The teratology of autism
Tara L. Arndt, Christopher J. Stodgell, Patricia M. Rodier*
Department of Obstetrics and Gynecology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
Received 9 September 2004; received in revised form 22 November 2004; accepted 22 November 2004
Autism spectrum disorders affect behaviors that emerge at ages when typically developing children become increasingly social and
communicative, but many lines of evidence suggest that the underlying alterations in the brain occur long before the period when symptoms
become obvious. Studies of the behavior of children in the first year of life demonstrate that symptoms are often detectable in the first 6
months. The environmental factors known to increase the risk of autism have critical periods of action during embryogenesis. Minor
malformations that occur frequently in people with autism are known to arise in the same stages of development. Anomalies reported from
somatic that originate early in the first trimester. In addition, it is possible to duplicate a number of anatomic and behavioral features
characteristic of human cases by exposing rat embryos to a teratogenic dose of valproic acid at the time of neural tube closure.
# 2004 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Autism; Autism spectrum disorders (ASDs); Valproic acid; Eyeblink conditioning; Congenital malformations
Teratology is the study of congenital anomalies and their
causes, whether they are genetic or environmental in origin.
Over most of the period since autism was first described
there has been debate over whether the disorder was
acquired in utero or whether it was acquired closer to the
time when symptoms become obvious, typically around age
two. Hintsthat a change in the course of development occurs
long before the diagnostic symptoms appear have been
available for many years, but it is only in the last decade that
many lines of evidence have come together to indicate that
autism spectrum disorders (ASDs) have their origins in early
prenatal life. This paper summarizes several different kinds
of evidences that address the time when development is
altered and focuses on the anatomical and behavioral
parallelism between human cases and animals exposed in
utero to valproic acid (VPA).
1. How early can symptoms be observed?
It is natural to suspect recent events when symptoms of
any disorder appear, but there are many examples of
neurological disorders in which symptoms begin long after
the precipitating event. It is easy to understand why this
would be true in disorders caused by a gradual loss of
neurons (e.g., Parkinsonism and amyotrophic lateral
sclerosis) or the buildup of some injurious product (e.g.,
phenylketourea). However, even with a single discreet
injury, symptoms may appear long after the fact. A classic
example is the role of dorsolateral prefrontal cortex in
delayed response tasks in primates. Using cryogenic
(1977) demonstrated that the reversible lesion had no effect
on performance when animals were 9 to 16-month-old. In
contrast, a significant impairment of performance was
subjected to cooling between 34 and 36 months of age the
impairment was much greater. Presumably, the late-
developing dorsolateral prefrontal cortex plays little or no
role in the performance of the task until some time in the
second year of life, and its full contribution is not
Int. J. Devl Neuroscience 23 (2005) 189–199
Abbreviations: VPA, valproic acid; ASD, autism spectrum disorder;
SLOS, Smith–Lemli–Opitz syndrome; RARE, retinoic acid responsive
element; RA, retinoic acid; CS, conditioned stimulus; US, unconditioned
stimulus; CR, conditioned response; UR, unconditioned response; PKCg,
protein kinase C–gamma isoform knockout
* Corresponding author. Tel.: +1 585 275 4789; fax: +1 58 5244 2209.
E-mail address: Patricia_Rodier@urmc.rochester.edu (P.M. Rodier).
0736-5748/$30.00 # 2004 ISDN. Published by Elsevier Ltd. All rights reserved.
demonstrable until shortly before puberty. Thus, the onset of
symptoms is a far from perfect guide to when a disorder
began. However, the onset of symptoms cannot precede the
initial injury, so the time when symptoms of autism can be
recognized is an important issue.
An early study to determine when the first symptoms of
autism are expressed used responses of parents to a
questionnaire. Ornitz et al. (1977) compared the responses
of parents of 74 children with autism to the responses of
parents of 38 age-matched typically-developing children.
