Tsc2, which lie
org). The information in the site includes the nature of the tar- downstream of Pten in regulating mTORC1 activity, show autistic
geting construct, the background strain and a thorough summary traits. Rapamycin reversed disturbed social behavior in Knock out
of the phenotypic features of the mice that are most relevant to mice of either Tsc1 or Tsc2 [31] and prevented the pathological and
autism. ASD candidate genes are divided to those for which a rig- behavioral decits among mice with selective cerebellar Purkinje
orous statistical comparison between cases and controls has been cells Tsc2 knock out [30].
performed in a series of independent studies, yielding genome-
2.1.4. Serotonin
wide statistical signicance of ASD association (categories 1 and
2) and Genes with suggestive or minimal evidence of ASD associ-
Serotonin (5-hydroxytryptamine) signaling mediates neuro-
ation for which only small/unreplicated genetic studies have been
genesis, cell migration and survival, synaptogenesis, and synaptic
performed (categories 3 and 4).
plasticity. Elevated levels of serotonin in blood have been reported
for up to 45% of ASD subjects [5,58]. Serotonin knock out mouse
2.1.1. Neural pathways evaluated by animal models (Tables 13) pups displayed ultrasonic communication impairment during early
development that may contribute to decient mother-infant inter-
The different genetic animal models often demonstrate abnor- action, presumably contributing to growth retardation phenotype
mal neural and synaptic pathways which are difcult to study in [59]. Serotonin is accumulated in blood almost exclusively in
human ASD. Hence, in that respect these models reveal valuable platelets, via the serotonin transporter. Linkage studies impli-
information that might have direct reection on the pathogenesis cated the 17q11.2 region containing the serotonin transporter gene
of human ASD. The following are the more common pathways that SLC6A4 to be associated with ASD. Conversion of a single amino acid,
were studied: Gly56 to Ala56 in the transport gene resulted in autistic pheno-
type in conjunction with increased p38- Mitogen-activated protein
2.1.2. The synapse: excitatory/inhibitory neurotransmission kinases (MAPK)sensitive basal phosphorylation. It also resulted
in enhanced hippocampal serotonin clearance rates and hyper-
Studies from Animal models show that the primary decit serotonemia. Additionally, a decrease in the ring rate under basal
occurs early in neural development and in the development of conditions was followed by serotonin1A and serotonin2A receptor
synaptic function resulting in abnormal development in the super- hypersensitivity [60].
cial cortical layers and in the cerebellum. Continuous synapse
formation occurs throughout development, with synaptogenesis 2.2. The future: treatment modalities (Tables 13)
dominating early in development, followed by synapse pruning
which lasts through adolescence. Intact glutamate signaling is crit- The development and evaluation of treatments that are effec-
ical for sensory processing and cognitive function. Calcium inux tive in human diseases is one of the main goals of animal models.
results in release of glutamate and its concentration is regulated The lack of objective clinical endpoints or biomarkers to appraise
in the synaptic cleft by the neurons and neighboring astro- treatment modalities complicates the development and evaluation
cytes. The spines, the postsynaptic dendritic protrusions contain of medications. Since ASD is a developmental disorder, diagnosis
scaffolding proteins, receptors, and signaling molecules. Gluta- at earlier ages will allow treatment during development while the
mate works through ionotropic glutamate receptors (iGluRs), and neurons in the brain are still organizing, possibly before irreversible
glutamate-activated G proteincoupled receptors (mGluRs). iGluRs 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 Minocycline, a microglial modulator was used in a model of pater-
behavior and perseveration in a Cntnap2/ cortical dysplasia focal nal duplication (patDp/+) corresponding to human chromosome
epilepsy syndrome [46]. 15q11-q13. Perinatal treatment with minocycline restored the Iba1
Tianeptine, an antidepressant structurally similar to tricyclic expression and reduced the anxiety related behavior of patDp+ mice
antidepressants, but not Fluoxetine that increases synaptic sero- [49].
