Histological and Magnetic Resonance Imaging

Assessment of Cortical Layering and Thickness

in Autism Spectrum Disorders
Jeffrey J. Hutsler, Tiffany Love, and Hong Zhang
Background: Qualitative reports of the cerebral cortex in a small number of autism spectrum disorder (ASD) cases have suggested
an increase in thickness and disruptions in migration and lamination patterns.
Methods: We examined postmortem ASD individuals and age-matched controls using magnetic resonance imaging (MRI) to evaluate
total cortical thickness, and histological samples to evaluate the pattern of cortical layering.
Results: Overall, thickness measures from ASD subjects were equivalent to control cases. Individual regions showed marginal but
nonsignificant thickness differences in the temporal lobes. Cortical thickness values in ASD subjects decreased significantly with age.
Quantitative examination of proportional layer thickness in histological sections indicated that the pattern of cortical layering was
largely undisturbed, while qualitative examination of these same samples revealed evidence of cell clustering and supernumerary cells
in layer I and the subplate. These features were not severe and were never found in a majority of cases.
Conclusions: These findings support limited disturbances in cortical cell patterning, but do not indicate a major deficit in the orderly
migration of cortical neuroblasts during development, or their subsequent aggregation into the laminar pattern found in typically
developing individuals.

Key Words: Frontal lobe, morphometry, neuropathology, parietal 1996). Additionally, MRI studies reporting polymicrogyria, macro-
lobe, postmortem, temporal lobe gyria, and schizencephaly in small subgroups of ASD subjects point
toward a potential deficit in neuronal migration (Piven et al 1990).
Given the qualitative reports of laminar disturbances and

A
utism spectrum disorders (ASD) comprise a family of devel-
opmental disabilities characterized by behavioral abnormal-
ities that include language delays, difficulties with social
interactions, and repetitive or stereotyped behaviors (DSM-IV-TR;
American Psychiatric Association 2000). The underlying genetic and
biological bases of these disorders are widely accepted although
ill-specified (for reviews see Bailey et al 1995; Gutknecht 2001;
Lauritsen and Ewald 2001; Muhle et al 2004; Rutter 2000; Trottier et
al 1999; Veenstra-VanderWeele and Cook 2004). Alterations to the
neural structure in individuals with ASD have been identified in
several postmortem studies with the majority of these changes
found in noncortical regions, particularly the hippocampus, cere-
bellum, and medial temporal lobes (Bauman and Kemper 1985;
Bauman 1991; Bauman and Kemper 1994; Fatemi et al 2002;
Hashimoto et al 1995; Kemper and Bauman 1998; Palmen et al 2004;
Raymond et al 1996). Reports of microanatomical abnormalities in
the organization of cerebral cortex have been reported less fre-
quently, although there are indications of possible cortical thicken-
ing, layering abnormalities, and alterations to both inter- and
intracolumnar spacing (Bailey et al 1998; Casanova et al 2002a,
2002b; Kemper and Bauman 1993; Mukaetova-Ladinska et al 2004;
Williams et al 1980).
Abnormalities in cortical structure in individuals with ASD
have been found more frequently in magnetic resonance imag-
ing (MRI) studies, but conflicting results are not uncommon (for
reviews see Brambilla et al 2003; Palmen and Van Engeland
2004). Increased brain volumes have been reported repeatedly
and this result is especially consistent during early development
(Cody et al 2002; Courchesne et al 2001, 2003; Piven et al 1995,
From the Department of Psychology (JJH, HZ) and Program in Neuroscience
(JJH, TL), University of Michigan, Ann Arbor, Michigan.
Address reprint requests to Jeffrey J. Hutsler, Ph.D., Psychology Department,
530 Church Street, University of Michigan, Ann Arbor, MI 48109-1043;
E-mail: hutsler@umich.edu.
Received March 9, 2005; revised November 10, 2005; accepted January 9,
2006.
cortical thickening in ASD (Bailey et al 1998; Mukaetova-Ladin-
ska et al 2004), as well as suggestions of migration difficulties
(Piven et al 1990), the present study sought to quantitatively
evaluate cortical layering and thickness using two parallel meth-
odological approaches. Structural MRIs of postmortem brains
were used to assess total cortical thickness in multiple locations
sampled from frontal, parietal, and temporal lobes. In addition,
histological sections were used to quantitatively assess the
pattern of cortical layering in the superior frontal gyrus (Brod-
mann’s Area [BA] 9), the superior parietal lobule (BA 7), and the
middle temporal gyrus (BA 21).
Methods and Materials
All subject material utilized in these studies was acquired with
the assistance of the Autism Tissue Program (ATP), and the
procedures for tissue collection, case identification, and process-
ing were approved by the Medical Internal Review Board at the
University of Michigan. Cortical tissue samples were acquired
from eight postmortem ASD males and eight age- and sex-
matched control subjects from the Harvard Brain Tissue Resource
Center, the Brain and Tissue Bank for Developmental Disorders
at the University of Miami, and the Brain and Tissue Bank at the
University of Maryland. In addition, MRI scans of postmortem
brains from eight ASD subjects and eight age- and sex-matched
controls were also acquired from the University of California,
Davis (Schumann et al 2001). In five of the individuals with ASD
and three of the control cases, both MRI image sets and
histological samples from the appropriate cortical locations were
available (see Table 1). Identification of the ASD cases was made
based upon available medical and psychological records, and the
diagnosis was subsequently confirmed with a Revised Autism
Diagnostic Inventory (ADI-R; Lord et al 1994) administered to the
parents or primary caregiver following tissue donation. In the
present group, all cases met the criteria for a diagnosis of autism
except for case 008 (see Table 1), who did not meet the cutoff in the
area of communication and was classified as Asperger’s syndrome.