The questionnaire queried many aspects of early develop-
ment, including motor function, speech, language, and
perception. The results indicated that children who were
later diagnosed with autism exhibited developmental delays
in every area of function, with some landmarks delayed as
early as the second or third month of life. About half of
families were concerned that something was wrong by the
time their child was 14-month-old. Obviously, having
recently received their child’s diagnosis could have
influenced parents as they tried to remember their child’s
early development, but soon investigators came up with a
way around the problems of retrospective recall. Rosenthal
et al. (1980) reported a study of home movies from families
with children later diagnosed with psychoses and movies of
children with typical development. They used a scale of
sensorimotor stages described by Piaget to rate the age
appropriateness of behavioral development in tapes from the
first two years of life. Children in the group with psychoses
differed significantly from controls in showing fewer age-
Better-controlled studies have examined movies of
specific events, such as first birthday parties, and shown
that most children later diagnosed with autism can be
distinguished from controls at one year of age by such
anomalies asfailuretopointtoobjects andfailure torespond
to their name (Osterling and Dawson, 1994). In a more
recent study, investigators blind to diagnosis evaluated
movies from the first six months of life (Maestro et al.,
2002). Even at this age, children who would later be
diagnosed with autism differed significantly from controls
on eight items related to social attention (e.g., looking at
people) and social behavior (e.g., smiling at people and
vocalizing to people), while they did not differ from controls
on most measures related to objects (e.g., looking at objects
and smiling at objects). Several groups are now studying
infant siblings of children with autism, and their preliminary
reports suggest that many symptoms are present at 12
months and a few as early as six months in siblings who later
receive a diagnosis (e.g., Bryson et al., 2004; Zwaigenbaum
et al., 2004). Taken together, studies of behavior prior to
diagnosis suggest that most, if not all, children who will be
diagnosed with autism show symptoms long before the age
when symptoms become obvious.
In a study of heel-stick blood collected from newborns,
would later be diagnosed with either autism or mental
retardation were distinguished by anomalous concentrations
of neuron-related products in blood. Recycling immunoaf-
finity chromatography was used to measure the neuropep-
tides substance P (SP), vasoactive intestinal peptide (VIP),
calcitonin gene-related peptide (CGRP), and the neurotro-
phins, nerve growth factor (NGF), brain-derived neuro-
trophic factor (BDNF), neurotrophin 3 (NT3), and
neurotrophin 4/5 (NT4/5). Of these, VIP, CGRP, BDNF,
and NT4/5 were elevated in neonates who would later be
diagnosed with autism or mental retardation. Each group
differed significantly from a group of typically-developing
children, but the two clinical groups did not differ from each
other. A fourth group, later diagnosed with cerebral palsy,
resembled the controls. It is difficult to interpret why such
differences would be present in blood, where the bulk of the
products measured must have been generated in the enteric
nervous system. Nonetheless, the results suggest that
children with autism are already different from typically-
developing children at the time of birth.
2. What is the exposure period when teratogens that
increase the risk of autism act?
The critical period for exposure to teratogens shown to
increase the risk of autism is early in the first trimester. Five
teratogens related to autism risk have been identified in
? maternal rubella infection (Chess, 1971);
? ethanol (Nanson, 1992);
? thalidomide (Stro ¨mland et al., 1994);
? valproic acid (Moore et al., 2000);
? misoprostol (Bandim et al., 2003).
Of these, thalidomide and misoprostal have been disc-
ussed extensively in the review by Miller et al. (2005). The
timing of the thalidomide critical period was deduced from
accompanying somatic defects to be days 20–24 postcon-
ception (Stro ¨mland et al., 1994). The timing of the miso-
prostol exposures was determined by questioning mothers
and was in the sixth week postconception (Bandim et al.,
Fortunately, the critical period for the other three
teratogens can be estimated from existing data, as well.
The epidemiological sample used to identify the increased
risk for autism after rubella infection did not include data on
time of onset of the rash that heralds rubella, but the
investigators did note that all the children with an autism
outcome had multiple symptoms of rubella injury (Chess
and Fernandez, 1980). In a study specifically designed to
identify the critical periods for eye defects, deafness, mental
retardation, and heart malformations after rubella exposure,
Ueda et al. (1979) found that cases with multiple symptoms
came mainly from those exposed within the first eight weeks
T.L. Arndt et al./Int. J. Devl Neuroscience 23 (2005) 189–199190
postconception. The same study showed that mothers whose
offspring had severe mental retardation had onset of rash in
the second to fifth week postconception.
The evidence for increased risk with ethanol exposure is
not as strong as that for the other environmental factors on
the list. Indeed, Fombonne (2002) has concluded that it is
insufficient to determine whether or not the risk is actually
elevated. What makes the studies interesting for the topic
under discussion is that the suspected connection is not
between autism and ethanol exposure per se, but between
autism and Fetal Alcohol Syndrome (Nanson, 1992; Harris
et al., 1995; Aronson et al., 1997). Fetal Alcohol Syndrome
is distinguished from Fetal Alcohol Effects by the presence
of minor congenital anomalies such as epicanthal folds,
short palpebral fissures, and a flattened maxillary area, and
by growth retardation, in addition to the behavioral
anomalies associated with both conditions (Jones and
Smith, 1973; IOM, 1996). Studies in animals suggest that
the physical features that define Fetal Alcohol Syndrome are
established in the third to fifth weeks postconception (Sulik
et al., 1986).
The timing for the teratogenic effect of valproic acid that
increases the risk for autism cannot be estimated directly, as
the drug is typically taken throughout the entire pregnancy.