tonin, abolished signs of anhedonia and depression in mice
overexpressing Grik4, a gene coding for a glutamate receptor sub- 2.2.5. Antisense oligonucleotides
unit of the kainate type in the forebrain [63]. Clonazepine, an
anti-psychotic drug that targets serotonin and dopamine recep- Antisense oligonucleotides (ASO) are small, modied nucleic
tors recovered the defects in sensorimotor gating function, but acids that can selectively hybridize with messenger RNA tran-
not in fear-conditioned memory among Pax6 heterozygous mutant scribed from a target gene and silence it. ASOs designed to bind
rats that had decreased brain and serum serotonin levels. Muta- several regions of the human MECP2 precursor mRNA (pre-mRNA)
tions in the Pax6 gene were described in both syndromic and so as to reduce the levels of both alternatively spliced MECP2
non-syndromic ASD patients [64]. isoforms, MECP2-e1 and MECP2-e2, were successfully used in
Mecp2-overexpressing mouse model and corrected MECP2 levels
2.2.2. Behavioral therapy in lymphoblastoid cells, abolished abnormal EEG discharges and
eliminated behavioral phenotype and electrographic seizure spikes
environmental stimulation by housing pups from birth until [28].
weaning with the mother and an additional non-lactating female
rescued the behavioral decits displayed in adulthood by Fmr1- 2.2.6. Summary
knock out mice including hyperactivity, reduced social interactions,
and cognitive decits. Abnormalities in the morphology of hip- Genetic animal models are helpful to elucidate mechanisms
pocampal and amygdala dendritic spines, improved as well. related to the genetic etiology and the pathogenesis of ASD. In
The manipulation affected motherinfant interaction, resulting in recent years these models are also used to evaluate the possible
enhanced physical contact with the pups, by the nursing and success of different treatment modalities; this is of special impor-
non-nursing postures [20]. Enriched environment cages improved tance as it may close a gap in our understanding of the effects of
behavioral and neuronal abnormalities in adult Fmr1 knock out therapy and help in the search for new and effective ways for the
mice as well [61] supporting the paradigm of environmental stim- treatment of ASD. The fact that many different genetic modica-
ulation. tions result in a similar phenotype, point to the complexity of the
gene environment interaction in the etiology of ASD.
2.2.3. Synaptic modulation
3. Animal models for ASD induced by chemicals
GABAergic transmission mostly in the amygdala, striatum or
cerebral cortex is central to fragile X behavioral abnormalities. 3.1. Exposure to VPA (Tables 46)
Enhancing GABAergic transmission by the GABA A receptor agonist
THIP (gaboxadol) corrected attenuated hyperactivity and reduced VPA is a frequently used antiepileptic drug, also being used for
prepulse inhibition (evaluated by the Auditory Startle Testing) treatment of bipolar disorders, migrain. headaches and neuropathic
of the Fmr1 knock out mouse [23]. The metabotropic gluta- pain. VPA is well known for its teratogenic effects, including neural
mate receptor subtype 5 (mGluR5), which modulates excitatory tube defects, cardiovascular anomalies, limb anomalies, cranio-
neurotransmission and mediates signaling and protein synthesis facial abnormalities and neurodevelopmental delay [91,92]. Its
is increased in fragile X knockout mice. The selective negative main mechanisms of action are the inhibition of histone deacety-
allosteric modulator of the mGluR5 receptor GRN-529 reduced lase, being an epigenetic modulator and the ability to increase
repetitive behaviors and spontaneous stereotyped jumping and oxidative stress. Prospective and retrospective human studies have
improved sociability [65]. D-cycloserine, a partial agonist at the demonstrated that prenatal exposure to VPA, especially during the
glycine-binding site of NMDARs, recovered the NMDA/AMPA ratio rst trimester of pregnancy, is also associated with reduced cogni-
and improved social interaction in a Shank2/ knock out mouse tive function and high risk for ASD among the offspring [6,7,9396].
model [66]. D-serine, a co-agonist of the glycine modulatory Supporting data to the human studies have been seen in VPA rodent
sites on the NMDAR fully rescued the NMDAR-dependent LTD models. Exposure of mice and rats to VPA can lead to behav-
impairment and signicantly improved social recognition at P- ioral abnormalities that are similar to those observed in autistic
Rex1 knock out mice in the CA1 region of the hippocampus patients, including decreased social interactions, increased repet-
[67]. Based on VU0155041, a positive mGluR4 Allosteric Modula- itive or stereotypic behaviors, lower sensitivity to pain, impaired
tor improved autistic-like symptoms and normalized immediate sensorimotor gating or eye blink conditioning, increased anxiety,
early gene expression induced by social exposure in the brain of reduced exploratory behavior and abnormally high and longer
Mu Opioid Receptor Null Mice [62]. Treatment with a negative lasting fear memories [9799]. Moreover, Along with the behav-
allosteric modulator of mGluR5 2-chloro-4-((2,5-dimethyl-1-(4- ioral phenotype, VPA-exposed rodents present neuroanatomical
(triuoromethoxy)phenyl)-1H-imidazol-4-yl) ethynyl) pyridine and cellular changes similar to human ASD including reduced num-
(CTEP) (NAM), reversed the cognitive decit in a mouse model for ber of motor cells in cranial nerve motor nuclei in the brain stem,
human chr16p11.2 microdeletion [68]. decreased cerebellar volume and reduced number of Purkinje cells
Rapamycin reversed impaired social behavior [30] and was asso- in cerebellar vermal lobules, as well as enhanced synaptic plastic-
ciated with inhibition of mTOR signaling in a mouse model of ity of the prefrontal cortex. Signicant changes in gene expression
Tuberous sclerosis [31]. following prenatal VPA exposure have also been reported [100].
