Cerebral Cortex Advance Access published February 27, 2008
Cerebral Cortex
doi:10.1093/cercor/bhn019
Cytoarchitecture and Transcriptional
Profiles of Neocortical Malformations in
Inbred Mice
Raddy L. Ramos1, Phoebe T. Smith2, Christopher DeCola1,
Danny Tam1, Oscar Corzo1 and Joshua C. Brumberg1
Malformations of neocortical development are associated with
cognitive dysfunction and increased susceptibility to epileptogenesis. Rodent models are widely used to study neocortical
malformations and have revealed important genetic and environmental mechanisms that contribute to neocortical development.
Interestingly, several inbred mice strains commonly used in
behavioral, anatomical, and/or physiological studies display neocortical malformations. In the present report we examine the
cytoarchitecture and myeloarchitecture of the neocortex of 11
inbred mouse strains and identified malformations of cortical
development, including molecular layer heterotopia, in all but one
strain. We used in silico methods to confirm our observations and
determined the transcriptional profiles of cells found within
heterotopia. These data indicate cellular and transcriptional
diversity present in cells in malformations. Furthermore, the
presence of dysplasia in nearly every inbred strain examined
suggests that malformations of neocortical development are
a common feature in the neocortex of inbred mice.
channel and neurotransmitter receptor expression remains
unknown.
A number of inbred mouse strains with congenital autoimmunity, exhibit neocortical heterotopia with striking similarity
to those found in the dyslexic brain (Sherman et al. 1985, 1987;
Sherman, Morrison, et al. 1990). Supporting the link between
deficits in neuronal migration with cognitive disruption, the
presence of heterotopia in these mice is associated with
impaired performance on spatial and nonspatial memory tasks
(Denenberg et al. 1991; Boehm et al. 1996; Balogh et al. 1998) as
well as deficits in sensory processing (Clark et al. 2000; Frenkel
et al. 2000). Interestingly, mice with heterotopia were recently
found to have lower thresholds for convulsant-induced seizures
(Gabel and LoTurco 2002). Thus, cognitive deficits and increased seizure susceptibility in mice with heterotopia is
suggestive of circuit reorganization in neocortex.
Given the widespread use of inbred mice in the investigation
of the genetic basis of brain development (Seecharan et al. 2003;
Wahlsten et al. 2006), cognitive function (Bolivar et al. 2001;
Wahlsten et al. 2005), and sensation and perception (Zheng et al.
1999; Wong and Brown 2006), we sought to determine whether
heterotopia or other malformations of neocortical development
are widely present in the inbred neocortex. We examined brains
of 11 different inbred and 2 outbred strains of mice including
several priority strains defined by the Mouse Phenome Database
(http://www.jax.org/phenome). Surprisingly, we observed dysplasia of the neocortex in 10 of 11 inbred strains of mice and in
the present report, we describe the cytoarchitecture and areal
distribution of these malformations. Malformations were found
in early postnatal brains examined. No malformations were
found in outbred mice. In order to describe the transcriptional
profiles of cells within neocortical malformations in C57BL/6J
mice, we used the Allan Brain Atlas, a database containing in situ
hybridization data assayed for >20 000 genes (Lein et al. 2007).
Our database search identified the expression of a number of
cell-type specific genes, ion channel and neurotransmitter
receptors genes, and cytoskeletal and transcription factor
genes present in cells found in malformations. These data
provide greater insight into the transcriptional diversity and
cellular constituents of malformations of neocortical development. Moreover, the presence of neocortical dysplasia in many
different inbred mice strains and in early postnatal brains,
suggests that deficits in neocortical development are a characteristic feature of the inbred neocortex.
Keywords: development, ectopia, inbred, neocortex
Introduction
Neocortical lamination emerges from the precisely orchestrated migration of newly generated neurons away from
proliferative regions of the embryonic forebrain (Angevine
and Sidman 1961; Rakic 1974). During corticogenesis and early
postnatal periods, migrating neurons are sensitive to a number
of genetic (Gleeson and Walsh 2000; Bielas et al. 2004) as well
as environmental (Schmidt and Lent 1987; Ang et al. 2006)
perturbations, which alter their migratory routes and affect
their final positioning. Malformations of neocortical development resulting from altered migration include phenotypes with
fewer neuronal lamina, inverted lamina, band or periventricular
heterotopia, or phenotypes lacking lamination (reviewed in
Gupta et al. 2002; Olson and Walsh 2002).
