Anatomy and Aging of Amygdala and Hippocampus in ASD

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RESEARCH ARTICLE

Anatomy and Aging of the Amygdala and Hippocampus in AutismSpectrum Disorder: An In Vivo Magnetic Resonance Imaging Studyof 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 ofthe amygdala and hippocampus (AH). However, previous reports are inconsistent, perhaps because they included peopleof different ASD diagnoses, ages, and health. We compared, using magnetic resonance imaging, the in vivo anatomy ofthe AH in 32 healthy individuals with Asperger syndrome (1247 years) and 32 healthy controls who did not differsignificantly 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 investigatedage-related differences. We compared differences in anatomy before, and after, correcting for whole brain size. There wasno significant between group differences in whole brain volume. However, individuals with Asperger syndrome had asignificantly larger raw bulk volume of total (Po0.01), right (Po0.01), and left amygdala (Po0.05); and when correctedfor 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(r50.486, Po0.01, and r50.007, P50.97, z51.995). There were no significant group differences in volume or age-related effects in hippocampus. Individuals with Asperger syndrome have significant differences from controls in bulkvolume and aging of the amygdala. Autism Res 2011,4:xxxxxx. & 2011 International Society for Autism Research,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 abnorm-

alities in the amygdala and hippocampus [Baron-Cohen,

Ring et al., 2000; Schultz, 2005]. To date, however, researchfindings 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 amygda-

la 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 fusiform-

extrastriate cortices [Deeley, Daly et al., 2007]. The

amygdala also interacts reciprocally with the hippocam-

pus to encode emotional memories [Richardson, Strangeet 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

INSAR Autism Research 4: 110, 2011 1

Received October 6, 2010; accepted for publication July 20, 2011

Published online in Wiley Online Library (wileyonlinelibrary.com)

DOI: 10.1002/aur.227

&2011 International Society for Autism Research, Wiley Periodicals, Inc.

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.)

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: [email protected]

Grant sponsor: Medical Research Council.

35: 312, 2012 3

21 September 2011 in Wiley Online Library (wileyonlinelibrary.com)

2012,5:312. 2011 International Society for Autism Research,


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Controls were recruited locally by advertisement.

Individuals with Asperger syndrome were recruited withthe 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 ofPsychiatry 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 syn-

drome) possibly affecting brain function. Participants were

excluded if they had a comorbid psychiatric or medical

disorder affecting brain development (e.g. epilepsy or

psychosis), history of head injury, genetic disorder asso-

ciated with autistic spectrum disorder (e.g. tuberous

sclerosis or Fragile X syndrome), or an IQo70. Intelligence

quotients were measured using the Wechsler Adult Intelli-

gence 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 time513.8 msec, echo time52.8msec,

inversion time5450 msec, 256192 acquisition matrix,

reconstructed as a 256256 matrix, over a 220220 field of

view, 1241.5-mm slices) were obtained from all subjects.

ROI Approach

Manual tracing of the bulk volume (i.e. both gray andwhite 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 defini-

tions 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 hippo-

campus 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 plusright) and individual left and right amygdala and

hippocampus were measured. As manually traced regio-

nal 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 thick-

ness. 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

Table I. Participant Demographics

Controls,N5 32

(31 male, 1

female)

Asperger syndrome,

N5 32 (30 male, 2

female)

Age in years, mean7SD (range) 23711 (1049) 23711 (1247)

FSIQ, mean7SD 111715 108713

Autistic Diagnostic Interview (ADI), N532 (30 male, 2 female)

Social (mean7SD) 1976

Communication (mean7SD) 1375

Restricted, repetitive behavior

(mean7SD)

673

Autistic Diagnostic Observation

Schedule (ADOS), N56 (5

male, 1 female)

Communication 571

Social 872

Total (social and communication) 1373

Stereotyped behaviors and

restricted interests

272

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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 normalizedbrain 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 (P50.0038)

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 ofstatistical significance ofPo0.05.

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 differ-

ences in age-related correlations, the data were tested for

(i) linearity, (ii) independence, (iii) homoscedasticity, and(iv) normality error distribution. None of these assump-

tions 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 enhancedthe effect size of our correlation coefficients.

ResultsEffect of Group: Asperger Syndrome (N532) and Controls(N532)

There was no significant between group difference in:

age, Full-Scale IQ, WBV, or bulk volume of hippocampus.