However, the timing of injury to the developing nervous
system can be estimated from accompanying somatic
features in exposed children. Children with prenatal
exposure to VPA exhibit similar patterns of physical
malformations as those exposed to thalidomide in utero,
but with a decreased severity of symptoms. These include
dysmorphic features indicative of injury around the time of
neural tube closure (e.g., neural tube defects, congenital
heartdisease,craniofacial abnormalities, abnormally shaped
or posteriorly rotated ears, genital abnormalities, and limb
defects; Mo and Ladusans, 1999; Kozma, 2001). Based on
the pattern of abnormalities we can estimate that VPA and
thalidomide injure the developing embryo at similar times
The fact that each of these teratogens appears to act
during the embryonic period (the first eight weeks of life)
at other stages of development, or that later influences could
add to the effects of an early injury. However, the
coincidence of critical periods for the first five environ-
mental risk factors identified is strong evidence that autism
arises very early in development.
3. Dysmorphic features suggest early injury in
Dysmorphic facial features have been reported in
populations of children with idiopathic autism (e.g., Rodier
et al., 1997; Miles and Hillman, 2000). The dysmorphic
features reported in idiopathic autism resemble those in
cases of autism following exposure to thalidomide and VPA
in utero. These types of dysmorphology cannot be induced
postnatally, and most minor anomalies arise in the first eight
weeks postconception. While dysmorphic features are not a
part of the diagnosis of autism, they are observed in
idiopathic and teratogen-exposed populations at a very high
rate. Rodier et al. (1997) described minor physical
malformations linked to autism in a population in Nova
Scotia, Canada. Posterior rotation of the external ears was
the feature most characteristic of children with autism
compared to their unaffected siblings and non-autistic
children with developmental delay. These types of ear
abnormalities have also been found in children with autism
following exposure to thalidomide or VPA (Miller and
Stro ¨mland, 1993; Stro ¨mland et al., 1994; Moore et al.,
2000). Examined together, these studies provide two
extremely important pieces of information—dysmorphic
features in autism occur across geographical and ethnic
boundaries, and these features occur in idiopathic cases as
well as those with exposure to known teratogens.
Miles and Hillman (2000) also reported an increased rate
of physical malformations in children with autism. To be
identified as ‘‘abnormal’’, children had to possess at least six
abnormalities in the following categories: (1) Minor
(2) Measurement abnormalities (beyond two standard
deviations from the mean, such as macrocephaly and
hypertelorism). (3) Descriptive traits (abnormalities that
occur in >4% of the population and are seen in families, but
are difficult to measure, such as deep set eyes or high
forehead). (4) Malformations, such as cleft lip, renal or
cardiac defects. Among 94 children with a confirmed
diagnosis of an ASD, 88 had ‘‘idiopathic autism’’ (they did
not have a known genetic disorder such as Fragile X
Syndrome). Of these children, only 56% were classified as
phenotypically normal. Of the remaining children, 22% had
clear abnormalities and 20% had equivocal results. These
findings indicate that some abnormal physical features are
common in ASDs.
This is not to say that all children with ASDs have
physical anomalies. They do not. Further, none of the
physical anomalies reported in autism are seen exclusively
in that disorder. Rather, all are seen in children with other
developmental disabilities, and in the population of
typically-developing children, as well. The importance of
these features is simply that they speak to the issue of the
stage of development when ASDs are initiated.
4. Neuroanatomical evidence for early injury in
Studies of neuroanatomy at the histological level in the
brains of people with autism provide a number of arguments
in favor of a very early alteration of development as part of
the etiology of ASDs. For example, the brains studied by
Bailey et al. (1998) included one with extra tracts running
T.L. Arndt et al./Int. J. Devl Neuroscience 23 (2005) 189–199191
through the pontine tegmentum and two with oddities of the
pyramidal tracts. In one case, the pyramidal tracts appeared
small and in the other, they did not exhibit the sharp
separation from each other that is typical. Because the basic
tracts running up and down the neuroaxis are well-
established before parturition, it is impossible to reconcile
such anomalies with a disturbance of postnatal brain
A brain from a patient with autism, studied by Rodier
neurons in the facial nucleus. In the normal brain used for
comparison, this nucleus is outlined by a capsule formed by
passing fibers as they skirt the nucleus. In the brain with few
facial neurons, no capsule was present. This indicates that
the neurons were not in position as passing fibers made their
way by them. If the facial neurons had been lost in late
gestation or postnatal life, the capsule would mark its
boundaries. In this case, the obvious conclusion is that the
nucleus failed to form, or were lost very early, before they
had a chance to influence the passing fibers.