Developmental disruption of neocortical lamination is often
accompanied by seizure susceptibility and/or cognitive deficits
(Schwartzkroin and Walsh 2000; Schwartzkroin et al. 2004). For
example, a defining feature of brains of dyslexic patients is the
presence of neocortical heterotopia consisting of clusters of
misplaced neurons found in the molecular layer (layer I;
Galaburda and Kemper 1979; Galaburda et al. 1985; Humphreys
et al. 1990). Cognitive disruption and learning impairment,
defining phenotypes of dyslexia, may therefore result from
altered neuronal migration and cortical circuit reorganization
(reviewed in Galaburda 2005; Galaburda et al. 2006). Whether
molecular layer heterotopia is also accompanied by other
anatomical deficits such as dendritic and spine growth or ion
The Author 2008. Published by Oxford University Press. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org
1
Department of Psychology, Queens College, CUNY, Flushing,
NY 11367, USA and 2Department of Math & Science, Suffolk
County Community College, SUNY, Riverhead, NY 11901, USA
Methods
Neocortical Histology of Inbred/Outbred Mice Brains
Adult mice (outbred CD1 and CFW, Charles River, Wilmington, MA;
inbred, Jackson Laboratories, Bar Harbor, ME; Table 1) were deeply
Table 1
Inbred and outbred mouse strains used and incidences of malformations
Strain
N 5 male/female
Total (N)
Males w/dysplasia
% Males
Females w/dysplasia
% Females
Combined w/dysplasia
Total (%)
129P3/J
A/J
AKR/j
Balb/cJ
C3H/HeJ
C57BL/6J
C57BL/10J
CBA/J
CD-1 (outbred)
CFW (outbred)
DBA/J
SWR/J
SJL/J
Total
8/5
9/4
4/4
11/5
10/3
13/0
11/0
4/4
11/0
12/0
7/6
12/0
10/0
122/31
13
13
8
16
13
13
11
8
11
12
13
12
10
153
3/8
3/9
2/4
3/11
2/10
5/13
9/11
2/4
0/11
0/12
0/7
0/12
2/10
31/122
37.50
33.33
50.00
27.27
20.00
38.46
81.82
50.00
0.00
0.00
0.00
0.00
20.00
27.57
3/5
2/4
2/4
0/5
3/3
—
—
0/4
—
—
1/6
—
—
11/31
60.00
50.00
50.00
0.00
100.00
—
—
0.00
—
—
16.67
—
—
39.52
6/13
5/13
4/8
3/16
5/13
5/13
9/11
2/8
0/11
0/12
1/13
0/12
2/10
42/97
46.15
38.46
50.00
18.75
38.46
38.46
81.82
25.00
0.00
0.00
7.69
0.00
20.00
28.06
anesthetized with pentobarbital and perfused through the heart with
0.9% saline followed by a phosphate-buffered (PB; 0.1 M) fixative
containing 4% paraformaldehyde. Brains were removed from the skull
and postfixed with 4% paraformaldehyde at 4 C. Brains were sectioned
(50--60 lm) in the coronal plane on a vibratome and free-floating
sections were then collected in PB. Sections were mounted onto
gelatin-coated slides and dried overnight. Following Nissl staining,
slides were dehydrated and coverslipped with Permount. Myelin
staining was performed on floating sections with gold-chloride
according to Wahlsten et al. (2003).
Three breeding pairs of C57BL10/J mice were purchased from
Jackson Laboratories and maintained in our colony. Eleven pups from 3
litters were euthanized and whole-heads were placed in 4% paraformaldehyde for 3 days. Heads were transferred to 30% sucrose for an
additional 5 days and then cut on a cryostat at 40 lm. Every section was
collected, mounted onto gelatin-coated slides and dried overnight.
Following Nissl staining, slides were dehydrated and coverslipped with
Permount.
Immunocytochemistry was used to reveal the presence of neurons in
heterotopia as previously described (Ramos et al. 2006). Briefly, freefloating sections were collected into different wells and washed with
phosphate-buffered saline (PBS; 3 times). Sections were permeablized
and blocked in 5% normal goat serum (NGS) and 0.2% Triton X-100 for
1 h. Sections were incubated with antineuron-specific enolase (raised
in rabbit; Chemicon, Temecula, CA) in 0.1% Triton X-100, 2.5% NGS,
and PBS at 4 C overnight. For use with light microscopy, sections were
rinsed several times with 2.5% NGS in PBS and then incubated in
biotinylated secondary antibodies (biotinylated goat anti-mouse, goat
anti-rabbit; 1:200, Vector Labs, Burlingame, CA) for 2 h at room
temperature. Sections were rinsed 3 times with PBS and then incubated
for 1 h in an avidin--horseradish peroxidase mixture. Sections were
rinsed in PBS 3 times and then reacted with 0.05% diaminobenzidine in
the presence of 0.0015% H2O2. Sections were collected onto gelatincoated slides, dried for several hours, and coverslipped with Permount.
