RESEARCH ARTICLE Anatomy and Aging of the Amygdala and Hippocampus in Autism Spectrum Disorder: An In Vivo Magnetic Resonance

Imaging Study of Asperger Syndrome

Clodagh M. Murphy, Q. Deeley, E.M. Daly, C. Ecker, F.M. OBrien, B. Hallahan, E. Loth, F. Toal, S. Reed, S. Hales, D.M. Robertson, M.C. Craig, D. Mullins, G.J. Barker, T. Lavender, P. Johnston, K.C. Murphy, and D.G. Murphy
It has been proposed that people with autism spectrum disorder (ASD) have abnormal morphometry and development of the amygdala and hippocampus (AH). However, previous reports are inconsistent, perhaps because they included people of different ASD diagnoses, ages, and health. We compared, using magnetic resonance imaging, the in vivo anatomy of the AH in 32 healthy individuals with Asperger syndrome (1247 years) and 32 healthy controls who did not differ significantly in age or IQ. We measured bulk (gray1white matter) volume of the AH using manual tracing (MEASURE). We first compared the volume of AH between individuals with Asperger syndrome and controls and then investigated age-related differences. We compared differences in anatomy before, and after, correcting for whole brain size. There was no significant between group differences in whole brain volume. However, individuals with Asperger syndrome had a significantly larger raw bulk volume of total (Po0.01), right (Po0.01), and left amygdala (Po0.05); and when corrected for overall brain size, total (Po0.05), and right amygdala (Po0.01). There was a significant group difference in aging of left amygdala; controls, but not individuals with Asperger syndrome, had a significant age-related increase in volume (r 5 0.486, Po0.01, and r 5 0.007, P 5 0.97, z 5 1.995). There were no significant group differences in volume or agerelated effects in hippocampus. Individuals with Asperger syndrome have significant differences from controls in bulk volume and aging of the amygdala. Autism Res 2011,4:xxxxxx 2011International International Society Society for for Autism Autism Research, 2012, 5 : 312. . &2011 Wiley Periodicals, Inc. Keywords: Asperger syndrome; autism; amygdala; hippocampus; age

Introduction
It has been suggested that the biological basis of autism spectrum disorder (ASD) may include anatomical abnormalities in the amygdala and hippocampus [Baron-Cohen, Ring et al., 2000; Schultz, 2005]. To date, however, research findings have been variable [Cody, Pelphrey et al., 2002]. Classic lesion studies [e.g. see Kluver, 1939; Rosvold, Mirsky et al., 1954] and a review [Bachevalier, 1994] have demonstrated a crucial role of the amygdala in primate social and emotional behaviors. In humans, the amygdala contributes to reward and motivation [Baxter & Murray, 2002; Gottfried, ODoherty et al., 2003; Holland & Gallagher, 2004], processing of faces [Haxby, Hoffman, et al., 2002], recognition of emotions [Adolphs, 2003], emotional memory [McGaugh, 2004], detecting threat

[Amaral, 2003; Amaral & Bauman, 2003], fear and anxiety [Davis, Walker et al., 2003; LeDoux, 2003], and modulates social brain regions [such as fusiformextrastriate cortices [Deeley, Daly et al., 2007]. The amygdala also interacts reciprocally with the hippocampus to encode emotional memories [Richardson, Strange et al., 2004]. The hippocampus, in addition to its role in emotional processing and memory [Bannerman, Rawlins et al., 2004; Vargha-Khadem, Gadian et al., 1997], is also involved in spatial learning [Maguire, Gadian et al., 2000] and verbal novelty detection [Grunwald, Lehnertz et al., 1998]. Given the difficulties of individuals with ASD in responding to, and processing, socioemotional cues, it has been suggested that its biological basis includes abnormalities in the development and function of limbic structures, including the amygdala and