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doi:10.1016/j.biopsych.2006.01.015 © 2007 Society of Biological Psychiatry
450 BIOL PSYCHIATRY 2007;61:449 – 457 J.J. Hutsler et al

Table 1. Case Information

Case ID Group Age PMI (hrs) Brain Weight (g) Cause of Death

MRI
001 ASD 15 4 nab Hepatic Disease-Suspected
002 ASD 16 48 1990 Coronary Arrest
003a ASD 20 24 1140 Motor Vehicle Accident
004a ASD 25 26 1220 Probable Seizure
005a ASD 27 8 1720 Drowning
006 ASD 29 30 1420 Neuroleptic Malignant Synd.
007a ASD 44 31 1530 Myocardial Infarction
008a ASD 45 20 1370 Cardiac Arrest
Means 27.63 23.88 1484.29
009 Control 14 18 1440 Asphyxiation
010 Control 17 22 1440 Motor Vehicle Accident
011 Control 18 na 1520 Drowning
012 Control 24 19 1570 Gunshot Wound
013a Control 27 16 na Arteriosclerosis
014a Control 27 21 1330 Asphyxiation
015a Control 43 24 1450 Cardiovascular Disease
016 Control 45 11 1520 Cardiac Arrest
Means 26.88 18.71 1467.14
Cortical Tissue
017 ASD 10 30 1000 Drowning
003a ASD 20 24 1140 Motor Vehicle Accident
004a ASD 25 26 1220 Probable Seizure
005a ASD 27 8 1720 Drowning
018 ASD 29 24 1340 Cardiac Arrest
019 ASD 32 21 1710 Congestive Heart Failure
007a ASD 44 31 1530 Myocardial Infarction
008a ASD 45 20 1370 Cardiac Arrest
Means 29.00 23.00 1378.75
020 Control 11 20 1420 Internal Bleeding
021 Control 17 7 na Motor Vehicle Accident
022 Control 22 24 1580 Motor Vehicle Accident
013a Control 27 21 1330 Asphyxiation
014a Control 27 16 na Arteriosclerosis
023 Control 43 24 1350 Gunshot Wound
015a Control 43 24 1450 Cardiovascular Disease
024 Control 51 24 1600 Coronary Thrombosis
Means 30.13 19.94 1455.00
PMI, postmortein interval; MRI, magnetic resonance imaging; ASD, autistic spectrum disorder.
a
Indicates cases used in both the MRI and histology studies.
b
na - total brain weight was not available.

MRI Procedures by request) running on a Mac OSX workstation. Briefly, the

Coronal proton density (PD)-weighted fast spin-echo images
(repetition time [TR] ⫽ 6700 msec, time-to-echo [TE] ⫽ 8.23
msec, 16-cm field of view [FOV], 1.6 mm slice thickness) were
acquired using a General Electric 1.5 T Signa System (GE Medical
Systems, Milwaukee, Wisconsin). The image sets were trans-
ferred to a Pentium III workstation and processed using the
ANALYZE™ software package (Robb and Hanson 1991). Data
sets were resliced into isotropic voxels (.625 mm3) and then
realigned and filtered to correct for RF inhomogeneity (Brink-
mann et al 1996) and noise (Sapiro and Tannenbaum 1993).
Since skull-stripping procedures are not required in postmortem
cases, a semi-automated thresholding procedure was utilized to
extract brain from nonbrain regions. To segment the cortical gray
matter from the resulting images, a thresholding value that
represented the minima between the white matter and gray
matter peaks was acquired from an intensity histogram in each
case (Pham et al 2000). Cortical thickness measurements were
collected using software developed in our laboratory (available
operator, who was blind to diagnosis, selected a point on the
crown of the gyrus of interest. The thickness of the cortex at
the selected point is the shortest distance to a pixel at the
gray/white matter boundary in three dimensions. Highly
convoluted cortical regions were avoided. The linear distance
between the outer edges of the two resulting voxels, as well as
the Cartesian coordinates of the surface voxel and gray/white
boundary voxel, were recorded.
In most postmortem cases, scans were only taken from a
single hemisphere. For both the control and ASD groups, mea-
surements were taken from the right hemisphere in three cases
and the left hemisphere in five cases. Measures were taken from
three frontal locations, four parietal locations, and three temporal
lobe locations in the axial plane. Multiple measurements were
taken from each gyrus throughout its extent in five to seven
different planes spaced 5 mm apart. Locations were identified
based upon morphology and the center of the gyral crown as it
appears on the right hemisphere of the Talairach brain are given

