Cerebellar contributions to autism spectrum disorders

Clinical Neuroscience Research 6 (2006) 195–207 www.elsevier.com/locate/clires

Cerebellar contributions to autism spectrum disorders Greg Allen * Department of Psychiatry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9127, USA Program in Cognition and Neuroscience, School of Behavioral and Brain Sciences, The University of Texas at Dallas, USA

Abstract The pathophysiology of autism appears to encompass a number of different brain structures and systems. However, the most consistent site of neural abnormality in autism is the cerebellum. Postmortem investigations have reported a variety of anomalies, most notably a reduction in the number of Purkinje neurons. Additionally, structural neuroimaging studies have shown volumetric changes in the cerebellum, including decreases in gray matter and increases in white matter. Emerging evidence for cerebellar abnormality in autism was paralleled by a revolution in our understanding of normal cerebellar function and cerebellar connectivity, such that the importance of elucidating the contributions of the cerebellum to autism is now clear. In fact, recent brain-behavior correlation studies suggest that cerebellar abnormality may play a more central role in autism than previously thought. At present, it is crucial that we increase our understanding of cerebellar functioning in autism, and functional neuroimaging studies are just beginning to reveal the possible role(s) of cerebellar dysfunction in this disorder. In this review, evidence for cerebellar anatomic and functional abnormality in autism will be delineated. This will be followed by consideration of the implications of cerebellar abnormality in autism. Two major questions will be addressed: (1) how might dysfunction of the cerebellum impact the development of connectivity between the cerebellum and other brain systems, and (2) how might cerebellar dysfunction impact behavior and the symptoms of autism. The paper concludes with a discussion of how cerebellar abnormalities might inform our understanding of the etiology of autism spectrum disorders. Ó 2006 Association for Research in Nervous and Mental Disease. Published by Elsevier B.V. All rights reserved. Keywords: Autism; Cerebellum; Connectivity; Development; FMRI; MRI; Purkinje cells

1. Introduction A crucial step in determining the cause of autism is to understand its underlying neurobiology. Abnormalities in a variety of brain regions and systems have been proposed to contribute to the pathophysiology of this disorder (e.g., [1–11]), and there are data to support involvement of a number of these regions. However, this review will focus on one of the most consistent sites of neuroanatomic abnormality in autism – the cerebellum. Emerging evidence for cerebellar abnormality in autism has been paralleled by a revolution in our understanding of normal cerebellar function, such that the importance of elucidating the contributions of the cerebellum to autism is now clear. The purpose of this review is to delineate cerebellar findings *

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in autism and discuss their implications for understanding the disorder. 2. Cerebellar anatomic abnormality in autism: postmortem evidence In postmortem studies of the brains of individuals with autism, the cerebellum has been the most consistently observed site of pathology. The most frequently reported pathology in these studies is a reduction in the normal number of Purkinje neurons (Fig. 1), which has been reported in nearly every case in which the cerebellum was examined [10,12–18]. The extent of Purkinje neuron reduction may vary from case to case, but one recent study showed an overall 41% decrease in Purkinje cells in a group of eight cases with autism compared to eight controls [17]. The precise anatomic distribution of Purkinje neuron reduction has yet to be decisively determined. While some

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Fig. 1. Purkinje neuron reduction in autism. (A) Control cerebellum normally populated with Purkinje neurons. (B) Patchy reduction of neurons in the Purkinje cell layer (adapted from [16]).

studies described it as diffuse or widespread [10,15,18], others reported it to be more prominent in the posterolateral neocerebellar cortex, with less involvement of the vermis and anterior hemispheres [14,19]. Variability in the localization of Purkinje cell reduction was further shown by those studies that provided more detailed descriptions of their individual cases. For instance, Bailey et al. [12] reported Purkinje neuron reduction in five cases. Of these, three demonstrated diffuse reduction, while two showed greater reduction in the hemispheres than the vermis, consistent with the reports of Bauman and Kemper. In contrast, Lee and colleagues reported on two cases with Purkinje neuron reduction, one of which showed greater reduction in the vermis than the hemispheres [10]. Variable reduction across the cerebellum is developmentally plausible, given that Purkinje cells in the vermis and anterior and posterior hemispheres have different times of peak neurogenesis, different migratory patterns, and different times of settling [20]. Clearly, more studies are required to determine the anatomic distribution of Purkinje cell reduction, and this is an important question, given that different regions of the cerebellum have distinct connections with other brain regions and appear to be preferentially involved in different functions. While it remains the single most consistent finding in postmortem autism studies, Purkinje cell reduction is not the only cerebellar pathology to be reported. For instance, reduced numbers of granule cells have also been found [16,21]. Bauman and Kemper also reported reduced numbers of cells in the fastigial, globose, and emboliform nuclei, but not the dentate nucleus [14]. This group has also reported that in their younger cases, neurons in all of the deep cerebellar nuclei were abnormally large in size but adequate in number. Moreover, Fatemi and colleagues have shown that Purkinje neurons are not only reduced in number, but also reduced in size [22]. Changes in neurotransmitter systems have also been seen in postmortem studies. These include glutamatergic [23] and cholinergic