Additional Supporting Information may be found in the online version of this article. Clodagh M. Murphy and Q. Deeley are first authors. From the Kings College London, Institute of Psychiatry, Department of Forensic and Neurodevelopmental Science, London, United Kingdom (C.M.M., Q.D., E.M.D., C.E., B.H., E.L., F.T., S.R., S.H., D.M.R., M.C.C., D.M., T.L., P.J., D.G.M.); Royal College of Surgeons in Ireland, Dublin, Ireland (F.M.O., K.C.M.); Department of Clinical Neuroscience, Kings College London, Institute of Psychiatry, Centre for Neuroimaging Sciences, London, United Kingdom (G.J.B.) Received October 6, 2010; accepted for publication July 20, 2011 Address for correspondence and reprints: Clodagh M. Murphy, Department of Forensic and Neurodevelopmental Science, Institute of Psychiatry, De Crespigny Park, PO Box 50, Denmark Hill, London SE5 8AF, UK. E-mail: clodagh.m.murphy@kcl.ac.uk Grant sponsor: Medical Research Council. September 2011 in Wiley Online Library (wileyonlinelibrary.com) Published online 21 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/aur.227 & 2011 International Society for Autism Research, Wiley Periodicals, Inc.

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hippocampus [Amaral et al., 2003; Baron-Cohen et al., 2000; Damasio & Maurer, 1978; Salmond, Ashburner et al., 2005; Schultz, 2005; Sweeten, Posey, et al., 2002]. Some postmortem studies reported that people with ASD have increased neuronal cell density, but reduced cell size, in both amygdala and hippocampus [e.g. see reviews by Bauman & Kemper, 2005; Palmen, van Engeland et al., 2004], and reduced dendritic branching in hippocampal CA4 and CA1 neurons [Raymond, Bauman et al., 1996]. However, it has been suggested that neuronal density is not the most sensitive indicator of neuropathology, and may measure postmortem changes in tissue volume rather than actual cell number [Haug, Goldner et al., 1994; West, Slomianka et al., 1991]. Hence, stereological microscopy has been used to quantify the number, volume, and area of neurons in postmortem amygdala [Schumann & Amaral, 2005]. In the only postmortem stereological investigation of amygdala in autism, no differences were detected in amygdala volume or cell size, but a significant reduction in number of neurons was noted [Schumann & Amaral, 2006]. While this awaits replication, it is suggestive of quantifiable differences in the amygdala of individuals with ASD. These neuropathological studies were crucial first steps, but are subject to potential confounds, such as postmortem delay. Hence, other investigations have used magnetic resonance imaging (MRI) to measure in vivo anatomy. However, previous MRI studies have yielded conflicting results. In comparison with healthy controls, the volume of amygdala in people with ASD has been reported as: not different [Bigler, Tate et al., 2003; Haznedar, Buchsbaum et al., 2000; Palmen, Durston et al., 2006], decreased [Aylward, Minshew et al., 1999; Pierce, Muller et al., 2001], and increased [Schumann, Hamstra et al., 2004; Sparks, Friedman et al., 2002]. Likewise, hippocampal volume has been reported as: not different [Bigler et al., 2003; Haznedar et al., 2000], decreased [Aylward et al., 1999; Herbert, Ziegler et al., 2003; Nicolson, DeVito et al., 2006; Saitoh, Karns, et al., 2001], and increased [Schumann et al., 2004; Sparks et al., 2002]. Studies of the volume of amygdala and hippocampus in family members of individuals with ASD have also yielded conflicting results: with reports of no difference in temporal lobe volume [Palmen, Hulshoff Pol et al., 2005], a larger hippocampus [Rojas, Smith et al., 2004], and a smaller amygdala [Dalton, Nacewicz et al., 2007]. The results of the previous in vivo volumetric MRI studies may be inconsistent as they included relatively small and clinically heterogeneous groups, which differed in age, autistic subtype, IQ, exclusion criteria (e.g. presence/absence of epilepsy), and methods of measurement [Ecker, Marquand et al., 2010]. For example, age may be an important confound, as there is preliminary evidence for age-specific limbic brain abnormalities in people with ASD. This includes reports that young