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J.J. Hutsler et al
for each region. Prefrontal cortex measures were taken from
dorsal axial images, where the superior frontal gyrus (Talairach:
15, 52, 35), the superior frontal sulcus, and the middle frontal
gyrus (32, 41, 35), could easily be identified. The precentral (51,
⫺2, 45) and postcentral gyrus (51, ⫺17, 45) were sampled from
axial planes located 15 to 45 mm from the dorsal surface of the
brain where the central sulcus could be visualized. Measures of
the superior parietal lobule (30, ⫺66, 45) were collected 20 to 45
mm from the dorsal boundary of the cortex and posterior to the
intraparietal sulcus. The supramarginal (55, ⫺48, 35) and angular
(50, ⫺62, 35) gyrus measures were inferior to these sampling
sites at locations anterior and posterior to the intraparietal sulcus.
Temporal lobe measures from the superior (35, 14, ⫺32), middle
(44, 6, ⫺32), and inferior (49, ⫺7, ⫺32) temporal gyri were
collected from axial images 15 to 40 millimeters from the ventral
surface of the cortex near the anterior boundary of the temporal
lobe. Where regional identification varied from standardized
conventions, three-dimensional models of the cortical surface
were used to verify the sample locations.
Microanatomical Procedures
Cortical tissue samples from the lateral surface of the superior
temporal gyrus (BA 21), dorsolateral frontal lobes (BA9), and
dorsal parietal lobes (BA7) were used to evaluate cortical layer-
ing. These blocks correspond to the middle temporal gyrus, the
superior frontal gyrus, and the superior parietal lobule in our MRI
sample. Blocks 5 to 20 mm thick were recut orthogonal to the
cortical layers from larger blocks acquired from regional tissue
banks. Blocks were initially stored in 10% formalin and trans-
ferred to fresh 4% paraformaldehyde for a period of two weeks
prior to processing. Blocks were cryoprotected in 25% sucrose
for 72 hours and cut into 50 ␮m sections on a freezing mic-
rotome. Every sixth tissue section was mounted onto a gelatin-
subbed slide and air dried prior to Nissl staining. Sections were
dehydrated in a graded series of alcohols and cleared in toluene.
Sections were then rehydrated and transferred for ten min into
BIOL PSYCHIATRY 2007;61:449 – 457 451
Figure 1. Photomicrographs of Nissl-stained sec-
tions and examples of typical cell patterning abnor-
malities in autistic spectrum disorder (ASD) individ-
uals. (A) Typical appearance and boundaries of
layers I – VI in the superior frontal gyrus (Brodmann’s
Area [BA]9) of one individual with ASD. (B) An exam-
ple of supernumerary neurons in Layer I and an un-
even or “sawtooth” boundary with layer II. (C) Layers
III, IV, and V showing uneven lamination due to the
presence of cell clumping/dysplasia (outlined re-
gion). (D) Higher magnification view of region out-
lined in panel C. Such examples of neuronal clump-
ing were one of the more frequently encountered
patterning abnormalities in these ASD subjects.
(E) Supernumerary neurons within the subplate re-
sult in an indistinct lower boundary in layer VI. Scale
bars: A and B ⫽ 100 ␮m; C, D, and E ⫽ 50 ␮m.
.25% thionin and .4% sodium acetate diluted in .1M phosphate
buffer. Excess stain was cleared in water prior to dehydrating and
clearing in toluene. All sections were coverslipped in Permount.
To quantitatively assess cortical layering in each subject, four
equally spaced sections were selected from each block with a
random starting order. Measurements were taken from cortical
locations that were neither obviously deformed by bending or by
a tangential plane of section. Cortical layering was evaluated
using a Leica microscope (Leica Microsystems, Bannockburn,
Illinois) at a final magnification of 25⫻. All measures were taken
blind to the diagnosis of the subject and the region being
evaluated. After selecting a location, the operator traced a line
perpendicular to the surface of the cortex and parallel to the
cortical columns; this line was then segmented according to the
boundaries of the cortical layers (Figure 1A). Since these lines are
perpendicular to the cortical surface and laminae only within the
plane of the tissue section, they will typically overestimate
cortical thickness. Thus, raw thickness measures of individual
layers were transformed into proportions of total cortical thick-
ness. Prior to directly comparing the raw thickness measures
with those obtained from MRIs, histological measures were
corrected for shrinkage by assessing the distance between two
calibration marks made in each block prior to sectioning. Such
corrections can accommodate changes in tissue dimension dur-
ing processing, but not changes that may occur during the
postmortem interval prior to fixation.
Qualitative Analysis
Each individual case was examined for indications of focal
dysplasias, cell clumping within the layers, disturbances in
lamination boundaries, supernumerary neurons in layer I, an
uneven (“sawtooth”) boundary between layers I and II, and
supernumerary cells in the subplate region that might be associ-
ated with an indistinct gray/white matter boundary. If such
abnormalities were observed, judgments were made as to
whether they were mild (subsuming only small, circumscribed
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452 BIOL PSYCHIATRY 2007;61:449 – 457 J.J. Hutsler et al