Rodier et al. [101] were the rst to develop an animal model of
2.2.4. Microglial modulator 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
Since several studies have demonstrated microglial distur- stage of neural tube closure and brain stem nuclei formation, pro-
bances in different brain regions of individuals with ASD, duced damage to the motor cranial nerve nuclei and reduced the
Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140 131

number of their neurons. Since then, and especially within the last and decreased number of social behaviors, in addition to delayed
decade, the number of studies using the prenatal or early postna- maturation and motor development. Following these ndings,
tal VPA exposure animal model is increasing, demonstrating many many other researchers found this model to be reliable and demon-
of the structural and behavioral features that can be observed in strated autistic like behaviors in VPA offspring using different
ASD patients [8,92,97,98,102,103]. Prenatal administration mimics behavioral tools. Decrease in social interaction and social prefer-
VPA exposure during different stages of human pregnancy, while ence was demonstrated in prenatally VPA-treated offspring using
postnatal models investigate the effect of VPA exposure during the different tools including the 3 chamber test [108,124], open eld
third trimester of pregnancy and after, in the rst several months test [99,113,117] and play behavior measurements [125]. Decits
after birth. in social communication was demonstrated by ultrasonic vocaliza-
tions [117], nest seeking response [124] or olfactory habituation.
3.2. Prenatal rodent models of VPAinduced ASD (Tables 45) Increased repetitive, stereotyped behaviors were demonstrated by
open eld [99], self-grooming, marble burying [118] or Y maze tests
3.2.1. Exposure time points [109]. Additional behavioral impairments as found in subsets of
To mimic human intra uterine exposure to VPA, several time ASD children were also detected in rodents, including enhanced
points for VPA exposure in rodents have been suggested. In most anxiety by elevated plus maze, lower sensitivity to pain and learn-
studies animals were exposed during organogenesis, a develop- ing and memory disabilities by radial maze and open eld test. VPA
mental period during which exposure to teratogens may cause offspring demonstrated faster eye-blink conditioning, consistent
specic abnormalities according to the embryonic developmental with studies in autistic children [107]. Taken together, these stud-
stage at time of exposure, occurring in mice and rats during embry- ies demonstrate a relatively good similarity between the behavioral
onic days 815. Therefore, the most common time points for VPA phenotype observed in ASD individuals and in prenatally VPA
exposure are days 9 [104106], 12 [107,108] and 12.5 [99,109112] exposed rodents strengthening the validity of the different studies
in rats, and 12.5 [113116] and 13 [102,117119] in mice, around on VPAinduced ASD. However, we should remember that some
the time of neural tube formation that complete at approximately of the tests measure behaviors that are not necessarily typical to
gestational days 10.5-11 in these animals. the behavior of children with ASD, and they therefore should be
The most sensitive time for ASD induction by VPA was assessed considered questionable. Moreover, since there are obvious varia-
by Kataoka et al. [113] and Kim et al. [108] who compared behav- tions in the behavior of different offspring in the same litter, these
ioral phenotypes to different time points of prenatal exposure. variations were not considered in most studies .