children with autism [15 years old, Sparks et al., 2002; Schumann, Barnes et al., 2009; 7.512.5 years old, Schumann et al., 2004] have an enlarged amygdala, but adolescents (12.7518.5 years old) with autism do not [Schumann et al., 2004]. This putative amygdala enlargement in autism at an early stage of development contrasts with the later amygdala enlargement seen in typically developing adolescents [Giedd, Vaituzis et al., 1996]. Initial investigations of age-related differences in the hippocampus of people with autism have also produced variable findings, with reports of an enlarged hippocampus in children and adolescents [Schumann et al., 2004; Groen, Teluij et al., 2010] but significant hypoplasia of the Area Dentate in adults and children [Saitoh et al., 2001]. Taken together, these preliminary findings suggest the importance of investigating the relationship between age and volume of the amygdala and hippocampus in individuals with ASD. In summary, it has been suggested that individuals with ASD have abnormal development of the amygdala and hippocampus. However, results from previous volumetric studies are variable. Included among the factors accounting for the variability in previous findings are the autistic subtype, age, IQ, and exclusion criteria (e.g. the presence/absence of epilepsy) of the people investigated. Also, no studies have compared (in vivo) age-related differences in the volume of amygdala and hippocampus in individuals with ASD from childhood into adulthood taking a continuous approach. That is, previous reports investigated the effect of age by comparing groups of people above and below particular ages; and only included children and adolescents. To overcome these difficulties, in this study we compared bulk volume and age-related differences in the amygdala and hippocampus in typically developing controls, with nonmentally retarded, physically healthy individuals with ASD aged 1247 years who were diagnosed using International Classification of Diseases, 10th Edition (ICD-10) research and Autism Diagnostic InterviewRevised (ADI-R) [Lord, Rutter et al., 1994] criteria. Moreover, we restricted the subtype of people with ASD that we investigated to those with Asperger syndrome and no history of epilepsy. We used hand tracing MEASURE [Barta, Dhingra et al., 1997] to measure the bulk volume (gray and white matter) of the amygdala and hippocampus. We tested the null hypotheses that both bulk volume and age-related changes in the amygdala and hippocampus do not differ significantly between typically developing controls and physically healthy individuals with Asperger syndrome.

Methods and Materials

Participants Participant demographics are summarized in Table I.

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Table I.

Participant Demographics
Controls, N 5 32 Asperger syndrome, (31 male, 1 N 5 32 (30 male, 2 female) female)

Age in years, mean7SD (range) 23711 (1049) 23711 (1247) FSIQ, mean7SD 111715 108713 Autistic Diagnostic Interview (ADI), N 5 32 (30 male, 2 female) Social (mean7SD) 1976 Communication (mean7SD) 1375 Restricted, repetitive behavior 673 (mean7SD) Autistic Diagnostic Observation Schedule (ADOS), N 5 6 (5 male, 1 female) Communication 571 Social 872 Total (social and communication) 1373 Stereotyped behaviors and 272 restricted interests