Table 2. Frequency and Severity of Cortical Neuropathologies

ASD Subjects Control Subjects

Pathology Mild Moderate Severe Mild Moderate Severe

Supernumerary Layer I Neurons 1 (003) 2 (007,018) — 1 (013) — —

Cell Clumping/Dysplasia 4 (018,007,008,017) 2 (004,005) — 2 (014,024) — —
Lamination Disturbances 1 (017) 2 (007,005) — 2 (020,021) — —
Supernumerary Subplate Neurons 2 (005,008) 1 (018) — — 1 (013) —
Indistinct Cortical Boundary 2 (004) 1 (018) — 1 (013) — —
ASD, autistic spectrum disorder.

regions of cortex), moderate (found frequently along the cortical tween the groups. A multivariate analysis of variance comparing
ribbon), or severe (present in widespread regions of cortex). cortical thickness between ASD and control cases for the 10
Examinations were conducted on both ASD and control group individual cortical regions (see Figure 3) indicated that there was
subjects, and the examiner was blind to the diagnosis. no overall difference between the two subject groups (F10,5 ⫽
.789, ns). Since it is unclear whether the impact of ASD on cortex
Data Analysis is widespread or regional, we also examined the 10 regions
Measures of total cortical thickness taken from MRI images individually. Only regions from the middle temporal gyrus (F 1,14 ⫽
were evaluated with a multivariate analysis of variance [ANOVA] 5.98, p ⫽ .028) were found to be larger in individuals with ASD
on the ten cortical locations using diagnosis as the independent (see Figure 3A), but this effect was nonsignificant following a
variable. Proportional measures of cortical layering were ana- correction for multiple comparisons. Uncorrected values for the
lyzed with an ANOVA using diagnosis as a between groups two remaining temporal lobe locations showed modest, but
factor and both cortical region and cortical layer as repeated nonsignificant, differences compared to control subjects (inferior
measures. Thickness measures were also correlated with subject temporal gyrus, F1,14 ⫽ 2.45, p ⫽ .14; superior temporal gyrus,
variables that included age, brain weight, total brain volume as F1,14 ⫽ 2.49, p ⫽ .137; see Figure 3A). One control case showed
assessed by MRI, and postmortem interval (PMI). Raw measures thickness values that were two standard deviations greater than
from both MRI images and histology samples were directly those found in the remaining group of subjects. Removal of this
compared when they were available for the same individual case from the analysis left the pattern of results unchanged in
case. every cortical region.
We also evaluated the possibility that there might be a
Results consistent directional difference between age-matched subject
pairs by using a sign test where differences of less than 25 ␮m
Quality of Cortical Layering
Several types of abnormalities in cortical layering have been
previously reported in ASD cases and Table 2 shows a tally of the
abnormalities identified in both subject groups in the present
study. In general, the qualitative characteristics that appeared
substantially more often in the ASD group were dysplasias,
associated disturbances in lamination (see Figures 1C and 1D),
and a slightly less distinct gray/white matter boundary, which
could be attributed to supernumerary cell numbers in the
subplate region (see Figure 1E). Supernumerary layer I cells also
appeared slightly more frequently (Figure 1B). None of these
findings characterized the autism group as a whole or even a
majority of the autism-diagnosed subjects, and several of these
abnormalities were also evident in our control group. Addition-
ally, many of these abnormalities were patchy or infrequent and
not found widely in the regions examined. Seven of the eight
ASD subjects examined had at least one type of pathology and as
many as four, while six of the control subjects showed evidence
of pathologies with the maximum number of pathologies being
three. In addition, the pathologies present in the control group
were almost universally characterized as mild. Had the entire
hemisphere been available for study, more examples of such
limited pathologies might have been evident.