Kemper and Bauman (1993) have pointed out Taka-
shima’s (1982) studies, which demonstrate that reduced
Purkinje cell numbers result in retrograde loss of neurons in
the inferior olive if reduction occurs after the 30th week in
line of neurons, misplaced neurons, etc.) have been reported
in the brains of people with autism by Bauman and Kemper
asonewouldexpectifcellswere degenerating inresponseto
a late loss of Purkinje cells. Further, because the Purkinje
cell body is wrapped by processes of the neighboring basket
cells, the late loss of Purkinje cells is characterized by the
presence of ‘‘empty baskets’’. Bailey et al. (1998) found no
empty baskets in their cases. Each of these findings support
the conclusion that the neuroanatomy of people with autism
is altered prior to birth.
5. What do co-morbid syndromes tell us about the time
when autism begins?
In this volume, Miller et al. (2005) have reviewed several
congenital conditions in which high rates of autism occur.
These are Moebius sequence, the CHARGE association, and
Goldenhaar syndrome. While the causes of most cases are
unknown, features of each disorder indicate that they arise
from disruption of very early development. Here, wewish to
add several more co-morbid syndromes that also provide
information regarding the timing of autism’s origins.
Joubert syndrome is an extremely rare recessively
inherited disorder (Joubert et al., 1969). Most cases are
characterized by breathing difficulties, hypotonia, ataxia,
eyemovement anomalies, and other brain stem symptoms as
well as cognitive limitation. In one sample of 11 cases, four
met the criteria for ASDs and all had some symptoms
(Ozonoff et al., 1999). The brain in Joubert syndrome is
distinguished by failure of development of the cerebellar
vermis and the cranial nerve motor nuclei, and failure of the
superior cerebellar peduncles to cross (Yachnis and Rorke,
1999; Padgett et al., 2002). On MRI, there is apparent failure
ofthe pyramidal tracts tocross, andfunctional MRIsupports
this finding (Parisi et al., 2004). This is especially interesting
in light of one of the cases of autism reported by Baileyet al.
(1998) in whose brain the pyramids seemed to lack
separation. It may be that full or partial failure of the
decussation of the pyramids characterizes some idiopathic
cases of autism. The dysplasias of the brain stem nuclei and
cerebellar vermis in this syndrome suggest that development
has already gone off course in the fourth or fifth week
postconception, when those structures are forming.
causes of the developmental disorder, Cornelia de Lange
syndrome (Kranz et al., 2004; Tonkin et al., 2004). The
human gene is the homolog of the drosophila gene Nipped–
B, which is important in the regulation of the Notch
signaling pathway, which plays a major role in many
developmental events. The phenotype of the disorder
includes growth reduction and cognitive limitation, with
autism and/or self-injurious behavior (e.g., Jackson et al.,
1993). Some of the facial features are unusual, such as
eyebrows that are not separated in the midline and long
eyelashes, butmanyare ones also seen in peoplewith autism
from other genetic or environmental causes. Dysmorphic
features commonly found in de Lange syndrome include
epicanthal folds, ptosis, broad nasal bridge, short nose, long
upper lip, and micrognathia. Anomalies of the limbs, heart,
and gastrointestinal tract are commonly present, as well
(Jackson et al., 1993).
Smith–Limli–Opitz syndrome (SLOS) is caused by
mutations in the gene responsible for the product that
catalyzes the last step in cholesterol synthesis (Tint et al.,
1994). It is thought that the resulting low levels of
cholesterol interfere with the function of Sonic Hedgehog,
another early developmental gene. Mutations in Sonic
Hedgehog are a cause of holoprosencephaly (Roessler et al.,
1996; Belloni et al., 1996), the condition in which extreme
cases exhibit a forebrain with a single ventricle, and a
face with a single eye. The brain anomalies of SLOS are not
so severe, but on the Autism Diagnostic Interview, about
half of the cases meet the criteria for ASDs (Tierney et al.,
2001) and all are thought to have cognitive limitation (Smith
et al., 1964). The characteristic facial features of SLOS
include a broad, high forehead, hypertelorism, ptosis,
epicanthal folds, broad nasal bridge, short nose with
antiverted nares, and micrognathia (reviewed in Nowaczyk
and small. Other physical malformations that appear in the
syndrome include cleft palate, syndactyly, and genital
These syndromes are different in many ways. What they
have in common is that all appear to involve abnormal
development in the embryonic period, just as the CHARGE
T.L. Arndt et al./Int. J. Devl Neuroscience 23 (2005) 189–199192
association, Goldenhar syndrome,and Moebius sequencedo
(Miller et al., 2005).