[10,24] receptor abnormalities, as well as reduced levels of glutamic acid decarboxylase [25], which is necessary for the synthesis of the neurotransmitter GABA. Several additional abnormalities that may leave the cerebellum susceptible to aberrant development or neuronal loss have been discovered in recent years. These include reduced levels of several important neuroregulatory proteins in the cerebellum, such as Bcl-2, an inhibitor of apoptosis [26–28], Reelin, which is involved in correct layering of the developing brain as well as cell signaling and synaptic plasticity in the adult brain [28], and neural cell adhesion molecule (NCAM), a protein thought to be involved in several aspects of nervous system development [29]. Furthermore, a few recent studies have examined levels of neurotrophins and neuropeptides in blood samples from autistic children. For instance, Nelson and colleagues found increased concentrations of vasoactive intestinal peptide (VIP), neurotrophin 4/5 (NT4/5), calcitonin generelated peptide (CGRP), and brain-derived neurotrophic factor (BDNF) [30] and reduced levels of neurotrophin 3 (NT3) [31]. While some of these substances may lead to abnormally enhanced neural growth [32], elevated levels of BDNF and NT4 and reduced levels of NT3 can actually contribute to a loss of Purkinje neurons [33–35]. Thus, altered levels of several substances may contribute to cerebellar pathology. A key goal of future investigations should be to examine the relationships between levels of these various substances and neuroanatomic differences in individuals with autism. 3. Cerebellar abnormality in autism: when does it emerge? Postmortem studies have provided some clues regarding the time during development when Purkinje cell reduction occurs. The absence of reactive gliosis in early reports [14,15,18] and the lack of empty basket cells, which normally ensheath the Purkinje neuron cell bodies [12], have provided perhaps the strongest evidence for an early devel-

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opmental (i.e., prenatal) reduction of Purkinje neurons. This is also supported by the lack of neuronal loss in the inferior olive [14]. However, Bailey and colleagues reported increased numbers of Bergmann glia in some of their autopsy cases, in addition to increased glial fibrillary acidic protein (GFAP), both of which typically point to later acquired pathology [12]. More recently, Laurence and Fatemi replicated the finding of increased GFAP in cerebellar cortex [36] and Vargas and colleagues found increased expression of GFAP in the cerebella of cases with Purkinje neuron reduction. In the latter study, GFAP immunostaining revealed increased astroglial reactions including a marked reactivity of Bergmann glia in the Purkinje cell layer [16]. Moreover, Bailey et al. noted that Purkinje neuron reduction in the presence of a relatively normal appearing cerebellar cortex is atypical for pathology occurring early in development [12]. In other words, an early reduction of Purkinje neurons, which are known to play a central role in cerebellar development, would be thought to have more profound effects on the overall development of cerebellar cortex. Of course, it is important to note additional possibilities regarding the timing of Purkinje neuron reduction that are based on more recent findings. One is that autism may involve both an early developmental reduction and a later acquired loss of these cells. The study by Lee and colleagues [10], who conducted a histological examination of the cerebellum in two cases mentioned above, lends support to this notion. One case with a history of epilepsy demonstrated severe Purkinje neuron reduction, with Bergmann gliosis in the Purkinje cell layer and additional glial clusters in the molecular layer. In the other case, with no history of epilepsy, the reduction was less severe and not accompanied by gliosis. These cases suggest that both developmental reduction and acquired loss of Purkinje neurons may indeed co-exist in autism. A second intriguing possibility is that pathological changes in the cerebellum involve a chronic, ongoing process of neuroinflammation, and neurodegeneration that extends well beyond early neurodevelopment and may actually continue into the late stages of life in individuals with autism [16]. This notion is based Vargas and colleagues’ findings of marked immune responses in the cerebellum that were closely associated with degenerating Purkinje cells, granule cells, and axons. These findings were reportedly similar to those seen in neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amytrophic lateral sclerosis.