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 3.2.4. Anatomical and cellular changes in the brain
and 15 in rats. E day 12.5 for mice and 12 for rats were found to be Prenatal exposure to VPA was associated with anatomical and
the most vulnerable to VPA exposure. morphological alterations similar to those observed in the brains
of ASD patients, such as reduced size of cerebellar hemispheres,
3.2.2. Doses diminished number of cerebellar Purkinje cells and cranial neu-
The VPA doses ranged between 300 and 800 mg/kg, while most rons [103], enhanced synaptic plasticity of the prefrontal cortex and
of the studies used the amount of 600 mg/kg. In clinical populations amygdala, alterations in monoamines levels and amino acid neu-
the dose of VPA administered is 2003600 mg/day, equivalent to rotransmission [104,106] and lower cortical expression of BDNF
355 mg/kg for an average female that weight 65 kg [111], while [124]. Immunological alterations such as decreased splenocytes
adverse effects were common in association with plasma lev- proliferative in response to concanavalin A have also been reported
els above 100 micrograms/ml [120]. VPA doses administered to in this model [126].
rodents are approximately 1020 times higher than the human
therapeutic dose, and results in a peak level of 900 mg/ml in mater- 3.3. Postnatal rodent models (Table 6)
nal plasma in less than 1 h [121]. Only few studies addressed these
differences and explained them by species differences in the phar- Most studies that used postnatal VPA models, exposed mice
macokinetics of VPA, human higher bioavailability of VPA [111,122] and rats to VPA on postnatal day 14. This time point has been
and rapid elimination of VPA in rodents, with enhanced reduction chosen because it is a sensitive period during which cerebellar
in the average plasma concentrations [123]. Furthermore, most of organization and neuronal proliferation are essentially complete,
the studies used a single time exposure to VPA while in human it but neuronal differentiation, myelination, synaptogenesis and glia
is generally an accumulated effect . genesis are continuing in the cerebellum, striatum and hippocam-
Lucchina et al. [114] compared between prenatal exposure to pus. Wagner et al. [102] examined male and female mice exposed
400 or 600 mg/kg VPA. They found that mice prenatally exposed to prenatally to VPA or males exposed on postnatal day 14 (P14).
maternal doses of 600 mg/kg VPA had signicantly impaired social They found that in utero exposure to VPA resulted in develop-
interactions and increased anxiety behavior while mice prenatally mental retardation, with delayed appearance in the maturation
exposed to 400 mg/kg had much lower behavioral effect. Most of of surface and mid-air righting as well as negative geotaxis, grip
the investigators injected a single dose of VPA dissolved in nor- strength, motor activity and water maze performances. Postnatal
mal saline; several studies administered the VPA orally by using an VPA administration caused similar retardation in the maturation
infant feeding tube [104,105] or mix of peanut butter [124]. of negative geotaxis and water maze performance. Regression of
acquired skills (mid-air righting) was also demonstrated following
3.2.3. Behavioral phenotype postnatal VPA exposure. They concluded that a single postna-
Studies in rodents demonstrated behaviors analogous to those tal exposure to VPA also has long-lasting effects mimicking ASD.
observed clinically, including the core symptoms of human ASD: Moreover, this study has shown that critical cerebellar-mediated
decits in social interaction and communication and presence of behaviors of mid-air righting and negative geotaxis mature rst
restricted, repetitive behavior. Schneider and Przewlocki [69] eval- appear on P14 in mice. Following these ndings, Yochum et al. [127]
uated the behavioral aberrations of prenatally VPA exposed rats examined BALB/c mice (male and female) exposed to single VPA
using a large battery of tests in order to validate the VPA animal injection on post-natal day 14. They found decreased ano-genital
model of autism. They found lower sensitivity to pain, locomo- snifng, crawl-under/over behavior and allogrooming in pairs of
tor and repetitive/stereotypic-like hyperactivity, increased anxiety, 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- manifestation between males and females, as observed in human
lum and hippocampus. Earlier postnatal VPA exposure described by studies [138]. The lack of gender differences in the VPA induced
Wang et al. [128] demonstrated anxious, depressive, and impaired ASD can be explained by the fact that the specic effects of VPA on
social behaviors in addition to changes in neural cell proliferation the brain during different stages of brain development are appar-
and differentiation in male rats exposed to VPA on post-natal day ently not related to the genetic differences between genders and in
7, that is equivalent to a fetus at 3640 weeks gestation. that case gender differences play only a minimal role.
Taken together, these studies demonstrate that postnatal VPA
exposure results in anxiety-related and depressive behaviors and 3.5. Summary
fewer social interactions along with diminished number of Purkinje
cells and enhanced cell death in the cerebellum and hippocampus We should remember that rodents are not man, and the
[102,127,129]. It should be emphasized, however, that many of the observed neurobehavioral decits, although mimicking human
behavioral changes described in these studies are not necessarily ASD, may not be exactly the same. Moreover, these studies also
typical of ASD like behaviors . 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
3.4. Gender-specic differences
on early postnatal VPAinduced ASD like symptoms in ICR albino
mice (Ornoy et al., personal communication), we know that there
ASD is 45 times more common in male children than in females.