6. What can VPA teach us about autism?
Of the environmental agents linked to ASDs, valproic
acid has been studied the most extensively. Current
indications for VPA (Depakote) include: epilepsy (complex
partial and absence seizures) (Rimmer and Richens, 1985;
Beydoun et al., 1997), mania (Puzynski and Klosiewicz,
et al., 2002). Valproic acid, which crosses the placenta and
also can cross into breast milk, has been given a category
‘‘D’’ classification by the FDA for use during pregnancy.
This classification is given to drugs for which positive
evidence of human pregnancy risk exists but which may be
used in certain situations where the benefits to the mother
outweigh the risks to the embryo or fetus. These include
taking the drug and for which safer drugs are not efficacious
Many children exposed in utero to VPA exhibit Fetal
Valproate Syndrome, a syndrome characterized by a
constellation of major and minor malformations and
developmental and behavioral delays (DiLiberti et al.,
1984; Jager-Roman et al., 1986; Ardinger et al., 1988;
Kozma, 2001). Fetal Valproate Syndrome has been reported
in a number of sibling pairs (DiLiberti et al., 1984; Winter
et al., 1987; Clayton-Smith and Donnai, 1990; Christianson
et al., 1994; Janas et al., 1998; Kozma, 2001; Malm et al.,
2002), with different constellations of features and severity
of abnormalities among affected siblings. Common facial
features of fetal valproate syndrome include epicanthal
folds, broad nasal bridge, short nose with antiverted nares,
long upper lip, and low set, posteriorly rotated ears. The tips
of the fingers appear pinched.
Autism was first reported to be one of the behavioral
outcomes of VPA exposure through case reports (Chris-
tianson et al., 1994; Williams and Hersh, 1997; Williams
et al., 2001). Moore et al. (2000) presented the first
epidemiological study ofthe riskof autism in57 offspring of
women taking anti-seizure medications. Combining mono-
therapy and polytherapy cases exposed to valproate, the rate
of ASDs in their sample was about 11%, with even more
children reported to have symptoms short of a diagnosis.
7. VPA-exposure as an animal model of autism
Utilizing evidence of early teratogenic insult from the
thalidomideandVPAstudies, Rodier etal.(1997) developed
an animal model of autism by exposing rats to VPA in utero.
Thalidomide exposure would be a valuable model if it had
the same teratogenic effects in rodents that it has in humans
and other primates (Hendrickx et al., 1966; Hendrickx,
1973). Unfortunately,thalidomide does not produce its well-
known constellation of somatic abnormalities in rodents
at any dose (Schumacher et al., 1968). In contrast, VPA
exposure induces similar patterns of abnormal development
across species. Skeletal abnormalities have been reported in
mice (Brown et al., 1980; Bruckner et al., 1983), rats
(Mengola et al., 1998), rabbits (Petrere et al., 1986), and
rhesus monkeys(at doses 10-fold above therapeutic levels in
humans) (Mast et al., 1986). Cardiac abnormalities (e.g.,
Sonoda et al., 1993) and neural tube defects, including
induction of spina bifida in mice (Ehlers et al., 1992) and
cranial neural tube defects in rats (Turner et al., 1990) have
alsobeen reported inanimal models.Most importantly,VPA
exposure in utero also leads to behavioral abnormalities in
rats (e.g., Vorhees, 1987).
Current hypotheses for the mechanism for VPA involve
retinoic acid responsive elements (RAREs) and alteration of
gene expression of early developmental genes, especially
Hox genes. Animals exposed to retinoic acid (RA) in utero
have a similar pattern of craniofacial, limb, and heart defects
as those seen after exposure to VPA (Ehlers et al., 1992).
Retinoic acid is the endogenous ligand for the RAREs that
initiate Hox gene expression (Langston and Gudas, 1992;
Conlon and Rossant, 1992).
Studies have demonstrated the effect of VPA and RA on
RAREs and expression of Hoxa1 and other genes. Stodgell
et al. (2003) found that VPA exposure may increase the
transport of RA into the nucleus, by showing an
approximately 50-fold increase in the expression of cellular
retinoic acid binding protein in exposed rat embryos using
affymetrix microarrays. In the same study, genes important
for cholesterol synthesis and transport were also affected by
VPA exposure. Another study (Stodgell et al., 2001)
demonstrated that in utero VPA exposure elevates expres-
sion of Hoxa1 more rapidly and to a greater extent than does
the expression of Hoxa1 at time periods before and after the
normaltimewindow forits expression. Itispossible that this
effect is mediated through the inhibitory effect of VPA on
histone deacetylase (Phiel et al., 2001). Inhibition of histone
deacetylase has been estimated to cause approximately 2%
of transcriptionally inactive genes to become available for
transcription via its effect on chromatin (Van Lint et al.,
1996). As VPA exposureis sufficient toinduce expression of
Hoxa1 when it is normally transcriptionally silent, the effect
may be mediated via this mechanism.