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matter volumes were significantly and substantially (i.e., nearly 40%) increased [37]. Studies that did not separate gray and white matter also reported significant increases in total cerebellar volume. This has been shown in 3- to 4-year-olds [38], 7- to 11-year-olds [39], and 6- to 14-yearolds [40] with autism spectrum disorders. However, in light of the findings of Courchesne and colleagues, these increases in overall volume may be attributable to increased white matter. Courchesne and colleagues also showed that early adolescence appears to be the time when differences in cerebellar white matter are diminishing (Fig. 2A), while gray matter differences are becoming evident (Fig. 2B), such that in older adolescents with autism, cerebellar gray matter volume was reduced relative to normal [37]. This discrepancy between gray and white matter effects calls for the separate analysis of gray and white matter in future studies and highlights the need for very careful tissue characterization in MRI studies of cerebellar volume in autism. A newer approach to examining brain anatomy with MRI is voxel-based morphometry (VBM). VBM, which is an anatomical derivative of the statistical parametric mapping approach to analyzing functional brain imaging data, involves the calculation of voxel-wise statistics on grouped MRI data. VBM analyses report localized differences in tissue concentration rather than the actual volume of a structure. Because it involves the averaging of anatomy within a group, the technique may be problematic when

4. Cerebellar anatomic abnormality in autism: in vivo evidence The anatomic study of the cerebellum in vivo using magnetic resonance imaging (MRI) has provided a wealth of evidence for cerebellar abnormality in autism spectrum disorders. In very young individuals with autism (2–3 years of age), gray matter volumes in the whole cerebellum did not differ significantly from normal [37], but cerebellar white

Fig. 2. Volumes of cerebellar white matter (A) and gray matter (B) in 2- to 16-year-old boys plotted with their best-fit curves (adapted from [37]).

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data are imperfectly registered across subjects [41]. Perfect registration is very difficult to achieve, particularly in a group with known neuroanatomic heterogeneity such as the autism population. Thus, VBM may produce some results that are difficult to interpret. A few autism studies have employed VBM, including three that reported differences in the cerebellum. Abell and colleagues [42] demonstrated focal areas of increased tissue concentration in the cerebellar hemispheres and vermis in young adults with Asperger’s disorder. Another group, which examined children and adolescents with autism, reported decreased white matter concentration in the left cerebellar hemisphere [43]. Similarly, McAlonan and colleagues [44] reported bilateral reductions in white matter concentration in the cerebella of high-functioning adolescents with autism. These latter two studies stand in contrast to a study using more traditional methods of cerebellar volume quantification that reported increases in cerebellar white matter volume [37]. Further investigations are required to clarify the status of white matter in the cerebellum in autism and how it might differ across the autism spectrum. In terms of cerebellar sub-regions, reduced size of one or more regions in the cerebellar vermis, which is predominantly gray matter, is frequently reported in autism MRI studies (Fig. 3) [37,45–56]. In the largest MRI study of autism yet conducted, Hashimoto and colleagues studied over 100 patients ranging from 6 months to 20 years of age. These investigators showed that vermis measures were smaller than normal at all ages [52]. A localized reduction in the size of cerebellar vermis lobules VI–VII has also been reported in several studies, including an investigation of sixty 2- to 16-year-old boys with autism [37]. Furthermore, in a study of 3- to 9-year-old children, this finding was specific to autism when compared to normal children and children with fragile X or Down syndrome [53]. While reduced size of posterior vermis lobules VI–VII is frequently reported, a similar reduction in anterior vermis lobules I–V is much less common. This discrepancy was noted in the earliest study to report cerebellar vermis differences in autism [48], and subsequent studies have con-

