The teratology of autism

Int. J. Devl Neuroscience 23 (2005) 189–199 www.elsevier.com/locate/ijdevneu

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

Abstract 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 histological studies of the brain are consistent with an early alteration of development. Congenital syndromes with high rates of autism include 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. Hints that 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). 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: [email protected] (P.M. Rodier).

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 depression of dorsolateral function, Goldman and Alexander (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 observed in monkeys tested at 19–31 months, and in subjects subjected to cooling between 34 and 36 months of age the impairment was much greater. Presumably, the latedeveloping 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

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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 development, 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 ageappropriate behaviors. 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 as failure to point to objects and failure to respond 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, Nelson et al. (2001) reported that samples from children who would later be diagnosed with either autism or mental

retardation were distinguished by anomalous concentrations of neuron-related products in blood. Recycling immunoaffinity chromatography was used to measure the neuropeptides substance P (SP), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), calcitonin gene-related peptide (CGRP), and the neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic 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 typicallydeveloping 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 epidemiological studies:     

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 discussed 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 postconception (Stro¨ mland et al., 1994). The timing of the misoprostol exposures was determined by questioning mothers and was in the sixth week postconception (Bandim et al., 2003). 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

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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 heart disease, 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 during embryogenesis. The fact that each of these teratogens appears to act during the embryonic period (the first eight weeks of life) does not rule out the possibility that autism could be initiated 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 environmental risk factors identified is strong evidence that autism arises very early in development.

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 anomalies (abnormalities present in <5% of the population). (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.

3. Dysmorphic features suggest early injury in idiopathic autism

4. Neuroanatomical evidence for early injury in idiopathic autism

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

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

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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 wellestablished before parturition, it is impossible to reconcile such anomalies with a disturbance of postnatal brain development. A brain from a patient with autism, studied by Rodier et al. (1996), had a massive reduction in the number of motor 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 Takashima’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 utero. While abnormalities of the inferior olive (breaks in the line of neurons, misplaced neurons, etc.) have been reported in the brains of people with autism by Bauman and Kemper (1994) and Bailey et al. (1998), the nucleus is not as cell poor as one would expect if cells were degenerating in response to 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, we wish 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, eye movement 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 of the pyramidal tracts to cross, and functional MRI supports this finding (Parisi et al., 2004). This is especially interesting in light of one of the cases of autism reported by Bailey et 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. This year, mutations in the gene NIPBL were identified as 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, but many are ones also seen in people with 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 et al., 1998). The same authors report that the ears are low set and small. Other physical malformations that appear in the syndrome include cleft palate, syndactyly, and genital anomalies. 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

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association, Goldenhar syndrome, and Moebius sequence do (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, 1984; Kmetz et al., 1997), and migraine prophylaxis (Freitag 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 situations where the mother’s health would be at risk without taking the drug and for which safer drugs are not efficacious or available. 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 (Christianson et al., 1994; Williams and Hersh, 1997; Williams et al., 2001). Moore et al. (2000) presented the first epidemiological study of the risk of autism in 57 offspring of women taking anti-seizure medications. Combining monotherapy 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 thalidomide and VPA studies, Rodier et al. (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,

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1973). Unfortunately, thalidomide does not produce its wellknown 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 also been reported in animal 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 expression of Hoxa1 more rapidly and to a greater extent than does RA. It was also shown that VPA exposure in utero can induce the expression of Hoxa1 at time periods before and after the normal time window for its expression. It is possible 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 exposure is sufficient to induce 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 human cases of autism and rats exposed to VPA in utero (e.g., Rodier et al., 1996, 1997; Ingram et al., 2000; Arndt et al., 2003). Following exposure to VPA during early brain stem development, the rats survived into adulthood and exhibited persisting neuroanatomic abnormalities. Changes in the timing of exposure were used to produce different injuries. A single intraperitoneal dose of

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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 et al., 1997; Hardan et al., 2001). A stereological study of the inferior olive demonstrated reductions in neuron number and the volume 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 weight), which could account for most of the reduction in the size of the dentate, but is unlikely to account for the larger reduction in the size of the interpositus nucleus. Interestingly, 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 levels in 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 similar behavior? In addition to neuroanatomical similarities between children with autism and rats exposed to VPA in utero, there is evidence of behavioral similarities. An active area 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 subject learns to associate the CS and US such that they blink 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. ProcessingofthetoneCSbeginsinthecochlearnuclei,from which projections are made into the basilar pontine nuclei. Mossy fibers from the pontine nuclei are sent to the cerebellum, 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 directed to the trigeminal nucleus and then to the inferior olive. Climbing fibers from the inferior olive then travel to the cerebellum where they synapse directly on Purkinje cells with collaterals to the deep nuclei. Additionally, fibers from the trigeminal nucleus are sent to the reticular formation and then the facial and abducens nuclei, which is the pathway by which an unconditioned response (UR) occurs (Steinmetz, 2000; Thompson, 2000). The association between the CS and US takes place in the globose and emboliform nuclei (homologous 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 which projections contact the facial motor nucleus. The facial 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 species.

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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). Postmortem 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 hemispheres, 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 cells in the deep nuclei relative to controls, but the neurons in 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 opportunities to those studying human neurological and psychiatric disorders. Because the neural circuitry that underlies eyeblink conditioning has been so well described, researchers 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), schizophrenia (e.g., Marenco et al., 2003; Hofer et al., 2001), 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

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autism (age 7–22) and 11 age, gender, and IQ-comparable typically developing controls. Each subject underwent two sessions of delay conditioning followed by 60 unpaired trials 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), age was 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 and amplitude of conditioned eyeblinks were also evaluated. The autism group displayed rapid acquisition of the conditioned responses, but the timing of 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 demonstrated 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 conditioned responses. It is surprising that children with autism and rats with exposure to VPA in utero display an enhancement of conditioning despite having cerebellar abnormalities. 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

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environmental agents (e.g., ethanol) or by genetic manipulation (Purkinje cell degeneration mice, in which genetically altered mice lose all of their Purkinje cells after birth) causes a striking impairment in conditioning (Freeman et al., 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. PKCg knockout mice retain multiple climbing fiber inputs to 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 corresponding neuroanatomical abnormalities in rats exposed to VPA in utero, we hope to clarify which structural abnormalities are involved in the enhancement of acquisition during eyeblink conditioning.

9. Conclusion Autism spectrum disorders are unusual among psychiatric 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.

Acknowledgements Many of the studies described in this review were funded by U19HD35466, a Collaborative Program of Excellence in Autism. We are also grateful for the support of U54MH066397, a STAART Center.

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