Rodier et al. (1996) exposed timed pregnant rats to VPA
during the sensitive window identified by the thalidomide
cases. To date, several studies have reported neuroanatomic
similarities between humancases of autism and rats exposed
2000; Arndtet al., 2003). Following exposure toVPAduring
early brain stem development, the rats survived into
adulthood and exhibited persisting neuroanatomic abnorm-
alities. Changes in the timing of exposure were used to
produce different injuries. A single intraperitoneal dose of
T.L. Arndt et al./Int. J. Devl Neuroscience 23 (2005) 189–199193
350 mg/kg VPA resulted in a significant reduction in the
trigeminal and hypoglossal nuclei following exposure on
day E11.5. Additional structures were found to be abnormal
after later exposures—exposure on day E12 resulted in
abnormalities of the abducens, trigeminal, and hypoglossal
nuclei, and exposure on day E12.5 resulted in reductions of
neurons in the oculomotor, abducens, trigeminal, and
hypoglossal nuclei. Interestingly, deficits of the facial
nucleus were not demonstrated, despite the presence of
abnormalities of this nucleus in some human cases of
autism. Whether repeated or later exposures alter the facial
nucleus is not known.
Cerebellar abnormalities consistent with human cases of
autism were found following exposure of 600 mg/kg sodium
valproate on day E12.5. Purkinje cell numbers in some
lobules (VI–VIII and X) of the vermis were reduced, but
were normal in the anterior lobes (IV–V). MRI studies have
shown decreased size of the posterior cerebellar vermis in
autism (Courchesne et al., 1994, 1988; Hashimoto et al.,
1995). It should be noted that some studies have found no
difference in the size of the cerebellar vermis when only
high-functioning subjects with autism are studied (Piven
etal., 1997; Hardan etal.,2001).Astereological study ofthe
thevolume of the nucleus (Rodier and LaPoint, 2001). Some
of the deep nuclei of the cerebellum were also examined
(Arndt et al., 2003). The interpositus nucleus corresponds to
the globose and emboliform nuclei in humans. In human
cases, the globose and emboliform nuclei are much more
severely affected than the dentate nucleus (Bauman and
Kemper, 1994). Likewise, in the animal model, the
interpositus nucleus showed a 62% reduction in volume,
while the dentate showed a 30% reduction. The overall brain
volume in the treated animals was reduced (18% by brain
size of the dentate, but is unlikely to account for the larger
reduction in the size of the interpositus nucleus. Interest-
ingly, because no cerebellar neurons are present at day
E12.5, the abnormalities in the cerebellum must have been
secondary to injury to the inferior olive, which forms at the
time the animals were exposed.
Narita et al., 2002 have also reported similarities between
rats exposed to VPA in utero and children with autism. After
exposure to VPA to Sprague–Dawley rats on embryonic day
9 (neural plate stage) exposed pups had increased serotonin
levelsin the hippocampus, increased dopamine in the frontal
cortex, and hyperserotonemia. Exposure on E9 also resulted
in a shift of serotonergic neurons in the dorsal raphe nucleus
(Miyazaki et al., 2005). Maturational differences were also
seen in serotonergic neurons with in vitro administration of
VPA. These studies imply that exposure to VPA in utero
could impact the development of the serotonergic system in
offspring. Hyperserotonemia has been reported in the whole
blood of some patients with autism (Anderson et al., 1990).
The clinical and developmental significance of altered
serotonin levels in children with autism is not known.
8. Do VPA-exposed rats and children with autism share
In addition to neuroanatomical similarities between
children with autism and rats exposed to VPA in utero,
there is evidence of behavioral similarities. An activearea of
investigation in several laboratories is the search for
behavioral tests that can distinguish individuals with autism
from both controls and other clinical groups, and that can be
tested in the rat. Preliminary evidence suggests that
Pavlovian (classical) eyeblink conditioning may fulfill both
of these criteria.