MRI Cortical Thickness Measures

Averaged cortical thickness values were similar between
individuals with ASD and control subjects (see Figure 2). Overall,
cortical thickness was only 2.5 percent greater in ASD subjects as
compared to controls. Frontal and parietal regions showed only
an average 2 percent difference between the groups, while
temporal areas showed an average 2.5 percent difference be-
Figure 2. Cortical thickness as assessed from magnetic resonance images
aggregated by lobe. Although slight overall differences occurred between
autistic spectrum disorder (ASD) and control subjects on the order of 100
␮m, these were nonsignificant (see text).

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J.J. Hutsler et al BIOL PSYCHIATRY 2007;61:449 – 457 453

Figure 4. Average cortical thickness for each subject as a function of age.

Cortical thickness decreased with increasing age in autistic spectrum disor-
der (ASD) subjects (r ⫽ ⫺.822, p ⫽ .012).

ship using this approach, moving from a difference in average

Figure 3. Cortical thickness as assessed from magnetic resonance imaging
for each of the 10 individual locations examined (A) and averaged across the
ten individual regions for each age-matched pair (B). Only regions the
middle temporal gyrus (*p ⫽ .028) were found to be larger in individuals
with autistic spectrum disorder (ASD), but this effect was nonsignificant
following a correction for multiple comparisons.
were considered ties. ASD subjects tended to show directionally
thicker average values relative to their age-matched control (p ⫽
.0625; see Figure 3B), but this result was nonsignificant. In
summary, although cortical thickness values tended to be direc-
tionally larger in ASD subjects, the magnitude of this difference
was small and nonsignificant.
cortical thickness of approximately 500 ␮m down to essentially
no difference by 45 years of age, but two age-matched pairs did
not fit this pattern (see Figure 3B).
As expected, whole-brain volumes calculated from MRI cor-
related strongly with brain weights collected at autopsy (r ⫽ .92,
p ⬍ .001). Nonsignificant negative relationships were found
between age and brain weight (r ⫽ ⫺.20, p ⫽ .493) and age and
hemispheric volume (r ⫽ ⫺.102, p ⫽ .718) for the entire group
of subjects. Postmortem interval (PMI) was unrelated to measures
of average cortical thickness (r ⫽ .086, p ⫽ .760), brain weight
(r ⫽ .355, p ⫽ .234), and hemispheric volume (r ⫽ .189, p ⫽
.517). Additionally, there were no significant differences between
the groups in either length of post-mortem interval (PMI) (t ⫽
.948, df ⫽ 13, p ⫽ .360) or age (t ⫽ .065, df ⫽ 14, p ⫽ .949; see
Table 1).

Cortical Thickness and Subject Characteristics

When pooled together, both groups showed decreasing
cortical thickness measures with age, but this relationship was
not significant (r ⫽ ⫺.49, p ⫽ .054). When divided by diagnosis,
the magnitude of this relationship was significant in the ASD
group (r ⫽ ⫺.822, p ⫽ .012), but not the control group (r ⫽
⫺.326, p ⫽ .430; see Figure 4). The difference between these
group correlation values was nonsignificant (z ⫽ ⫺1.30, p ⫽
.096). Given the magnitude of the relationship between age and
total cortical thickness in our ASD subjects, we evaluated the
possibility that cortical thickness might differ between ASD and
control subjects in the younger age ranges but not in the older
subjects (see, for example, Courchesne et al 2001, 2003). The
relationship between cortical thickness differences of the Figure 5. Thickness differences between age-matched subject pairs as a
matched pairs trended downward with age but was nonsignifi- function of age. In older ages thickness differences were quite small, while
cant (r ⫽ ⫺.183, p ⫽ .664). Interestingly, as can been seen in four of the six subjects under the age of 30 demonstrated more pronounced
Figure 5, six of the eight subject pairs showed a linear relation- differences.