excluded if they had a comorbid psychiatric or medical disorder affecting brain development (e.g. epilepsy or psychosis), history of head injury, genetic disorder associated with autistic spectrum disorder (e.g. tuberous sclerosis or Fragile X syndrome), or an IQo70. Intelligence quotients were measured using the Wechsler Adult Intelligence ScaleRevised (WAIS-R) short form [Weschler, 1999]. MRI Data Acquisition MRI data were obtained using a GE Sigma 1.5T Neuro-optimised MR system (General Electric, Milwaukee, Wisconsin). Whole head coronal three-dimensional (3D) Inversion recovery prepared spoiled gradient echo (IR-SPGR images) (repetition time 5 13.8 msec, echo time 5 2.8 msec, inversion time 5 450 msec, 256 192 acquisition matrix, reconstructed as a 256 256 matrix, over a 220 220 field of view, 124 1.5-mm slices) were obtained from all subjects. ROI Approach Manual tracing of the bulk volume (i.e. both gray and white matter) of total, left, and right amygdala and hippocampus was carried out as previously described [Cutter, Daly et al., 2006] by a single rater. In brief, manual tracing was performed on IR-SPGR data sets, using both MEASURE Image Analysis software [Barta et al., 1997] (Johns Hopkins University, Baltimore, Maryland) and published anatomical definitions [van Amelsvoort, Daly et al., 2001]. The anatomical definitions of the regions were defined using a modified version of Watsons guidelines [Watson, Andermann et al., 1992]. Images were realigned parallel to the sylvian fissure. The most posterior hippocampus measurement started on the slice displaying the aqueduct of sylvius. Continuing anteriorly, the superior border of the hippocampus merges with the inferior border of the amygdala and the regions are delineated by white matter and the temporal horn of the lateral ventricle. If the delineation is unclear, the inferior border of the posterior amygdala is arbitrarily marked as a horizontal line drawn medially from the head of the temporal stem to the medial border of the amygdala; the hippocampus taken to be gray matter inferior to the line. The anterior boundary of the amygdala measurement was made on the slice with closure of the lateral sulcus (i.e. temporal lobe is joined to frontal lobe by the limen insulae). Total (i.e. left plus right) and individual left and right amygdala and hippocampus were measured. As manually traced regional brain volumes are affected by brain size, we also measured total whole brain volume (WBV). The volume of each region was calculated by multiplying the summed pixel cross-sectional areas measured by the slice thickness. The rater was blind to subject status and intrarater reliabilities were obtained for all regions identified. On a separate test data set of ten scans, the rater had achieved

Controls were recruited locally by advertisement. Individuals with Asperger syndrome were recruited with the support of the Medical Research Council (MRC) UK Autism Imaging Multicentre Study (A.I.M.S) (MRC AIMS) program. Diagnosis of Asperger syndrome was made by two Consultant Psychiatrists (D.G.M. and D.R.) and a Nurse Specialist (S.R.), using ICD-10 research diagnostic criteria [1994]. All individuals fulfiled the diagnostic criteria of childhood autism, except that they did not have a history of language delay; they were therefore subtyped as having Asperger syndrome (International Classification of Diseases, 10th Edition ICD-10, F84.5). Where permission was given by individuals with Asperger syndrome, additional diagnostic measures were sought. The Autism Diagnostic Interview-Revised (ADI-R) [Lord et al., 1994] was completed for all 32 individuals with Asperger syndrome (13 young people and 19 adults). Thirty of the 32 reached the ADI algorithm cut-offs for autism in all three domains (social, communication, and restricted and stereotyped behaviors). One adult and one young person just failed to reach the ADI Communication algorithm cut-off for autism by one point. Six adults with Asperger syndrome agreed to complete the Autism Diagnostic Observation Schedule (ADOS) [Lord, Rutter et al., 1989], and were above cut-off for autism on all ADOS domains. Ethical approval was obtained from the Institute of Psychiatry and Bethlem and Maudsley Hospitals Trust Research Ethics Committee. All participants gave written informed consent, including each childs parent (where relevant). All participants underwent a structured physical and psychiatric examination to exclude comorbid medical and psychiatric disorders, and biochemical, hematologic or chromosomal abnormalities (including Fragile X syndrome) possibly affecting brain function. Participants were

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Table II.

Summary of Results: Mean AH Volumes and Significant Differences

Controls (N 5 32), mean7SD Asperger syndrome (N 5 32), mean7SD 1,117.507134.12 5.5870.66a 0.5070.07a 2.7870.36a 0.2570.04a 2.8070.34a 0.2570.03 5.8070.86 0.5270.07 3.0270.47 0.2770.04 2.7870.43 0.2570.04 t (62) 5 0.720, P 5 0.474 t (62) 5 2.863, P 5 0.006a,b t (62) 5 2.306, P 5 0.024a,b t (62) 5 3.300, P 5 0.002a,c t (62) 5 2.719, P 5 0.008a,b t (62) 5 2.053, P 5 0.044a,b t (62) 5 1.516, P 5 0.135 t (62) 5 0.623, P 5 0.535 t (62) 5 0.253, P 5 0.801 t (62) 5 0.691, P 5 0.492 t (62) 5 0.223, P 5 0.824 t (62) 5 0.456, P 5 0.650 t (62) 5 0.069, P 5 0.946