are signicant variations in the behavioral abnormalities among the
However, in children born to women treated during pregnancy
animals of the same litter and between males and females. Hence,
with VPA there seems to be no gender difference in the rate of
in spite of the difculties, it is important to try and assess all bio-
ASD [96]. Most animal studies, which used prenatal VPA exposure,
chemical, morphological and molecular changes in relation to the
did not report any differences between genders in ASD behavioral
type and extent of the individual neurobehavioral decits. The fact
phenotype [104,106,107,109,117,118,124,125,130133]. Several
that prenatal exposure to VPA at different phases of brain develop-
investigators used only males to eliminate the possible sex differ-
ment, lead to a similar phenotype of ASD like behavior, implies that
ence [99,105,110,112,114,115,119]. Wagner et al. [102] compared
there might be a general mechanism of action of VPA not related
between males and females and found no difference in the behav-
to the developmental stages of the brain. Indeed, many of the envi-
ioral assessment. However, Kataoka et al. [113] and de Theije
ronmental factors associated with ASD in pregnant women would
et al. [134] reported a decrease in social interaction in male
increase the rate of ASD with no correlation to the specic time
VPAexposed offspring compared to females. In contrast, Farve
of exposure in pregnancy [4]. Hence, these agents might produce
et al. [111] demonstrated more signicant olfactory decit in
their damage through a different mechanism, perhaps by epige-
female offspring of VPA rats compared to males.
netic changes.
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- 3.6. Exposure to thalidomide
ences in the behavioral tests [135,136]. Two studies have shown
ndings that are consistent with the male preponderance of ASD; Thalidomide caused multiple birth defects such as limb
Oguchi-Katayama et al. [129] reported enhanced anxiety in post- reduction defects, ocular and cardiovascular anomalies. Today,
natal exposed males but not in females. Reynolds et al. [137] thalidomide is still used in the treatment of leprosy and mul-
found Gender-specic differences in sensory processing. While tiple myeloma [139]. Strmland et al. [140] have reported an
VPA exposed males had signicantly diminished auditory startle increased number of children with ASD following prenatal expo-
responses and altered sensory motor gating during both the juve- sure to thalidomide. Very few Animal studies have examined the
nile (P day 23) and the adolescence (P day 45) stages, female rats use of thalidomide exposure as a model of autism. Narita et al. [141]
have shown similar results only during adolescence. They sug- exposed rats to 500 mg/kg of thalidomide on embryonic day E2, E4,
gested that this may indicate differences in the timing of symptom E7, E9, and E11. They also exposed rats to 500 mg/kg of VPA on E day
134 Z. Ergaz et al. / Reproductive Toxicology 64 (2016) 116140

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 Exposure window and Behavioral tests Results/outcome Other Conclusion


dose tests/markers/treatment

Schneider et al. [99] 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.
Stanton et al. [107] 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
Tsujino et al. [104] 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
Markam et al. [109] 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
Dufour-Rainfray et al. [106] 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
Narita et al. [105] 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
Kim et al. [108] 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
Bringas et al. [110] 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 Shultz et al. [148] reported that exposure to PPA consistently
hippocampus, increase dopamine in the frontal cortex, and cause impaired social behavior measured as distance apart, proximity,
hyperserotonemia when given on E9. In a later study by Narita et al. and play behavior in rats.
[105] they examined the behavioral phenotype of rats exposed to MacFabe et al. [147] demonstrated that rats treated with PPA
VPA or thalidomide on E day 9. They reported impaired achieve- displayed restricted behavioral interest to a specic object among
ment of learning in both VPA and thalidomide exposed pups. They a group of objects, impaired social behavior, and impaired rever-
did not nd other behavioral effects typical of ASD. Hence, it seems sal in a T-maze task compared to controls, in addition to reactive
that thalidomide is not an appropriate model for ASD in rats that astrogliosis and activated microglia in the brain.
are generally insensitive to the teratogenic effects of that teratogen. 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.