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454 BIOL PSYCHIATRY 2007;61:449 – 457
Figure 6. Box and whisker plots of the proportional thickness measures of
each cortical layer for autistic spectrum disorder (ASD) and control individ-
uals. Layering patterns were remarkably similar between the two groups.
Histological Measures of Cortical Layering
Relative to the MRI measures, measures of cortical thickness
taken from histological sections have added error due to the
inability to verify whether a measure is orthogonal to the pial
surface of the brain in three dimensions. Thus, these measures
will tend to overestimate cortical thickness. Because of this there
was a lack of correlation between the raw measures collected
with the two methods in the eight cases where both histological
and MRI images were available (5 ASD subjects and 3 control
subjects; r ⫽ .183, p ⫽ .665), and a lack of correlation between
histological and MRI measures of thickness in each individual
region except for the middle temporal gyrus (r ⫽ .718, p ⫽ .045).
Proportional measures of individual laminae were utilized to
assess the pattern of layering in ASD and control cases (see
Figure 6). A repeated-measures ANOVA that treated the three
regions and the six layers within each region as within-subject
factors was used to compare the groups. Overall the pattern of
layering between ASD and control subjects did not differ, as was
evident from a nonsignficant interaction between layer and
diagnosis (F5,70 ⫽ 1.739, p ⫽ .137). Additionally, there was no
difference in the pattern of layering between subject groups
across the three cortical regions examined (layer ⫻ region ⫻
diagnosis; F10,140 ⫽ 1.00, ns). Cortical regions did not interact
with either layer (F10,140 ⫽ .551, ns) or diagnosis (F2,14 ⫽ 1.10, p
⫽ .347). As expected, the proportional measures of the individ-
ual layers differed from each other (F5,70 ⫽ 179.94, p ⬍ .001), but
there was neither a main effect of diagnosis (F1,14 ⫽ 1.23, p ⫽
.285) or region (F2,28 ⫽ 1.189, p ⫽ .319). In sum, the pattern of
cortical layering as assessed by proportional measures revealed
no regional or overall differences between the ASD and control
groups. As with the MRI groups, there were no significant
differences between the groups in either the length of the PMI
(t ⫽ ⫺.917, df ⫽ 14, p ⫽ .375) or the age of the subjects (t ⫽ .174,
df ⫽ 14, p ⫽ .864; see Table 1).
ASD Subject Characteristics
Three of the 11 cases included in the present study had a
history of seizures that were controlled by medication (i.e.,
phenytoin sodium, carbamazepine, and valproic acid). Within
this group, seizures typically occurred early in development and
were classified as either generalized or Jacksonian. Petit mal
seizures were suspected, but not confirmed, in another case, and
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J.J. Hutsler et al
one case had a first seizure on the day of death. Other medica-
tions, prescribed for various behavioral symptoms, included
thioridazine hydrochloride, chlorpromazine, lorazepam, and flu-
razepam.
Based upon available patient records, three of the cases were
described as having either severe or moderate mental retardation
(IQ ⬍ 50), while five cases were classified as being mildly
mentally retarded (IQ ⬎ 50). Two cases were not classified as
mentally retarded, while in one case insufficient information was
available to assess the overall level of cognitive functioning.
There were no apparent relationships between average measures
of cortical thickness and seizure reports, antiepileptic medica-
tions, or overall cognitive level. Nor was there a relationship
between the individual temporal gyrus locations and these
factors.
Megalencephaly
Two ASD cases from the MRI sample and two ASD cases from
the histological sample (one overlapping case) had brain weights in
excess of one standard deviation of published norms (Courchesne
et al 1999; see Table 1). Neither of the MRI cases showed lobular
absolute thickness measures that differed substantially from the
group averages. In the case of proportional layer measurements,
one of the subjects (1720 g) showed enlarged layer III thickness
values that were 2.5 standard deviations greater than the average
for both the control and ASD groups, while the other case
(1710 g) showed no differences in layering pattern.
Discussion
The present study finds cortical thickness values that are very
similar between ASD and control subjects. While temporal lobe
locations showed the largest differences between the two
groups, these effects were nonsignificant. ASD subjects tended to
show directionally thicker cortices when compared to their
age-matched control, but these results were also nonsignificant.
Few studies have directly examined cortical thickness measures
in these regions, although in (Bailey et al’s 1998) microanatom-
ical study of six ASD cases, one individual showed an apparent
increased thickness in the superior temporal gyrus in histological
sections. Voxel-based morphometry studies demonstrate bilater-
ally reduced gray matter values in the superior temporal gyrus
(Boddaert et al 2004), while volumetric studies have reported
increased volumes in the middle and inferior temporal gyri (Abel
et al 1999). Other studies have reported no volumetric differ-
ences in these regions (Pierce et al 2001) except for in megalen-
cephalic ASD cases (Bigler et al 2003).
Changes in a cortical region’s gray matter volume, thickness,
and surface area are related but may not always covary in the
same direction. For example, reduced volume may occur as a