Slides were examined and representative photomicrographs were
taken on a Nikon Eclipse E500 or Olympus Bx51 equipped with digital
cameras. Figures were prepared in Adobe Photoshop.
(Arc, Egr1, Fos, Jun), and cell-cycle proteins. Our list also included celltype specific genes (neuron vs. glia), neocortical layer-specific genes (I-VI), and genes enriched in neocortex provided by Lein et al. (2007).
Finally, we examined the list of genes with high expression levels
provided on the ABA (Brain Structure Browser; 240 genes). We
reviewed photomicrographs from brains found in the ABA, which were
cut in the coronal plane. A smaller number of brains cut in the sagittal
plane were also examined from this list. Supplementary Tables 1 and 2
lists all of the genes for which photomicrographs were examined.
According to methods described in the ABA homepage, approximately 8 series of sections were created per brain. However, at this
time we do not know how many different genes were assayed per
brain. In the present report, quantification of brains with/without
malformations was performed assuming that no 2 hybridized cases
were taken from the same brain. Thus, if multiple hybridized cases
containing malformations come from the same brains, our quantification represents overestimates. However, if multiple hybridized cases
without malformations come from the same brains, our quantification
represents underestimates. As described below, percentages calculated
from our histological material were similar to percentages found in the
ABA.
The ABA provides colorimetric quantification of gene expression for
every photomicrograph in the database. Eight different colors are used
to code lowest-to-highest (blue-to-red) levels of expression. Based on
this scale we could determine genes with high and low levels of
expression in neocortical malformations.
Hemispheric bias in the occurrence of neocortical malformations
was examined for those brains found in the ABA, which were cut along
the coronal plane. According to methods posted on the ABA web site
(http://community.brain-map.org/confluence/display/FORUM/Home;
posted by Chinh Dang on 05/07/2007; ABA Community Site/User
Forum), the left side of each photomicrograph displays the left side of
each brain.
Photomicrographs containing the section with the largest extent of
neocortical dysplasia were printed and archived. Representative
photomicrographs taken from the ABA were imported into Photoshop
for preparation of figures.
Allen Brain Atlas Database Search
The Allen Brain Atlas (ABA; www.brain-map.org) is a public database
containing brains from adult, male, C57BL/6J mice processed for in situ
hybridization according to methods described in Lein et al. (2007).
Serial coronal and/or sagittal cryostat sections are found in the ABA (25lm thickness) for each of over 20 000 genes (Lein et al. 2007). We used
the ABA in order to examine the expression patterns of a number of
genes involved in neocortical development and function. We first
examined the sample data set provided by the ABA (www.brainmap.org), which included 98 genes. We next created a list of ~1200
+
+
+
+
genes including (for example) those for ion channels (Na , K , Ca2 , Cl ,
etc.), neurotransmitter receptors (glutamate, GABA, acetylcholine,
etc.), cytoskeletal proteins (tubulin), apoptosis, and cell death
associated proteins (annexin, caspase, etc.), and early immediate genes
Results
Page 2 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
Malformations of Cortical Development in Inbred Mice
In order to determine the incidence of malformations in the
inbred mouse neocortex, we examined brains from 11 inbred
and 2 outbred mouse strains commonly used in behavioral and
physiological strain surveys including priority strains defined by
the Mouse Phenome Database (http://www.jax.org/phenome).
Nissl and myelin-stained vibratome sections taken from these
mice revealed malformations of cortical development, deficits
in callosal development, and/or hydrocephaly in all inbred
strains except for SWR/J mice. In contrast to our observations
in inbred mice, we did not find any malformations in brains
examined from CD-1 or CFW outbred mice.
The incidence of malformations among the inbred mice
varied. These data are summarized in Table 1. Among the
strains that did exhibit malformations, the C57BL/10J strain
(9/11 brains; 81.82%) exhibited the highest total incidence,
whereas the DBA/J strain exhibited the lowest incidence
(7.69%). In addition to neocortical malformations, we observed
varying incidences of callosal dysgenesis. The 129P3/J strain
exhibited the highest incidence of callosal dysgenesis (6/13
brains) followed by Balb/cJ (1/10 brains) and DBA2/J (1/13
brains) strains. We observed 3/11 C57BL/10J mouse brains,
which were hydrocephalic.