WBV Total WBV (ml) Amygdala Total Raw (ml) Corrected (%WBV) Right Raw (ml) Corrected (%WBV) Left Raw (ml) Corrected (%WBV) Hippocampus Total Raw (ml) Corrected (%WBV) Right Raw (ml) Corrected (%WBV) Left Raw (ml) Corrected (%WBV)

1,095.837104.80 5.1670.50a 0.4770.04a 2.5270.26a 0.2370.02a 2.6470.30a 0.2470.02 5.6770.78 0.5270.06 2.9470.44 0.2770.03 2.7370.38 0.2570.03

WBV, whole brain volume; AH, amygdala and hippocampal. a Significant between group difference on independent samples t-test. b Did not survive Bonferroni. c Survived Bonferroni.

intrarater and interrater reliabilities of r40.9 for all regions traced [Bartko & Carpenter, 1976]. In order to control for the relationship of brain volume and head size, amygdala and hippocampus volumes were expressed as raw (uncorrected) volumes, and when normalized, as a percentage of traced WBV. Statistical analyses were carried out on both raw and normalized brain volumes. Statistical Analysis SPSS 15.0 for Windows (SPSS Inc, Chicago, IL) was used for all statistical analyses. All demographic data (age and IQ) and brain volume measurements were normally distributed. We corrected for multiple comparisons (Bonferroni). Those regions that did survive a corrected Bonferroni (P 5 0.0038) are marked as a in Table II (Summary of results). Those regions that did not survive are marked as b. Level of statistical significance was defined as Po0.05. Results in the discussion are based on findings with a level of statistical significance of Po0.05. Because regional brain volumes may be affected by overall brain size, and some have suggested that people with ASD have differences in WBV (i.e. macrocephaly); statistical analyses were performed on both the raw bulk volume of the AHC, and after being expressed as a percentage of WBV (% WBV). To test our main hypothesis, we first compared people with Asperger syndrome with the controls. Between-group

differences in age, IQ, and brain volumes were calculated using independent-samples t-tests. We then investigated our subsidiary hypothesis, that there would be no group difference in the effect of age, using a correlational approach. Prior to comparing differences in age-related correlations, the data were tested for (i) linearity, (ii) independence, (iii) homoscedasticity, and (iv) normality error distribution. None of these assumptions was violated. Hence, we initially calculated Pearson productmoment correlations to investigate within-group age-related differences in AHC volumes. Then, in order to examine between-group differences in brain aging, we converted the relevant Pearsons r coefficient into Fishers Z-score to test the statistical significance of between group differences in correlations [Pallant, 2005]. In addition, we further investigated the relationship between age and total amygdala hippocampus volume by calculating a partial correlation to control for the effect of WBV. This did not change our results and even enhanced the effect size of our correlation coefficients.

Results
Effect of Group: Asperger Syndrome (N 5 32) and Controls (N 5 32) There was no significant between group difference in: age, Full-Scale IQ, WBV, or bulk volume of hippocampus.

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However, individuals with Asperger syndrome had a significantly larger uncorrected bulk volume of total (left plus right) (t (62) 5 2.863, Po0.01), right (t (62) 5 3.300, Po0.01), and left amygdala (t (62) 5 2.053, Po0.05) and in the corrected (% WBV) total (t (49.335) 5 2.306, Po0.05) and right amygdala (t (50.066) 5 2.719, Po0.01). Age and Volume of Amygdala and Hippocampus Increased corrected (% WBV) volume of total (r 5 0.483, Po0.01) and left amygdala (r 5 0.486, Po0.01) in controls, but not individuals with Asperger syndrome, was significantly positively correlated with increasing age. Further, controls had a significantly larger age-related increase in corrected (% WBV) volume of left amygdala than people with Asperger syndrome (z 5 1.995) (z 5 significant if r1.96 or Z1.96). No significant correlations between age and volume of hippocampus were identified in either group. Increasing age in controls, but not individuals with Asperger syndrome, was significantly negatively correlated with WBV (r 5 0.549, Po0.01). There was a trend toward controls having a significantly larger age-related decrease in WBV than people with Asperger syndrome (z 5 1.953; Fig. 1) (See Supplementary data Appendix Table A).