Pavlovian eyeblink conditioning can be used to test
associative learning in a number of mammalian species
including rodents, rabbits, and primates. During eyeblink
conditioning, a conditioned stimulus (CS, usually a tone) is
paired with an unconditioned stimulus (US, an airpuff or
stimulus to the eye). Initially, the US causes the subject to
blink, while the CS does not. With repeated pairings, the
when the CS alone is presented. The neural circuitry for
eyeblink conditioning has been well delineated through
animal studies (Steinmetz, 2000). This task is mediated
through a brainstem-cerebellar circuit, with forebrain
involvement on some task variants.
which projections are made into the basilar pontine nuclei.
where they synapse on parallel fibers with collaterals to the
deep nuclei. These parallel fibers synapse on Purkinje cells
(Steinmetz, 2000). Information regarding the air puff US is
Climbing fibers from the inferior olive then travel to the
collaterals to the deep nuclei. Additionally, fibers from the
an unconditioned response (UR) occurs (Steinmetz, 2000;
Thompson, 2000). The association between the CS and US
to the interpositus nucleus in rats), while the Purkinje cells
enhance the rate of acquisition and are important for learning
appropriate timing for the conditioned blinks. The final
pathway for the performance of the blink (the last step in the
pathway for the CR and UR) starts in the red nucleus, from
nerve then projects to the muscles of the eyelid where
contraction produces a blink (Steinmetz, 2000). Higher order
task variants require forebrain structures, such as the
hippocampus in trace conditioning (during which a delay is
introduced between the offset of the tone and the onset of
the airpuff) and discrimination reversal (the reversal period
for a learned discrimination between two tone stimuli).
This pathway is shared by all the mammals examined and
behavior on the task can be used to search for parallels across
T.L. Arndt et al./Int. J. Devl Neuroscience 23 (2005) 189–199194
Many of the regions that have been reported to be
abnormal in individuals with autism are involved in the
eyeblink conditioning pathway. As previously discussed,
cerebellar abnormalities have been reported consistently in
cases of autism (Williams et al., 1980; Ritvo et al., 1986;
Bauman and Kemper, 1994; Bailey et al., 1998). Post-
mortem analysis of nine brains from individuals with autism
revealed consistent cerebellar abnormalities (Bauman et al.,
1997). The most consistent findings were a marked
reduction in Purkinje cell number in the cerebellar hemi-
spheres, a reduction in granule cell numbers, and decreased
size of the deep cerebellar nuclei. The globose and
emboliform nuclei were the nuclei most severely affected,
while the dentate nucleus showed the least dramatic
reduction. The abnormalities noted in the deep nuclei were
age dependent: younger individuals tended to have large
the deep nuclei of brains from older cases were reduced in
size relative to controls.
Abnormalities in the hippocampus have been reported
less consistently. While some studies have suggested an
increased cell density in the hippocampus with small cell
bodies and decreased dendritic branching (Bauman and
Kemper, 1985; Raymond et al., 1989), other studies have
found no differences in the hippocampal formations of
autistic brains (Bailey et al., 1998). It is possible that the
individual variation in the hippocampal formation in autistic
brains represents subpopulations of individuals with autism.
However, it is also possible that there is a spectrum in the
density and cell types in the hippocampal formation and
these different studies have represented individuals at
different places along the spectrum.
Classical eyeblink conditioning provides unique oppor-
tunities to those studying human neurological and psychia-
tric disorders. Because the neural circuitry that underlies
eyeblink conditioning has been so well described, research-
ers can compare individuals with suspected functional
abnormalities in these areas. Many disorders have been
studied utilizing eyeblink conditioning. Some of these
include Alzheimer’s disease (e.g., Woodruff-Pak and Papka,
1996a), amnesia (e.g., McGlinchey-Berroth et al., 1995),
phobias (e.g., Martin et al., 1969; Sachs et al., 2003),
Huntington’s disease (Woodruff-Pak and Papka, 1996b),
alcoholism (e.g., McGlinchey-Berroth et al., 1995), ataxia
telangiectasia (Mostofsky et al., 1999), mental retardation
(e.g., Orlich and Ross, 1968; Lobb and Hardwick, 1976);
Down Syndrome (e.g., Papka et al., 1994; Woodruff-Pak
et al., 1994), dyslexia (Coffin and Boegle, 2000), Fragile X
Syndrome (Woodruff-Pak et al., 1994), and autism (Sears
et al., 1994; Sears and Steinmetz, 2000). Most disorders
studied show impairment in conditioning ability or similar
learning to control subjects.
Autism is unique among disorders studied in that an
increased rate of acquisition has been reported. Sears et al.
(1994) studied delay conditioning in 11 individuals with
autism (age 7–22) and 11 age, gender, and IQ-comparable
typically developing controls. Each subject underwent two
to evaluate extinction. Several outcome measures were
evaluated: acquisition and extinction rate, response timing
and amplitude. Acquisition was measured as time to reach a
learning criterion: individuals needed to produce CRs on
nine out of ten consecutive trials. Subjects with autism did
this in 34.5 trials, while controls required 56.1 trials. There
was also some indication of more rapid extinction in the
autism group. While both the autism and control groups had
individuals representing a wide age range (7–22 years in the
autism group and 6–23 years in controls), agewas correlated
with rate of acquisition and extinction in the control group
only. Acquisition in the control group was negatively
correlated with age, while extinction was positively
correlated with age.