consequence of cortical thinning in the absence of surface area
changes. Conversely, cortical column depletion, or addition,
could result in surface area and volume changes, while cortical
thickness might be conserved. The absence or presence of
thickness changes can serve as a useful tool in providing
preliminary evaluations of volumetric changes within the cortex.
Volumetric changes to the frontal lobes have been reported in a
subset of ASD cases (Carper et al 2002; Carper and Courchesne
2000), and if cortical thickness is unaffected, volumetric enlarge-
ments may instead be related to expansion of the cortical sheet.
This inference is partially supported by recent findings of in-
creased cortical folding in frontal lobe locations (Hardan et al
2004).
J.J. Hutsler et al
Cortical Layering Patterns
The establishment of proper cortical layering is dependent
upon numerous processes during ontogeny, including the pro-
duction of the correct number of cortical progenitor cells and
neuroblasts during prenatal neurogenesis (Caviness et al 1995).
Once generated, neuroblasts must migrate to their proper posi-
tion within the cortical plate and establish themselves into the
developing cortical circuits (Gleeson and Walsh 2000). If either
neurogenesis or migration is compromised, one might expect
disruptions in both cell distribution patterns and layering pat-
terns. Additionally, abnormalities in postmigratory neuronal mat-
uration could also affect cortical layering patterns, cortical thick-
ness, and the thickness of individual layers. In the present work,
we found that individuals with ASD show quantitative values for
the pattern of cortical layering that were indistinguishable from
control cases.
Defects in migration can result in heterotopias, lissencephaly,
and numerous other malformations. These can result from a
failure to initiate migration or a failure in the signals that guide a
neuroblast into its appropriate position within the cortical plate
(Pilz et al 2002; Uher and Golden 2000). Focal malformations of
cortical development (dysplasias) are characterized by the dis-
ruption of cortical lamination and the presence of neurons, or
groups of neurons, in the white matter. Previous studies in ASD
have reported dysplasias in the cerebral cortex as well as
disorganized pyramidal cell groups and mislamination (Kemper
and Bauman 1998). Such neuropathological findings are also
often present in subjects with epileptiform activity (Foldvary-
Schaefer et al 2004). In the present sample, only two cases in the
histology study had a history of epilepsy, and both had mild or
moderate dysplasias (the remaining four cases with dysplasias
did not have a history of seizures). Our results support the view
that dysplasias are commonly found in ASD but are neither
universal nor severe. At this time it is impossible to determine if
such pathologies characterize only distinct subgroups of individ-
uals with ASD or represent variation in neuropathological out-
comes arising from a common etiology early in development
(Keller and Persico 2003).
Bailey et al (1998) describes ectopic gray matter within the
white matter of two autistic subjects and an increased number of
individual neurons within the white matter of one additional
case. Although we did observe supernumerary cells within the
subcortical white matter (Figure 1E), we did not observe islands
or clusters of cells. Their absence is likely due to their nonuni-
versality and the limited sample of white matter in the present
study.
Neurochemicals and Lamination
Several neurochemicals that participate in migration and
subsequent cortical layer formation have been examined in ASD.
Deficits in serotonin expression have been found within the
central nervous system of both autistic children and adults
(Chugani et al 1997, 1999), but in animal studies, alterations to
serotonin levels early in development do not significantly impact
cortical layering and organization (Berger-Sweeney and Hohm-
ann 1997). Serotonin arising from fibers in the early marginal
zone may, however, play a role in the regulation of reelin
expression, which serves to guide migrating neuroblasts to their
proper position within the cortical plate (Janusonis et al 2004).
Reelin has also been studied in autism (Pilz et al 2002), and its
over-expression during development is associated with polymi-
crogyria (Eriksson et al 2001), a condition found in some
individuals with ASD (Piven et al 1990). Despite this, there is no
BIOL PSYCHIATRY 2007;61:449 – 457 455
strong evidence for a direct relationship between reelin polymor-
phisms and ASD (Bonora et al 2003; Li et al 2004). Additionally,
the abnormal layering patterns present in mice lacking reelin
expression are not present in autism. Still, there are occasional
cases (see Figure 1B and Bailey et al 1998) that show supernu-
merary neurons within layer I, indicating a problem with signal-
ing migrating neurons to stop at the boundary of layer I during
development. In our cases, the number of cells found within
layer I is relatively small.
Cortical Architecture in ASD
Although cortical layering is largely preserved in the present
study, there is substantial evidence that quantitative differences
of cortical organization exist in individuals with ASD. It is
estimated that approximately 20 percent of all autistic subjects
show evidence of megalencephaly (Bailey et al 1993; Filipek et al
1992; Piven et al 1992, 1995), although average brain sizes may
only be enlarged in ASD individuals between the ages of one and
seven years (Courchesne et al 1999, 2003). These early changes