firmed it. Only one investigation reported lobules I–V to be reduced in size [52], while two showed the size of these lobules to be increased [55,57]. This discrepancy between the anterior and posterior vermis somewhat parallels the reported anterior–posterior difference in Purkinje neuron reduction described by others [14,19]. It should be noted, however, that these latter studies described this discrepancy as a feature of the cerebellar hemispheres and not the vermis. Nevertheless, as the anterior and posterior lobes of the cerebellum develop from different portions of the rhombencephalon and have different times of neurogenesis [20], such a discrepancy is developmentally plausible. In addition to the findings in the cerebellar vermis, reduced size of the cerebellar hemispheres has been reported as well [58,59]. However, a study that only examined subjects with high-functioning autism reported that the total volume (gray and white matter combined) of the cerebellar hemispheres was significantly increased [60]. Again, the overall volume increases may have been due to the specific increases in white matter previously noted. Despite all of the supporting data, the issue of cerebellar involvement in autism has long been steeped in controversy. This controversy exists for several reasons, a major one being the fact that several studies failed to replicate the finding of cerebellar vermis size reduction [61–65]. The reasons for this are many, and they have been reviewed extensively [32,51]. One factor contributing to the lack of replication of vermis findings is the use of IQ as a matching variable or covariate. Several autism researchers have argued that controlling for IQ, either through matching subjects based on their intellectual abilities or using IQ as a covariate, is an essential step in the investigation of potential neuroanatomic differences in autism [55,62–66]. When these methods are employed, cerebellar differences are not always seen. The purpose of IQ matching is to control for possible group differences in general cognitive functioning. However, in autism, overall IQ is not necessarily a valid measure of such functioning given the atypical profile of subtest performance [67]. Moreover, for studies with anatomical or physiological dependent variables, ability

Fig. 3. Midsagittal T1-weighted MR images from a representative normal control subject and an individual with autism. Superimposed tracings highlight the difference in midsagittal vermis area between these two individuals (images courtesy of Eric Courchesne, Ph.D.).

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measures are not always appropriate matching variables. Factors chosen as matching variables are typically those that may influence the dependent variable in a manner that is not of interest to the experiment at hand (e.g., age and sex). Although IQ and other behavioral variables may certainly impact brain structure over time, one must also consider the well-established and likely stronger impact that brain structure has on IQ. Depending on the goal of a study, controlling for a variable that is influenced by the dependent variable in this manner may negatively impact the study’s results. Consider an investigation that examines whether two groups differ on a measure of brain anatomy. The anatomical region of interest also happens to be involved in a wide range of cognitive functions, including many of those indexed by IQ. In these two groups, there is a true difference in the size of this brain region. However, the variability in the region is also correlated with variability in IQ. Thus, removing the so-called ‘‘IQ effect’’ also removes true differences from normal that also happen to influence IQ. The cerebellum is just such a brain region. It is involved in a wide range of cognitive functions, including those indexed by IQ, and its anatomic size is known to be correlated with IQ [68,69]. Thus, in addition to removing the influence of IQ on cerebellar size, controlling for IQ may lead to the removal of anatomic differences that are important features of autism and that also happen to influence IQ. The relationship between cerebellar anatomy and IQ in autism is certainly interesting and important. Therefore, as an alternative to removing this relationship from the analyses, one may employ a different approach and directly examine the interrelationship among anatomy, ability, and autism severity. For instance, a reanalysis of vermis measures from multiple studies [51] showed that indeed, individuals who were the most impaired in terms of IQ showed the greatest reduction in vermis size. At the other end of the spectrum, Hardan and colleagues [60] showed no differences in cerebellar vermis area in individuals with high-functioning autism. And, in a study directly comparing low- and high-functioning children with autism, the area of vermis lobules VI–VII was significantly smaller in children with low-functioning autism, while the measures in normal control children were intermediate to these two groups [57]. Thus, evidence does support an important relationship between cerebellar anatomy and IQ in autism, and controlling for IQ is not incorrect per se. Rather, whether or not it is done appears to be a reflection of the particular goal of a group comparison. On the one hand, investigators who are primarily concerned with simply understanding how the brains of individuals with autism differ from normal may not be inclined to control for IQ, while investigators concerned with identifying abnormalities that are specific to autism will. Those who take the specificity approach have argued that because cerebellar abnormalities are also seen in other disorders of childhood, both developmental and acquired, such abnormalities may be less relevant to our