3.7. Exposure to misoprostol

Misoprostol is a prostaglandin analog drug commonly pre-


4. Animal models of infection and inammation in relation
scribed for the prevention and treatment of gastric ulcers [142],
to ASD
There is evidence that prenatal exposure to high doses of miso-
prostol during the rst or second trimesters of pregnancy can lead
Large population studies found increased rate of ASD in the
to the occurrence of Mbius Syndrome, a disorder characterized
offspring of women with fever and infection in pregnancy, espe-
by uni- or bilateral eye-face palsy due to damage to cranial nerve
cially when maternal infection was more severe and necessitated
nuclei, associated with muscle or skeletal malformations and a
hospitalization. This association is apparently related to maternal
high prevalence of autistic- like behavioral symptoms [4,143,144].
inammatory process. Maternal immune activation may play a role
Koenig et al. [145] were the only ones who examined the effect
in neuro-developmental perturbation [4].
of Misoprostol on autistic behavioral phenotype in mice using a
Animal models in which early viral infection results in behav-
battery of behavioral tests. They found no signicant neurodevel-
ioral changes later in life include the inuenza virus model in
opmental or behavioral effects of injection with 0.4, 4 or 40 g/kg
pregnant mice and the Borna disease virus model in newborn Lewis
misoprostol on postnatal day 7 in mice.
rats. Respiratory infection with human inuenza virus in pregnant
mice at mid-gestation resulted in behavioral abnormalities in some
3.8. Exposure to PPA of the offspring, mostly decits in social interaction and prepulse
inhibition (PPI) and in histological abnormalities in the hippocam-
Propionic acid (PPA) is a short chain fatty acid, a metabolic pus and cortex of the neonatal offspring [150]. Neonatal Borna
end-product of enteric bacteria in the gut, and a common food virus infection resulted in decreased play activity among pups and
preservative. A number of studies indicated that PPA can cause a stereotypic behavior with decrease in the exploratory activity
autistic like behaviors and a neuro-inammatory response in rats at adulthood. Immunohistochemistry was positive for the pres-
[146149]. ence of Borna virus throughout the brain. Pathological ndings
136
Table 6
Studies on postnatally VPA treated mice and rats: time of exposure and dose, tests used, outcome and conclusions from each study.

Reference Type and gender Exposure window and Behavioral tests Results/outcome Other Conclusion
dose tests/markers/treatment

Wagner et al. [102] BALB/c male mice P day 14, 200 or Mid-air Righting, Retardation in the no Postnatal VPA exposure
400 mg/kg hanging wire, negative maturation of negative cause developmental
(Subcutaneous geotaxis, balance geotaxis and water maze decits in an ontogenic
injection) beam, water maze, performance. Regression of fashion that parallels the
passive avoidance, acquired skills (mid-air clinical signs of autism
motor activity and righting)
self-injury behavior
Yochum et al. [127] BALB/c male mice P day 14, 400 mg/kg Social interactions and Decreased ano-genital Histopathological analysis, VPA- enhanced cell death
(Subcutaneous play behavior (open snifng, crawl-under/over TUNEL assay for apoptosis in the cerebellum and
injection) eld) behavior and allogrooming, hippocampus may be
no signicant difference for responsible for ASD like
nonsocial behavior such as behavior
self-grooming, decrease in
motor activity; enhanced
cell death in the

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


cerebellum and
hippocampus
Pragnya et al. [135] BALB/c mice (males P day 14, 400 mg/kg Negative geotaxis, Impaired performance on Malondialdehyde Postnatal VPA exposure
and females) (Subcutaneous nociception, locomotor negative geotaxis, elevated concentration, glutathione causes behavioral decit,
injection) activity, Rota rod test, plus maze and Morris and total nitrite levels, and increased oxidative stress,
Moris water maze, water maze tests. serotonin determination; increased serotonin levels
elevated plus maze Decreased motor pepirine treatment in the hippocampus, and
co-ordination, thermal diminished number of
nociception and social Purkinje cells in the
behavior increased cerebellum. Treatment
locomotor activity, with pepirine can
increase oxidative stress signicantly improve these
outcomes.
Oguchi- Katayama et al. [129] Wistar Hannover/Rcc P day 14, Elevated plus maze Impaired performance on microarray assay analysis The gene expression
rats (males and 400 mg/kg(Subcutaneous elevated plus maze in of changes in gene changes in the amygdala
females) injection) males expression in prole in the might be an important
amygdala cause for impaired social
behavior and enhanced
anxiety rather than
expression changes of
particular genes
Wang et al. [128] Sprague-Dawley male P day 7200 mg/kg Elevated plus maze, Depressive and Studies of differential VPA disrupts neuronal
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social interaction test impaired social interaction differentiation mainly in depressive, and impaired
the hippocampus social behavior and
involved in anxiety
disorder typical to autism
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testing including impairments in motor seen in individuals with
sunower seed eating speed and performance ASD.
task and a vermicelli
handling task
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