Independent In Silico Confirmation of Neocortical
Malformations in the C57BL6/J Inbred Mouse Strain
We preformed a database search of the ABA in order to confirm
our findings of neocortical malformations in inbred mice, with
the prediction that by chance, a percentage of mice used in the
preparation of the ABA (C57BL/6J) would also contain
malformations (Sherman and Holmes 1999). The ability to
view photomicrographs found in the ABA with extremely high
resolution, allowed us to examine material found in the ABA at
high magnification, even at the single-cell level. Our database
search of the ABA confirmed our observations of malformations
in C57BL/6J mice as well as similar dysplastic phenotypes
found in other strains (described below). From among 1244
hybridized cases we examined ( >5000 photomicrographs), 385
displayed some type of neocortical malformation (30.95%;
Supplementary Tables 1 and 2), a percentage similar to that
observed in this strain previously (Sherman et al. 1987;
Sherman and Holmes 1999). In light of the fact that our tissue
sections were prepared using a vibratome (50-lm sections) and
material found in the ABA were prepared using a cryostat
(25-lm sections), the observation of near-identical malformations from both sets of data argues strongly against sectioning/
histological artifact as the cause of the observed malformations.
Cytoarchitecture of Neocortical Malformations in
Inbred Mice
Two distinct types of malformations of neocortical development were present in inbred mice from our histological data
set, which were also present in material examined from the
ABA. The first malformation type was characterized by the
presence of clusters of cells in layer I disrupting the normal
border between the molecular layer and gray matter (molecular layer heterotopia). Besides heterotopia, we observed
a diverse class of malformations characterized by a more
complex disruption of white and gray matter and included
tissue invaginations or subduction of adjacent cortical areas.
Molecular Layer Heterotopia in Dorsal Neocortex
Molecular layer heterotopia was found in frontal, motor, and
somatosensory cortices and was characterized by radial
clusters of neurons in layer I extending up to the pial surface.
We observed molecular layer heterotopia in 129P3/J, A/J, AKR/
J, C57BL/6J, C57BL/10J, CBA/J, DBA/2J, and SJL/J strains. Large
heterotopia was visible without magnification as bumps on the
surface of the cortex. Representative photomicrographs of
brains from C57BL/10J mice containing 2 distinct heterotopia
are found in Figure 1A,B (arrows and arrowhead). As can be
Figure 1. Dorsal neocortical molecular layer heterotopia. (A, B) Representative
photomicrographs of heterotopia from adult C57BL/10J mice where 2 heterotopia can
be seen on the cortical surface of each brain (arrows and arrowhead). (C, D) Coronal
sections from same brain as in (B) stained for Nissl. Cells can be seen invading
the molecular layer. Heterotopia in (C) corresponds to rostral heterotopion in (B)
(arrowhead). Heterotopia in (D) corresponds to caudal ectopia in (B) (arrow). (E, F)
High-magnification micrographs of heterotopia shown in (C) and (D), respectively. (G,
H) Representative micrographs of a P2 C57BL/10J mouse brain with a molecular layer
heterotopia (arrow). Calibration in lm: (A, B) 2000; (C, D), 500; (E, F), 250; (G) 1000;
(H) 250.
seen, heterotopia can be found on either hemisphere, vary in
size, and generally have a circular shape. Coronal serial
sectioning of the brain shown in Figure 1B revealed 2
heterotopia found exactly where we saw the bumps on the
cortical surface (Fig. 1C,D). Examination of individual Nisslstained cells within heterotopia (Fig. 1E,F) suggested the
presence of both neurons and glia according to criteria used for
stereological analyses of Nissl-stained tissue including cell size
and shape (Ling et al. 1973; Satorre et al. 1986; Williams and
Rakic 1988a, 1988b; Seecharan et al. 2003; Schmitz and Hof
2005).
Not surprisingly, we observed many hybridized cases found
in the ABA also containing molecular layer heterotopia. From
among 385 cases in the ABA, which exhibited malformations, 324 of these cases displayed heterotopia (84.16%;
Cerebral Cortex Page 3 of 15
Supplementary Table 1; 114 dorsal cortical heterotopia; 210
midline heterotopia, see below).
Heterotopia was found in over much of the rostro-caudal
and medial--lateral axes of the neocortex. In order to
demonstrate the medial--lateral range of observed heterotopia,
we plotted the position of heterotopia on an illustration of
a coronal section at the level of sensory--motor cortices. As
shown in Figure 2, we observed heterotopia in all locations of
dorso-lateral neocortex including midline cortical areas (cingulate cortex, discussed below). In contrast, we did not
observe heterotopia in lateral cortical areas such as piriform,
perirhinal, and entorhinal cortices. As shown in Figure 2,
heterotopia had generally similar cytoarchitecture regardless of
location, with the exception of heterotopia found in midline
cortical areas (discussed below).