in brain size may be the result of a modified developmental
trajectory, which continues to have an impact on brain organi-
zation throughout adolescence and early adulthood resulting in
volumetric reductions as subjects age (Akshoomoff et al 2002;
Hardan et al 2004). Although the volume of cerebral cortex
accounts for only a portion of total brain volume (Palmen and
Van Engeland 2004), the present work supports this general
view, since cortical thinning appears to be related to increasing
age in our group of ASD cases.
Two ASD subjects showed abnormally high brain weights
(1720 and 1990 g), but neither showed abnormally large cortical
thickness measures. In addition, there was little relationship
across ASD cases between measures of cortical thickness and
measures of brain size. If cortical thickness is unrelated to brain
size, and if intercolumnar distance is either the same or smaller
than that found in typically developing controls (Casanova et al
2002a, 2002b), this could suggest that the number of cell columns
is greatly increased in individuals showing prominent megalen-
cephaly. On the other hand, studies in ASD subjects evaluating
cortical gray matter indicate that increased brain size may
depend upon changes in the volume of the underlying white
matter (Herbert et al 2002) rather than changes to cortical gray
matter volumes (see Palmen and Van Engeland 2004 for a
review).
Limitations
Future MRI assessments of thickness differences would obvi-
ously benefit from an in vivo whole-brain approach. In vitro
scanning, although necessary in the context of the present study,
is subject to several pragmatic shortcomings (see Schumann et al
2001).
Since the boundary between cortex and white matter is
sometimes indistinct in ASD due to supernumerary cells in the
subplate region, MRI measures could result in an overestimation
of cortical thickness due to a misplaced lower boundary. In
histological sections, one might expect that an indistinct bound-
ary would result in an increase in the proportional measures of
layer VI. This result was not evident in the individual layer VI
values in ASD subjects showing indistinct boundaries.
The present histological findings are qualified by several
factors. First, we did not exhaustively examine every cortical
region for evidence of deviations in cortical layering, but instead
focused on cortical regions representing samples of eulaminate
cortex from each of three lobes. Second, the establishment of
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456 BIOL PSYCHIATRY 2007;61:449 – 457
proper cortical layering does not address several other aspects of
cortical organization that might significantly impact functioning.
Third, the hemisphere from which the samples were taken could
not be held constant. Hemispheric asymmetries have been
reported in both control (Hutsler and Galulske 2003) and ASD
subjects in a number of contexts. For example, abnormal asym-
metries have been found in both the gross morphology of the
brain (Herbert et al 2005) and in the patterns of serotonin
synthesis (Chandana et al 2005). Although it would be preferable
to hold the hemisphere sampled constant or to include it as an
additional factor in the analysis, there is little available evidence
to indicate a widespread asymmetry in lamination in either
control groups or ASD subjects at the present time.
Finally, a greater number of subjects available for study could
increase the ability to detect group differences. Despite this,
average cortical thickness measures for each lobe were never
more than three percent greater than those found in controls, and
only a few regions, mainly those in the temporal lobes, showed
cortical thickness values exceeding five percent. Differences
between the layering patterns were also quite small.
The current findings demonstrate that cortical organization as
assessed both by measures of thickness and lamination is largely
intact in ASD individuals. Temporal lobe areas, unlike frontal and
parietal locations, show quantitative differences in thickness
between ASD and control subjects. In contrast, quantitative
evaluations of the layering patterns in ASD individuals are
remarkably normal and do not show evidence of regional
pathology. Qualitative examinations of the cortex confirm previ-
ous findings of circumscribed dysplasias, an indistinct layer VI
boundary with the white matter and, occasionally, supernumer-
ary cells in layer I. These pathologies occur at a higher frequency
than in control subjects, but no single pathology is evident in
more than a few members of our ASD group. Finally, cortical
thinning that occurs with age in typically developing subjects
appears to be accelerated in autistic individuals, suggesting the
presence of an altered trajectory in the dynamic reshaping of the
cortex that extends across the lifespan (Aylward et al 2002;
Boddaert and Zilbovicius 2002).
We would like to thank Dr. Jane Pickett of the Autism Tissue
Program; the Harvard Brain Tissue Resource Center; the Brain
and Tissue Bank for Developmental Disorders at the University of
Miami; and the Brain and Tissue Bank at the University of
Maryland, for their invaluable assistance in collecting the tissues
utilized in the present study. Additionally, we would like to thank
Dr. Cyndi Schumann and Dr. David Amaral for their efforts in
making magnetic resonance imaging scans from postmortem
tissue available for study.
This work and HZ were supported through the generosity of
the National Alliance for Autism Research.
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