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understanding of the neurobiological basis of autism [46,70,71]. This view has fostered, at least in part, the use of IQ matching, which in some cases leads to the inclusion of individuals with mental retardation or borderline intelligence in the comparison group [55,62,64,65]. Clearly, comparing such disparate groups will limit an individual study’s ability to observe how the brains of individuals with autism differ from normal. Furthermore, this type of matching may cloud our ability to understand the basis of cerebellar anatomic abnormalities in autism, for although multiple developmental disorders of different type and etiology may show abnormalities of the cerebellum, the morphologic patterns of these abnormalities can differ drastically [53], as can the actual cellular bases for such abnormalities [51]. Macroscopic deviations from normal can serve as important guides for subsequent microscopic studies, and limiting our ability to identify these deviations may slow our progress in identifying the true nature of neuroanatomic differences in autism. As will be discussed a bit more later, recent years have seen a revision in how we look at cerebellar function, such that the cerebellum is now seen as a structure involved in a wide range of functions, including many of the cognitive operations indexed by IQ. The relationship between cerebellar anatomy and IQ in autism is thought to be a manifestation of this broader role of the cerebellum. Furthering our understanding of this relationship is likely a key to better understanding the contribution of cerebellar dysfunction to autism. 5. Imaging cerebellar function in autism Functional neuroimaging data on the cerebellum in individuals with autism are relatively limited, with few studies designed specifically to address cerebellar function. Early investigations using positron emission tomography (PET) to measure resting glucose metabolism showed no difference from normal in the cerebellar vermis and hemispheres [72,73]. In contrast, serotonergic abnormalities in the cerebella of boys with autism were revealed using PET [74], and activation studies using the PET 15O method demonstrated less activity than normal while listening to sentences [75] or tones [76], and greater activity than normal while repeating sentences [75]. Abnormal cerebellar function was further supported by magnetic resonance spectroscopy studies, which exhibited lower levels of N-acetyl-aspartate, a putative marker of general neuronal function, in the cerebella of individuals with autism [77,78]. Early neurobehavioral studies established that the cerebellar anatomic abnormalities in autism are associated with deficits in specific attention operations. For instance, individuals with autism have difficulty rapidly reallocating attentional resources to new sensory modalities [79,80] and to new spatial locations [81–84]. Such findings are more prominent in those with greater abnormality of the cerebellum, and they are also seen in patients with acquired neocerebellar lesions [79,83,85]. These findings inspired a

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more direct investigation of cerebellar function in autism by Allen and colleagues using functional magnetic resonance imaging (FMRI) [86]. This study examined functional activation in the cerebella of patients with autism and normal comparison subjects performing a nonspatial visual selective attention task. Similar to previous findings [87], normal subjects showed bilateral superior posterior cerebellar hemisphere activation during the attention task. However, unlike their normal counterparts, subjects with autism showed minimal cerebellar activation. In addition to these findings, three other studies have reported reduced FMRI activation of the cerebellum in autism. One of these studies investigated brain activity during a task involving judgments of facial expression [88], another was a study of motor learning [89], and a third was an investigation of the detection of novel auditory stimuli [90]. In addition to examining cerebellar functioning in the context of attention, Allen and colleagues also studied patterns of activation in the cerebellum during a motor task [91]. For this task, each subject held a joystick in the dominant hand and repeatedly pressed a button with the thumb at a comfortable pace. During this simple motor task, individuals with autism showed substantially increased cerebellar activation relative to normal controls. Similarly, Mu¨ller and colleagues showed greater than normal cerebellar activity in subjects with autism during a finger movement task [92]. Overall, the results from functional neuroimaging studies are just beginning to reveal the role of cerebellar dysfunction in autism. However, a hint of a pattern can be seen in the collective findings. This pattern suggests that decreased cerebellar activation occurs with greater frequency during higher-level sensory and cognitive tasks (e.g., attention, learning, and decision making) [86,88,89], while abnormal increases in cerebellar activation appear more common during basic motor and speech processes [75,91,92]. The reasons for this dichotomy are not clear. However, an intriguing possibility is that it may be a functional reflection of a regional difference in Purkinje neuron reduction. Cognitive operations normally engage the posterior neocerebellar hemispheres, while motor tasks typically engage the anterior hemispheres and vermis. As noted above, the posterior neocerebellar hemispheres are reported to be the location of more prominent Purkinje neuron reduction, while the anterior hemispheres and vermis show less reduction. Reduced activation in the posterior cerebellar hemispheres correlates significantly with reduced size of these same regions [86] (Fig. 4), and it is likely that this relationship between function and structure extends to the microscopic level (i.e., the reduction in Purkinje neurons). Thus, PET and FMRI findings may be a functional manifestation of the anterior–posterior discrepancy seen in both postmortem and in vivo structural MRI studies. These functional neuroimaging findings call for a more detailed inquiry into the functional implications of cerebellar pathology.