Although the vast majority of hybridized cases we examined
from the ABA were restricted to those for which coronal
sections were available, we did review a number of cases cut
along the sagittal plane, which also contained heterotopia. In
order to demonstrate the rostro-caudal range over which
heterotopia was observed, we plotted the position of heterotopia on an illustration of a mid-sagittal section. As shown in
Figure 3, we observed heterotopia in much of neocortex
including frontal/prefrontal cortex extending to visual cortex.
Several interesting observations could be made by examining
cases in both the coronal and sagittal planes using our own
material and that found in the ABA. First, heterotopia often had
an inverted triangular shape with neurons becoming increas-
ingly more ‘‘fanned-out’’ as they approached the pial surface. In
addition, sections adjacent to those containing heterotopia
often displayed increases in the thickness of the molecular
layer.
We measured the size of heterotopia found in our histological database. Additionally, we estimated the rostro-caudal
extent of heterotopia (not including midline heterotopia, see
below) found in the ABA based on the thickness of sections
used in the preparation of the ABA (25 lm) as well as the
section number of each photomicrograph. Heterotopia ranged
in size from 50 to 800 lm (mean = 364 lm; standard error of
the mean [SEM] = 62.80 lm; measurements from 20 cases).
In order to determine whether malformations are present in
the early postnatal cortex, we prepared cryostat sections of P2
heads from the C57BL10/J strain in order to examine brains in
situ. We observed molecular layer heterotopia (detailed below)
in 4/11 brains (36.36%) with cytoarchitecture identical to that
observed in adult mice (described below). A representative
example of these data is shown in Figure 1 (G,H; see also
Supplementary Fig. 1). These data suggest that malformations in
inbred mice are the result of a defect in neocortical
development. These data are consistent with heterotopia
observed in perinatal brains of inbred New Zealand black
(NZB) mice as previously reported by Sherman et al. (1992).
Midline Molecular Layer Heterotopia
In the mammalian brain, midline cortical areas are characterized
by the apposition of the molecular layers of both hemispheres at
Figure 2. Medio-lateral extent of observed molecular layer heterotopia. (A) Illustration of a coronal mouse section. Arrowheads point to the aerial extent to which heterotopia
was observed. Small-labeled arrows in (A) refer to other panels with heterotopia found at different positions. (B--F) Representative examples of heterotopia observed in the ABA.
Hybridized gene listed next to each micrograph taken from ABA.
Page 4 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
surrounding heterotopic cells could be observed indicative of
tissue folding and invagination (arrows in Fig. 4C).
We measured the size of midline heterotopia we found in
our histological database and also estimated the rostro-caudal
extent of those found in the ABA as described above (see Fig.
4D). Midline heterotopia ranged in size from 200 lm to 4 mm
(average = 1.77 mm rostro-caudal; SEM = 103.43 lm; measurements from 46 cases).
Figure 3. Rostro-caudal extent of observed dorsal cortical heterotopia. (A)
Illustration of a mid-sagittal mouse section. Arrows point to the aerial extent to
which heterotopia was observed. (B--D) Representative examples of heterotopia
observed in the ABA. Hybridized gene listed next to each micrograph taken from ABA.
the interhemispheric fissure (IHF). In contrast to this normal
organization, we observed molecular layer heterotopia along
the IHF (Fig. 2F). Unlike dorsal cortical heterotopia, which
contained clusters of cells in the molecular layer, midline
heterotopia generally had diverse cytoarchitecture. Specifically,
midline heterotopia often contained cells in the molecular
layer as well as cells in between the molecular layers of each
respective hemisphere. Representative examples of such
heterotopia are found in Figure 4A (arrow). Heterotopia was
also observed, which was devoid of cells in the molecular layer
but had numerous cells in between the molecular layers of each
respective hemisphere forming ‘‘islands’’ of cells. Representative
examples of such heterotopia are found in Figure 4B where
white matter can be clearly seen surrounding heterotopic cells.
We also observed midline heterotopia with additional white
matter disruption. For example additional layers of white matter
Other Malformations of Cortical Development
In addition to molecular layer heterotopia we observed other
malformations of cortical development with diverse cytoarchitecture and location. The feature common among many brains
containing this type of dysplasia was the dramatic misalignment
of parenchyma and the molecular layer resulting in areas of
white matter in continuous register to gray matter. In some
cases cells were found outside the limits of the pial surface of
the normotopic cortex (subarachnoid space) resulting in gray
and white matter, which folded over/onto normotopic cortex.