Discussion
We compared the bulk volume and age-related differences of the amygdala and hippocampus in physically healthy individuals with Asperger syndrome and controls, who did not differ significantly in age and IQ. Volume of the Amygdala and Hippocampus Our finding that people with Asperger syndrome have a significantly larger volume of total, left, and right amygdala and no difference in volume of hippocampus, is in agreement with earlier findings of amygdala enlargement in individuals with ASD as measured using VBM [Abell, Krams et al., 1999], ANALYZE [Howard, Cowell et al., 2000], and manual tracing [Mosconi, Cody-Hazlett et al., 2009]. It is also in agreement with previous reports of no significant difference in hippocampal volume as measured using ANALYZE [Howard et al., 2000] and manual tracing [Piven, Bailey et al., 1998]. Furthermore, our finding of an enlarged amygdala was true for both uncorrected (total, left, and right amygdala) and corrected (total and right amygdala) values. Our results are, nevertheless, in contrast to others who found no significant difference in bulk volume of amygdala [Haznedar et al., 2000; Palmen et al., 2006], or reduced volume of amygdala [Nacewicz, Dalton et al., 2006] as measured using manual tracing. Palmens study cohort was only very slightly smaller than ours but it included much younger (725 years old) groups of mixed diagnoses (both autism and Asperger syndrome). Similarly, Nacewiczs study included a younger (825 years old) group of 11 males with autism and 5 with Asperger syndrome or pervasive developmental disorder. Likewise, Haznedars study included ten individuals with autism and seven individuals with Asperger syndrome, who ranged in IQ from 55 to 125. In contrast, we included a slightly larger sample of people with Asperger syndrome from a wider age group, who had an IQ above 70, and they did not significantly differ from controls in IQ or age. Distinct differences between brain anatomy of individuals with Asperger and autism have been reported [McAlonan, Cheung et al., 2009; McAlonan, Suckling et al., 2008]. Such differences in autistic subtypes, age, and IQ may partially explain the differences in our findings. Development of the Amygdala and Hippocampus There are relatively few in vivo neuroimaging studies of normal amydala and hippocampal development, and most previous studies have been cross-sectional [Giedd et al., 1996]. Nevertheless, those that are available suggest that amygdala and hippocampus volume in typical human development changes with age, and that the

Figure 1. The relationship between age and corrected bulk volume of the left amygdala. NB: Controls had a significantly larger age-related difference in corrected bulk volume of the left amygdala (significant between group difference: z 5 1.995). r 5 correlation co-efficient; correlation is significant at the 0.01 level; strength of relationship: r 5 0.10.29 5 small, r 5 0.300.49 5 medium, and r 5 0.501.0 5 large; z 5 significant if o1.96 or 41.96; AS, Asperger syndrome.