Fig. 4. Correlation between cerebellar anatomy and cerebellar attention activation. Percent ROI active in the right superior posterior cerebellar hemisphere (lobule VIIA) during the attention task (performancematched) plotted against summed anatomical areas (mm2) in that same ROI for normal subjects (r = 0.72; t = 2.54, df = 6, p < 0.05, two-tailed t test) and subjects with autism (r = 0.88; t = 4.54, df = 5, p < 0.05, twotailed t test) (adapted from [86]).

6. The implications of cerebellar pathology in autism When considering the implications of cerebellar pathology in a developmental disorder such as autism, it is necessary to consider two separate questions about cerebellar dysfunction: (1) how might dysfunction of the cerebellum in the context of a developing central nervous system impact connectivity between the cerebellum and other brain systems? and (2) how might cerebellar dysfunction impact behavior and the symptoms of autism? 6.1. Disruption of cerebellar connectivity An emerging theoretical view in the autism field considers the abnormal development of neural connectivity to be a characterizing feature of the brains of individuals with autism [93–96]. Cerebellar pathology is a likely contributor to aspects of this aberrant connectivity, as Purkinje neuron reduction will lead to a disruption of activity leaving the cerebellum for other brain regions. Normally, Purkinje neurons provide inhibitory modulation of excitatory output from the deep cerebellar nuclei. A reduction of Purkinje neurons will release the deep nuclei from inhibition, leading to aberrant activity along the cerebellum–thalamus–cerebral cortex circuit. Such activity may lead to the abnormal strengthening of anatomic connections between the cerebellum and other brain regions and the emergence of aberrant patterning of functional connectivity with cerebral sites [93] (Fig. 5). This abnormal connectivity may contribute to a variety of functional and anatomic abnormalities, including

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Fig. 5. Hypothetical increases in cerebellar connectivity in autism shown on an oblique T1-weighted MR image passing through cerebellum, thalamus, and frontal cortex. A reduction in Purkinje neurons is thought to result in the disinhibition of deep cerebellar nuclei projections (represented by the increased weight of the cerebello-thalamic connection) and the emergence of aberrant patterning of functional connectivity with cerebro-cortical sites.

variable functional topography in cerebral cortex [92,97], excessive growth of cerebro-cortical areas [45,98], impaired modulation of frontal event-related potentials [99], and overexcitation of thalamocortical projections [93,94], increasing central nervous system ‘‘noise’’ and decreasing efficient information processing. A major goal of our future research is to examine how abnormalities of cerebellar connectivity may affect overall brain functioning to ultimately impact behavior in autism spectrum disorders. 6.2. Disruption of cerebellar function The traditional view of the cerebellum was that it is a structure involved exclusively in motor coordination. This view may in fact have contributed to controversy surrounding the notion of cerebellar involvement in autism, for the symptoms that define autism, at least from a diagnostic standpoint, are decidedly non-motor. However, research has shown that motor signs are indeed a common feature of individuals with autism spectrum disorders. For instance, the earliest published account of autism described children who were ‘‘clumsy in gait and gross motor performances’’ [100]. Later investigations of autistic individuals’ motor functioning on standard neurological measures revealed abnormalities on a variety of cerebellar tests (e.g., gait, alternating and sequential movements, balance) [101,102], with an overall picture ‘‘indicative more of cerebellar dysfunction than of any other definable central nervous system disorder’’ [102], p. 1306. More recent investigations have employed a variety of motor paradigms to explore how motor control is affected in autism. Though the findings have been somewhat mixed, several studies have identified deficits that may be a reflection of impaired cerebellar function. These include oculomotor deficits [103,104], deficient postural adjustments