More rare cases exhibited areas with complex cytoarchitecture
including multiple neuronal layers separated by extra layers of
white matter, or subduction of adjacent cortical areas. Including our examination of data from the ABA, we observed
these types of malformations in 129P3/J, A/J, AKR/J, BALB/cJ,
C3H/HeJ, C57BL/6J, CBA/J, and SJL/J strains. From among 385
cases in the ABA, which exhibited malformations, 107 of these
cases displayed this type of malformation (27.79%; Supplementary Table 1).
Figure 5 contains representative examples of brains containing these diverse malformations of cortical development.
Examination of brains cut along sagittal and coronal planes
serves as an indicator that these malformations occur at
multiple orientations such as medial--lateral as well as rostral-caudal. Examination of adjacent sections (Fig. 5A--D, E--G, H,I)
clearly demonstrates the complex cytoarchitecture of these
malformations. Data shown in Supplementary Figure 2 demonstrate representative examples of brains where tissue folds are
evident containing labeled cells. Though often torn either
during the process of brain removal or tissue sectioning, a clear
pattern/trajectory of cells leading outside the molecular layer
limits (arrows) could be observed resulting in a fold of tissue on
top of the normotopic cortex. Adjacent sections are provided
for clarity and help to demonstrate the 3-dimensional features
of this complex malformation. Additional examples of malformations found in the ABA including phenotypes containing
cells surrounded by white matter are found in Supplementary
Figure 3. Closer examination of these cases indicates both gray
and white matter on top of normotopic cortex (arrowheads
point to pial surface).
Multiple Neocortical Malformations in Individual Brains
We observed that only a single malformation was present in
most brains in our data set. Similarly, the vast majority of brains
with neocortical malformations found in the ABA exhibited
only 1 discrete malformation. However, a small percentage did
display multiple malformations and generally exhibited 2 or
more molecular layer heterotopia. Cases exhibiting both dorsal
and midline heterotopia were found in 21 of 385 identified
cases (5.45%). Cases exhibiting both midline heterotopia and
other malformations were found in 15 of 385 identified cases
(3.9%). Cases exhibiting both dorsal heterotopia and other
Cerebral Cortex Page 5 of 15
Figure 4. Midline molecular layer heterotopia. (A) Representative examples of heterotopia observed in the ABA with cells found in the molecular layer as well as in between the
molecular layers of the 2 cortical hemispheres (arrow). (B) Representative examples of heterotopia observed in the ABA with cells in between the molecular layers of the 2 cortical
hemispheres but without any apparent cells in the molecular (arrowhead). (C) Representative examples of heterotopia observed in the ABA with cells in between the molecular layers
with additional alterations in white matter (arrows). (D) Serial sections from brain hybridized for Citron (same as in middle panel C) demonstrating cytoarchitecture and rostro-caudal
extent of heterotopia. Asterisk denotes position corresponding to micrograph found in panel (C). Hybridized gene listed next to each micrograph taken from ABA.
malformations were found in 7 of 385 identified cases (1.82%).
We observed 7 cases containing 2, distinct dorsal heterotopia.
We found only a single case containing all 3 malformations.
Representative photomicrographs of brains containing multiple
heterotopia (dorsal and midline) evident within individual
sections are shown in Figure 6. These data indicate that
multiple malformations can be found within individual brains.
Laterality of Malformations
In order to determine whether there exists a hemispheric
asymmetry in the incidence of neocortical malformation, we
recorded the hemispheric location of each of the observed
types of dysplasia found in the ABA data set. These data are
shown in Supplementary Figure 4 and revealed a right hemisphere bias (61.22%) for dorsal cortical heterotopia (n = 98
samples; v2 = 4.94, P < 0.05). In contrast we observed that
midline heterotopia occur almost exclusively from the left
hemisphere (99.54%; n = 219 samples; v2 = 216.01, P < 0.001).
Malformations with more complex cytoarchitecture were
found to have a right hemisphere bias (58.82%; n = 119
samples; v2 = 62.84, P < 0.001).
Transcriptional Profiles of Cells in Heterotopia
The identification of cases in the ABA containing neocortical
malformations provides a novel means of identifying the
Page 6 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
transcriptional profiles of cells within malformations. We
examined brains hybridized for established genes, which are
known to display cell-type specificity, in order to determine
the general cellular phenotypes in malformations such as
neurons versus glia. Furthermore, we examined brains hybridized for established genes with neuronal subtype-specific
expression, in order to identify the neuronal phenotypes in
malformations such as excitatory and inhibitory neurons as
well as neurons found in individual layers. Finally, we examined
brains hybridized for established genes for ion channels and
neurotransmitter receptors, in order to provide clues as to the
intrinsic and synaptic physiology of cells in malformations.