Timing andamplitude of conditioned eyeblinkswere also
evaluated. The autism group displayed rapid acquisition of
the conditioned responses, butthe timingof these blinks was
less precise than in the control group. Subjects with autism
displayed blinks of shorter onset and peak latency following
CS presentation. The authors describe a ‘‘double-peaked’’
response pattern in which subjects blinked too early, then
opened their eyes and blinked again following the US.
Subjects in the autism group had a higher mean amplitude of
CRs as compared to typically developing subjects.
Importantly, on US alone trials, there were no group
differences in amplitude, suggesting that the increased
amplitude blinks in the autism group were due to learning,
not larger amplitude blinks at baseline. No differences in the
overall number of blinks or alpha (startle) responses were
reported. CS alone trials were not presented prior to
conditioning, so the baseline response to the tone in the
groups could not be compared.
Preliminary studies (Stanton et al., 2001) have demon-
strated similar learning patterns between children with
autism and rats exposed to VPA in utero on the eyeblink
conditioning task. In these studies, rats exposed to 600 mg/
kg NaVP on embryonic day 12.5 underwent standard delay
eyeblink conditioning. Exposed animals showed a pattern of
conditioning very similar to that reported in Sears et al.
(1994). Valproate-exposed rats displayed no difference in
basic sensory and motor function (audition and reflex
eyeblink responses to the unconditioned stimulus), but
amplitude of conditioned blinks was exaggerated. Timing of
conditioned blinks was also altered. One difference between
children with autism and the VPA-exposed rats was that the
animals did not display a higher rate of acquisition of
It is surprising that children with autism and rats with
exposure to VPA in utero display an enhancement of
conditioning despite having
Exposure to VPA early in gestation is known to cause a
reduction in Purkinje cell number in rats (Ingram et al.,
2000). Postnatal destruction of Purkinje cells by exposure to
T.L. Arndt et al./Int. J. Devl Neuroscience 23 (2005) 189–199195
environmental agents (e.g., ethanol) or by genetic manip-
ulation (Purkinje cell degeneration mice, in which geneti-
cally altered mice lose all of their Purkinje cells after birth)
1995; Stanton and Goodlett, 1998; Chen et al., 1996). One
possibility for the lack of impairment of conditioning
following VPA exposure is the early timing of the injury.
Exposure to valproate occurs prior to the time the Purkinje
cells form. To our knowledge, only one other animal model
has shown an enhancement of eyeblink conditioning, and it
involves early abnormalities in gene expression. Protein
kinase C–gamma isoform (PKCg) knockout mice display an
increased rate of acquisition during eyeblink conditioning.
Purkinje cells rather than pruning multiple inputs before
adulthood (Chen et al., 1996). These animals display rapid
acquisition of the conditioned eyeblink and high amplitude
CRs. While their neuroanatomy does not resemble that of
individuals with autism, it is important because it shows
that developmental cerebellar abnormalities can cause
conditioning enhancement (Sears and Steinmetz, 2000).
Importantly, all of the injuries of Purkinje cells that lead
to a decreased rate of conditioning occur long after the
Purkinje cells are formed. Early injury may lead to
reorganization of brainstem–cerebellar circuitry that causes
enhanced performance on the eyeblink conditioning task.
Based on the behavioral similarities between these animals
and children with autism during eyeblink conditioning, a
similar pattern of reorganization following early injury may
occur in both VPA-exposed rats and children with autism.
The behavioral similarities between VPA-exposed rats
and children with autism on the eyeblink conditioning task
are being investigated intensively in our laboratory in
collaboration with Mark Stanton at the University of
Delaware. By investigating behavioral patterns and their
exposed to VPA in utero, we hope to clarify which structural
abnormalities are involved in the enhancement of acquisi-
tion during eyeblink conditioning.
Autism spectrum disorders are unusual among psychia-
tric diagnoses in that the risk of diagnosis has been
determined to be increased by a number of environmental
exposures. In addition, the rate of ASDs is elevated in a
number of syndromes that are distinguished by multiple
birth defects. A detailed examination of the critical periods
when environmental exposures lead to autism and the
physical anomalies that occur in people with autism
indicates that many cases arise in the embryonic period.
Studies of the neuroanatomy of people with autism also
suggest a prenatal origin for the disorder. Experimental
studies of an animal model based on a known risk factor—
exposure to VPA during neural tube closure—confirm that it
is possible to model both anatomic and behavioral features
of human cases by injuring the embryonic brain.
Many of the studies described in this review were funded
by U19HD35466, a Collaborative Program of Excellence in
Autism.We are also grateful
U54MH066397, a STAART Center.
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