[105], deficits of motor planning [106] and motor preparation [107], and gait abnormalities [108]. Abnormal patterns of cerebellar activation during simple motor tasks [75,91,97] provide added support for the notion that the motor deficiencies observed in many autistic individuals are related, at least in part, to cerebellar dysfunction. Motor impairments are not part of the formal diagnostic criteria for autism. However, they may be among the earliest distinguishing features, as they are observable long before most social or language deficits become evident [109,110]. These motor impairments may have an effect that goes beyond hampering locomotion and other forms of coordinated movement, for a child with such early problems of motor coordination will be at a great disadvantage in exploring, interacting with, and learning about his environment. However, there is a more central reason that cerebellar dysfunction will have broader consequences for individuals with autism, and that is that the cerebellum is simply more than just a motor structure. The cerebellum is one of the most widely connected brain structures, receiving projections from and/or sending projections to all major divisions of the central nervous system [111–118]. Furthermore, acquired cerebellar lesions can lead to a variety of cognitive deficits and affective changes [80,83,119–128]. This led Schmahmann and Sherman to outline a ‘‘cerebellar cognitive affective syndrome’’ characterized by language, visuospatial, and executive function deficits in addition to personality changes (e.g., inappropriate behavior and flattening of affect) [127]. Interestingly, a similar syndrome has been reported in children following surgical resection of cerebellar tumors [123,126], and in at least one of these pediatric cases, certain behavioral changes resembled classical symptoms of autism (e.g., gaze aversion, social withdrawal, and stereotyped movements) [126].

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In addition to the clinical findings, functional neuroimaging studies revealed that the cerebellum is involved in a variety of cognitive, social, and emotional functions [87,129–141]. Together, these findings not only significantly revised the traditional view of cerebellar function, but also indicated that developmental cerebellar pathology can effect a wide range of cognitive and affective deficits [142]. In concert with evidence for cerebellar anatomic abnormality in autism, this revised view of the cerebellum as a structure involved in a broad range of cognitive, social, and emotional functions indicates that it is not only reasonable, but imperative to examine the potential role(s) of cerebellar dysfunction in autism. Answering this question requires a clear understanding of normal cerebellar function, which unfortunately has yet to be accomplished in neuroscience. However, several theories describe the fundamental role of the cerebellum as predictive or anticipatory (e.g., [143– 149]). It is well known that the cerebellum is involved in learning (for review, see [147]). Furthermore, the cerebellum seems to have a special role in detecting signals in temporal sequences [150]. Several papers by Courchesne and colleagues propose that when presented with such sequences, the cerebellum predicts – based on prior learning – what is about to happen and initiates preparatory actions that alter neural responsiveness in whichever neural systems are to be needed in upcoming moments [80,87,147,151]. The cerebellum is thought to perform this fundamental function for all systems with which it maintains connections (e.g., sensory, motor, autonomic, memory, attention, affect, and language systems). When preparatory functioning is impaired, other neural systems can continue to perform, but will do so suboptimally in situations where prediction and preparation might aid performance. This can apply to any context in which successful performance requires rapid and efficient processing or production of coordinated sequences of events or actions (e.g., following the natural, sequential flow of conversations, and other social interactions or understanding the causal relationship between behaviors and their consequences). Within the three broad categories of autism symptoms [152], many can be conceptualized as impairments of preparatory functioning. Symptoms that fall under the categories of impaired social interaction and impaired communication may be due, at least in part, to difficulty processing and responding to unpredictable sequences of actions or sensory events (e.g., social overtures or conversations). Furthermore, restricted and repetitive interests and behaviors, resistance to change, and insistence on sameness may emerge from attempts to adapt to such unpredictable situations by reducing the environment to something more predictable, for in a world where the ability to prepare for change is impaired, repetition may be particularly reinforcing. Therefore, through its proposed role in preparatory functioning, cerebellar abnormality may not only be a common feature of autism, but a key contributor to some of its hallmark symptoms.