The large number of genes for which hybridized tissue
contained malformations precludes the description of them all
(Supplementary Table 1). Consequently, we describe data from
those brains where medium to high levels of expression
(colorimetric scale provided by the ABA) were observed.
Cell-Type Specific Genes Expression in Heterotopia
Heterotopia was found in a number of cases hybridized for
expression of established genes known to be enriched or
exclusively expressed in neurons (Lein et al. 2007) such as Chgb,
Disp2, Gpr162, Reps2. Figure 7 illustrates the expression of the
neuron-specific genes, Mtap1a, and the Tubulin b2a gene (Tubb2a).
Figure 5. Other observed malformations with complex cytoarchitecture. (A--D) Micrographs from a 129P3/J mouse with a complex malformation. Rostral--caudal, serial sections
(100-lm intervals) shown in (A--D). Low magnification of (B) shown in (B1). (E--K) Data from the ABA. (E--G1) Rostro-caudal, serial sections (200-lm intervals) of mouse brain
hybridized for Eno2. Low magnification of (G) shown in (G1). (H--K) Rostro-caudal, serial sections (200-lm intervals) of mouse brain hybridized for Plagl1. High magnification of (H)
and (J) shown in (I) and (K), respectively. Hybridized gene listed next to each micrograph taken from ABA.
Figure 6. Multiple heterotopia found in individual brains. Representative photomicrographs taken from hybridized tissue found in the ABA (A, B). Midline and dorsal cortical
heterotopia indicated by arrows and arrowheads, respectively. Hybridized gene listed next to each micrograph taken from ABA.
To further confirm the presence of neurons in heterotopia, we
performed immunocytochemisty for Neuron-specific enolase
(NSE) expression. As shown in Supplementary Figure 5, NSElabeled neurons were found in heterotopia. Together with our
observations of Nissl-stained material and data described below
demonstrating expression of neuron-specific ion channels and
neurotransmitter receptors genes, these data indicate that
neurons are found in neocortical heterotopia.
Cerebral Cortex Page 7 of 15
Figure 7. Cellular constituents of malformations include neurons. Representative photomicrographs taken from the ABA containing a dorsal (A) and midline (B) heterotopia and
hybridized for genes enriched in neurons. Magnified images found in middle (ISH) and right (colorimetric) panels. Hybridized gene listed next to each micrograph taken from ABA.
In order to test whether specific neuronal subtypes are
found in malformations, we examined cases found in the ABA
hybridized for expression of genes known to be enriched or
exclusively expressed in specific neuronal subtypes. For
example, a number of calcium-binding proteins and neuropeptides are exclusively found in GABAergic neocortical
neurons (reviewed in Wonders and Anderson 2006). With this
in mind, we included these genes in our search of the ABA.
Brains hybridized for Parvalbumin (Pvalb), Somatostatin (SST),
and Prodynorphin (Pdyn) were found to exhibit malformations
containing cells with expression of these transcripts (data not
shown), indicating that GABAergic neurons are found in
neocortical malformations.
Malformations were found in brains hybridized for expression of genes known to be enriched or exclusively expressed in
glia cells (Lein et al. 2007). For example, we observed midline
heterotopia in brains hybridized for the astrocyte-enriched
genes Gldc, S100b, Sox8, Sparc, and Sparcl1. We also observed
midline heterotopia in brains hybridized for oligodendrocyteenriched genes such as Car2, Fa2h, Mcam, Olig2, and Plekhb1.
Representative photomicrographs of astrocyte (Sparcl1) and
oligodendrocyte (Olig2) expression in heterotopia are shown
in Supplementary Figure 6.
A dorsal cortical heterotopion was observed in the brain
hybridized for the oligodendrocyte-enriched gene, Myelin basic
protein (Mbp). Interestingly, the expression profile of Mbp+
cells within the malformations revealed a pattern similar to our
results of the pattern of myelin staining found in heterotopia
following gold-chloride staining (Fig. 8).
These data indicate that the 2 major glia cell-types,
astrocytes and oligodendrocytes are found in heterotopia.
Moreover, these data indicate a change in the pattern of
oligodendrocytes and myelinated axons and suggest reorganization of axons entering and/or exiting heterotopia.