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developmental trajectory may differ between amygdala and hippocampus. For example, in boys left amygdala volume increases between 4 and 18 years of age [Giedd et al., 1996], but changes relatively little in early adulthood [Pruessner, Collins, et al., 2001]. In contrast, male hippocampal volume does not change during adolescence [Giedd et al., 1996; Schumann et al., 2004], increases in young adulthood (1921 years old, [Suzuki, Hagino et al., 2005] but decreases in later life and old age [Liu, Lemieux et al., 2003; Murphy, DeCarli et al., 1996; Pruessner et al., 2001; Raz, Lindenberger et al., 2005]. However, rates of volume loss may be significantly less marked than in parietal and frontal areas [Grieve, Clark et al., 2005] suggesting that the timing of age-related changes may differ across brain regions. Schumanns [Schumann et al., 2004, 2009] investigation of the amygdala and hippocampus further highlights the importance of age in amygdala hippocampal development. Schumanns [Schumann et al., 2004] study suggested that differences in bulk volume of the amygdala and hippocampus (measured using manual tracing) between people with ASD and controls vary by age, diagnostic subtype, and anatomical location. Schumanns group first investigated their sample as one age group (718 years old) and found amygdala enlargement only in young people with low-functioning autism and hippocampal enlargement in young people with both low- and high-functioning autism. No differences were found in young people with Asperger syndrome. In controls, age positively correlated with amygdala volume, whereas no correlation between age and volume was found for individuals with autism or Asperger syndrome. As such, Schumann et al. further investigated the relationship between age and amydala by dividing their sample into children and adolescents; amygdala enlargement was specific to young children (712 years old) with autism (both low and high functioning), although a trend toward enlarged right amygdala was found in young children with Asperger syndrome (N 5 11, P 5 0.06). No significant difference in amygdala volume was found between adolescents (12.7518.5 years old) with autism, Asperger syndrome (N 5 13), and controls (N 5 11). While this study was a very valuable first step, subjects were separated into categorical age groups (i.e. children and adolescents), rather than using a continuous approach. However, neuroimaging evidence suggests that postnatal phases of brain development do not begin, or end, at specific chronological ages, but extend across age ranges [Giedd et al., 1996]. Age and Volume of the Amygdala and Hippocampus We therefore investigated the continuous relationship between age and volume of the amygdala and hippocampus from childhood into adulthood. We found a significantly

stronger association of age and left amygdala volume in controls relative to people with Asperger syndrome. Other studies have previously reported the presence of amygdala enlargement at an early age in childhood autism [Mosconi et al., 2009; Schumann et al., 2004, 2009; Sparks et al., 2002] and in adolescence [Groen et al., 2010]. Our findings extend that work and suggest that amygdala enlargement in individuals with Asperger syndrome persists into adulthood. Also, they suggest that although the amygdala volume of people with Asperger syndrome may be significantly enlarged at an early age, it does not continue to increase in volume with age. In contrast, our finding of increasing amygdala volume with age in controls is in keeping with previous reports of typical amygdala development [Giedd et al., 1996; Schumann et al., 2004]. In sum, our findings support earlier reports of amygdala enlargement in young children and adolescents with ASD [Mosconi et al., 2009; Schumann et al., 2004, 2009; Sparks et al., 2002] and also suggest that, in adolescent and adult males with Asperger syndrome, the amygdala continues to be significantly larger compared with age-matched controls. Furthermore, the development of the amygdala appeared to be different across groups. The amygdala of controls continued to increase with age, whereas the amygdala of individuals with Asperger syndrome did not. Our results suggest that both the volume and aging of the amygdala is significantly different in individuals with Asperger syndrome from controls. Future longitudinal studies are required to help clarify the development and aging of the amygdala and hippocampus across the lifespan; and to determine if this differs across the diagnostic subtypes of ASD. Potential Causes The causes of the subtle differences in morphometry and development of the amygdala and hippocampus that we found are not known, but probably include a complex geneenvironment interaction. For example, the amygdala plays a central role in fear and anxiety [Davis et al., 2003; LeDoux, 2003]; people with an ASD have a significantly increased risk of experiencing stress and anxiety disorders [for review, see White, Oswald et al., 2009]; and stress in turn impacts on amygdala development [McEwen, 2007]. For instance, in animals, acute and chronic stress increases dendrite growth in amygdala [Vyas, Mitra et al., 2002] and it has been suggested that stress-related hypertrophy of amygdala may contribute to anxiety [Vyas, Bernal et al., 2003]. Hence, it is possible that increased stress in individuals with Asperger syndrome may have contributed to, or result from, the increased volume of amygdala we found. However, genetic influences may also contribute to differences in amygdala and hippocampus volume. Preliminary support for this suggestion is provided by studies that reported