This contribution of cerebellar dysfunction to autism symptoms may in fact be evidenced by a few recent investigations. For instance, in a study of children with autism exploring a structured environment, reduced exploration, and repetitive behaviors were significantly correlated with the size of cerebellar vermis lobules VI–VII (but not with total brain or frontal lobe volumes) [153]. Also, using discriminant function analysis, Akshoomoff and colleagues recently showed that cerebellar white matter volume and cerebellar vermis size were particularly strong predictors of diagnosis in the autism spectrum [57]. And in another recent MRI investigation that compared monozygotic twin pairs clinically concordant for autism to clinically discordant pairs, only the clinically concordant pairs showed concordance in cerebellar volume [154]. Thus, the degree of cerebellar abnormality may well be of critical importance to certain behavioral manifestations of autism. 7. The implications of cerebellar pathology for understanding the etiology of autism The etiology of autism is unknown. Family and twin studies suggest a strong yet complex genetic component [155], and linkage and association studies have pointed to an extensive list of candidate genes [156]. The confluence of evidence for cerebellar abnormality in autism suggests that investigators attempting to determine the genetic basis of autism might consider genes that are involved in cerebellar development. Mouse studies have identified a number of such genes [157], one of which is Engrailed 2 (En2). Misexpression or loss of function of the En2 gene in mice results in hypoplastic cerebella with reduced numbers of Purkinje neurons [158], suggesting a possible connection with autism. Gharani and colleagues thus examined En2 as a susceptibility locus for autism spectrum disorders, and found a significant family-based association in a sample of 167 families from the Autism Genetic Resource Exchange (AGRE) [159]. This finding was recently replicated with an additional sample of 222 AGRE families and 129 other families [160]. These finding suggests that genes that play a role in cerebellar development indeed may be contributing factors to the etiology of autism, and examination of other genes involved in cerebellar development is warranted. Despite strong evidence for genetic contributions to autism, environmental factors remain a concern, particularly in light of apparent increases in autism prevalence observed over the past decade [161]. These increases are at least partly attributable to increased awareness of autism spectrum disorders, evolving diagnostic criteria, and more complete ascertainment. Nevertheless, they have also raised suspicion that exposures to chemical or biological agents during critical periods of development, either prenatally or postnatally, may too have contributed to the apparent rise of autism. Agents that have raised particular concern include prenatal viral infection [162], mercury and other heavy metals [163,164], and pharmaceuticals such as thalidomide and valproic acid [163,165]. A full discussion of the data

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supporting or refuting the various environmental agents that may be involved in autism is beyond the scope of this review. Furthermore, as noted by Lawler et al. [163], our ability to understand the role of environmental factors in the etiology of autism is likely to be advanced if we transcend the gene-versus-environment argument and move toward investigations of how genetic aberrations may leave an individual susceptible to environmental factors that may be related to autism. In that context, it should be noted that Purkinje cells are highly vulnerable cells [166,167], and a wide range of abnormal physical conditions (e.g., hypoxia and ischemia) [168,169] and environmental insults (e.g., diseases, deficiencies, and toxic exposures) [170–176] can lead to a loss of these neurons. Although the cerebellum is not the only brain region impacted by these factors, it is still interesting to recognize the overlap between the agents that have raised concern due their apparent association with autism and those that are known to be toxic to the cerebellum; viral infection, mercury, and valproic acid have all been associated with Purkinje neuron reduction in animal studies [171,172,174,176]. Coupled with evidence that at least some Purkinje neuron reduction in autism may be occurring later in development than previously thought, these findings call for further studies exploring whether specific environmental factors play a role in the development of autism, and whether this relationship may be mediated by a loss of Purkinje neurons. 8. Conclusion The cerebellum is one of the most common sites of anatomic abnormality in autism. In recent years, neurofunctional investigations have revealed a pattern of cerebellar dysfunction that is compatible with previously observed anatomic defects. Cerebellar pathology in the context of a developing brain may influence the behaviors and symptoms of autism via at least two paths. Through a direct route, the pathology will lead to cerebellar dysfunction, which, due to the cerebellar role in diverse cognitive, social, and emotional functions, is a likely contributor to some of the hallmark symptoms of autism. Additionally, through an indirect route, cerebellar pathology is hypothesized to influence the abnormal development of anatomic and functional connections between the cerebellum and other brain regions and thereby impact functioning in diverse brain systems. This complex relationship among cerebellar pathology, abnormal cerebellar connectivity, and the behaviors and symptoms of autism is the target of our future investigations. It is our hope that findings from such studies will contribute to a richer understanding of the pathophysiology of autism. References [1] Anderson GM. Serotonin in autism. In: Bauman ML, Kemper TL, editors. The neurobiology of autism. Baltimore: The Johns Hopkins University Press; 2005. p. 303–18.

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