Table II. Summary of Results: Mean AH Volumes and Significant Differences

Controls (N5 32), mean7SD Asperger syndrome (N5 32), mean7SD

WBV

Total WBV (ml) 1,095.837104.80 1,117.507134.12 t(62)50.720, P5 0.474

Amygdala

Total

Raw (ml) 5.1670.50a 5.5870.66a t(62)52.863, P5 0.006a,b

Corrected (%WBV) 0.4770.04a 0.5070.07a t(62)52.306, P5 0.024a,b

Right

Raw (ml) 2.5270.26a 2.7870.36a t(62)53.300, P5 0.002a,c

Corrected (%WBV) 0.2370.02a 0.2570.04a t(62)52.719, P5 0.008a,b

Left

Raw (ml) 2.6470.30a 2.8070.34a t(62)52.053, P5 0.044a,b

Corrected (%WBV) 0.2470.02 0.2570.03 t(62)51.516, P5 0.135

Hippocampus

Total

Raw (ml) 5.6770.78 5.8070.86 t(62)50.623, P5 0.535

Corrected (%WBV) 0.5270.06 0.5270.07 t(62)50.253, P5 0.801

Right

Raw (ml) 2.9470.44 3.0270.47 t(62)50.691, P5 0.492

Corrected (%WBV) 0.2770.03 0.2770.04 t(62)50.223, P5 0.824

Left

Raw (ml) 2.7370.38 2.7870.43 t(62)50.456, P5 0.650

Corrected (%WBV) 0.2570.03 0.2570.04 t(62)50.069, P5 0.946

WBV, whole brain volume; AH, amygdala and hippocampal.aSignificant between group difference on independent samplest-test.bDid not survive Bonferroni.cSurvived Bonferroni.

4 Murphy et al./Anatomy and aging of the amygdala INSAR6


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However, individuals with Asperger syndrome had a

significantly larger uncorrected bulk volume of total

(left plus right) (t (62)52.863, Po0.01), right

(t (62)53.300, Po0.01), and left amygdala

(t(62)52.053, Po0.05) and in the corrected (% WBV)

total (t (49.335) 52.306, Po0.05) and right amygdala

(t(50.066)52.719, Po0.01).

Age and Volume of Amygdala and Hippocampus

Increased corrected (% WBV) volume of total (r50.483,

Po0.01) and left amygdala (r50.486, Po0.01) in con-

trols, 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 (z51.995) (z5 sig-

nificant ifr1.96 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 corre-lated with WBV (r50.549, Po0.01). There was a trend

toward controls having a significantly larger age-related

decrease in WBV than people with Asperger syndrome

(z51.953; Fig. 1) (See Supplementary data Appendix

Table A).

Discussion

We compared the bulk volume and age-related differ-

ences 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 hippo-

campal 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). Simi-

larly, Nacewiczs study included a younger (825 years

old) group of 11 males with autism and 5 with Aspergersyndrome 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: z51.995).

r5 correlation co-efficient; correlation is significant at the

0.01 level; strength of relationship: r50.10.295 small,

r50.300.495medium, and r50.501.05 large; z5 signifi-

cant if o1.96 or41.96; 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] investiga-

tion of the amygdala and hippocampus further high-

lights 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 manualtracing) 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 enlarge-

ment 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

(N511, P50.06). No significant difference in amygdala

volume was found between adolescents (12.7518.5 years

old) with autism, Asperger syndrome (N513), and

controls (N511). 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, neuroimagingevidence suggests that postnatal phases of brain devel-

opment 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 adult-

hood. Also, they suggest that although the amygdalavolume 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 develop-

ment [Giedd et al., 1996; Schumann et al., 2004]. In sum,

our findings support earlier reports of amygdala enlarge-

ment 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 syn-

drome 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 amyg-

dala 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 develop-

ment [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 syn-

drome 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 find-ings. 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,

medication nave adults with ASD have a significant

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 environ-

mental factors associated with differences in morphome-

try 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 hippo-

campus 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 relation-

ship 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 notgeneralize to others within the autism spectrum.Nevertheless, we were able to examine age-relateddifferences across an age-span of 39 years (1049 years ofage), which would not have been practically possible usinga longitudinal design. Also, the study was limited to apredominantly male sample and further studies are neededto ascertain whether these findings are also present infemales [Craig, Zaman et al., 2007]. ADOS assessmentswere not available for all subjects. However, ADI-Rs werecompleted on all subjects (N532), and ICD-10 researchdiagnoses were confirmed by two Consultant psychiatristsand 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 ofamygdala 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.

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Anatomy and Aging of Amygdala and Hippocampus in ASD