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differences in amygdala hippocampal volume within the relatives of individuals with ASD [Dalton et al., 2007; Rojas et al., 2004]. The specific genes/gene systems that may underpin the differences we found are unknown. Nevertheless, some have reported that cortical gray matter overgrowth, including within the temporal lobes, in young people [Wassink, Hazlett et al., 2007] (but not adults [Raznahan, Pugliese et al., 2009] with autism may be associated with functional variation in the serotonin transporter gene. This suggestion is supported by other work demonstrating that serotonin is crucial to brain development, including synaptic modeling, neurogenesis, dendritic organization and axon myelination [Whitaker-Azmitia, 2001], and abnormal brain serotonin synthesis has been found in children with autism [Chugani, Muzik et al., 1999]; and a significant reduction in brain 5-HT2A receptor density has been reported in adults with ASD [Murphy, Daly et al., 2006] and their relatives [Goldberg, Anderson et al., 2009]. Hence, genetically determined differences in serotonin metabolism may contribute toward our findings. There are however, other potential candidate genes. For example, there is increasing support from our group and others for an association between ASD and genetic variation in the glutamatergic and GABAergic systems; in nonautistic populations glutamate and GABA modify neuronal growth, connectivity and function, and we have previously reported that physically healthy, normal IQ, ve adults with ASD have a significant medication na increase in the glutamate/glutamine (Glx) concentration of amygdala and hippocampus complex [Page, Daly et al., 2006]. Future studies are required to replicate our work, and to investigate potential genetic and environmental factors associated with differences in morphometry of the amygdala and hippocampus. Finally, we do not suggest that people with Asperger syndrome only have differences in the anatomy of the amygdala and hippocampus, as both metabolic [Endo, Shioiri et al., 2007; Otsuka, Harada et al., 1999; Page et al., 2006] functional [Ashwin, Baron-Cohen, et al., 2007; Baron-Cohen, Ring et al., 1999; Critchley, Daly et al., 2000; Grelotti, Klin et al., 2005; Pierce et al., 2001], and subtle neural network differences [Ecker, Rocha-Rego et al., 2010] in the amygdala and hippocampus of people with ASD have also been reported. Hence, our findings add to the body of evidence that individuals with ASD have complex differences from controls in the structure, function, and metabolism of the amygdala and hippocampus. However, the relationship between these differences is unknown, and further research is required to clarify this. Limitations. This study was cross-sectional in design, and specific to individuals with Asperger syndrome. Hence, our findings describe age-related differences, not

individual changes across time, and our findings may not generalize to others within the autism spectrum. Nevertheless, we were able to examine age-related differences across an age-span of 39 years (1049 years of age), which would not have been practically possible using a longitudinal design. Also, the study was limited to a predominantly male sample and further studies are needed to ascertain whether these findings are also present in females [Craig, Zaman et al., 2007]. ADOS assessments were not available for all subjects. However, ADI-Rs were completed on all subjects (N 5 32), and ICD-10 research diagnoses were confirmed by two Consultant psychiatrists and a Nurse Specialist trained in the ADI and ADOS. We also carried out multiple statistical tests. However, we corrected (Bonferroni testing) for the increased risk of Type 1 errors due to multiple testing. Moreover, we used a relatively large sample of one clearly defined diagnostic group (healthy individuals with Asperger syndrome), who had no other medical history that may adversely affect brain development, and we controlled for head size. Finally, due to the nature of our study, we cannot determine if our findings of differences in volume of amygdala are a cause or a consequence of social communication difficulties and their associated stress and anxiety. Hence, our findings must be viewed as preliminary.

Conclusion
Individuals with Asperger syndrome have significant differences from controls in volume and aging of the amygdala. The cause of these differences is unknown, and most likely includes a complex interaction between a primary difference in brain development and abnormal interactions between the affected individual and their environment. Future studies of amygdala and hippocampus development, combining structural and functional imaging with clinical measures of behavior are warranted in ASD.

Acknowledgments We thank all the individuals and their families who participated in this study and our colleagues for their help in recruiting subjects. This study was supported by a grant from the Medical Research Council (MRC UK A.I.M.S research program). None of the authors reported any financial interests or potential conflicts of interests associated with this study. The authors declare no conflict of interest.

References
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