Ion Channels and Neurotransmitter Receptor Gene
Expression in Heterotopia
Our list of genes from which we examined the ABA contained
a number of ion channels and neurotransmitter receptors
Page 8 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
Figure 8. Changes in myelinated axons and oligodendrocytes found in dorsal
heterotopia. Representative photomicrographs of Nissl (A, B) and myelin-stained (C,
D) sections. Structural changes in myelinated axons found within a column extending
from the white matter up to the pial surface. (E--F) Images from ABA for case
hybridized for Mbp demonstrating labeled cells in a heterotopion.
genes known to play important roles in intrinsic electrophysiological phenotypes (e.g., voltage-gated K+ and Na+ channels),
synaptic transmission (e.g., GABA, acetylcholine, serotonin),
and synaptic plasticity (glutamatergic NMDA receptors). As
with all of the expression data found in the ABA, only
a percentage of brains hybridized for any given gene will
contain a malformation. Nevertheless, we identified a number
of cases hybridized for ion channels and neurotransmitter
receptors, which contained malformations. For example, we
identified expression of Ca2+, Cl–, K+, and Na+ channel genes in
cells within heterotopia, all of which are known to participate
in the electrophysiological function of neurons. Representative
photomicrographs demonstrating ion channel gene expression
in heterotopic cells (Ca2+, Cl–, K+) are shown in Supplementary
Figure 7. We also identified neurotransmitter receptors for
acetylcholine, corticotrophin releasing-hormone, dopamine,
GABA, glutamate (Glu), histamine, and serotonin in cells within
malformations. Representative photomicrographs demonstrating expression of GABA, Glu, and acetylcholine receptors are
shown in Figure 9. These data point to ion channel as well as
neurotransmitter receptor diversity present within neocortical
heterotopia.
Layer-Specific Gene Expression in Heterotopia
In order to estimate the neuronal birth dates of cells within
malformations, we looked for malformations in brains hybridized for genes expressed only in discrete neocortical layers.
This approach relies on the finding that 1) individual neocortical lamina are made up of neurons with similar birth dates
and that 2) early born neurons come to populate deep layers,
whereas subsequently generated neurons populate progressively more superficial layers (Angevine and Sidman 1961;
Caviness and Sidman 1973; Rakic 1974). For example,
examination of tissue in the ABA used to probe the Wolfram
syndrome 1 gene (Wfs1) revealed a midline heterotopion.
Closer examination and colorimetric analyses of Wfs1 expression revealed cells with strong expression exclusively in layer II
of normotopic neocortex as well as within the heterotopion
(Fig. 10A). Identical results were observed for tissue hybridized
for the Start-domain containing 8 gene (Stard8), Dendrin
(Ddn), and the RAS protein specific guanine nucleotide releasing factor 2 gene (Rasgrf2), which are preferentially found in
layer II cells (Fig. 10B). These data are shown in Figure 10 and
suggest that cells originally destined for layer II (late-born
neurons) are among the cells found in midline heterotopia.
In contrast to results described above, tissue used to assay
expression of genes found in deeper lamina were not observed
within midline heterotopia. For example, the case hybridized
for the Cysteine rich transmembrane BMP regulator 1 gene
(Crim1), which is found in layer III cells, contained a midline
heterotopion which lacked cells with detectable levels of
expression. Similar results were observed in tissue hybridized
for FEZ family zinc-finger 2 gene (Fezf2) and the chloridechannel gene, Clcn2, which are densely expressed in layer III
cells and only moderately expressed in layer II cells. We
observed that only lightly labeled Fezf2 or Clcn2 cells were
found in the heterotopia found in this tissue; heavily labeled
cells were not found in the malformation. These data are
illustrated in Supplementary Figure 8. Our results suggest that
layer III neurons (and perhaps deeper layer neurons) are not
found in midline heterotopia. Note that we did not find
Figure 9. Neurotransmitter receptor expression in heterotopia. Images from ABA for tissue hybridized for Glu (A), GABA (B), and Ach (C) receptors. Magnified images found in
middle (ISH) and bottom (colorimetric) panels.
Cerebral Cortex Page 9 of 15
Figure 10. Cellular constituents of midline heterotopia. (A--D) Representative
photomicrographs taken from the ABA containing midline heterotopia and hybridized
for genes enriched in layer II cells. Hybridization data shown on left and quantitative
colorimetric scale shown on right. Hybridized gene listed next to each micrograph
taken from ABA.
malformations in hybridized cases probed for other established
deep-layer genes (e.g. Ctip1, Er81, Otx1, Tbr1; reviewed in
Hevner et al. 2003, Molnar and Cheung 2006).
We performed a similar analysis in cases containing dorsal
cortical heterotopia hybridized for genes enriched in particular
neocortical layers, in order to determine the laminar origin of
cells found in heterotopia (Fig. 11). Genes specific to upper
and lower layer neurons were both found in dorsal cortical
heterotopia indicating that both early- and late-born neurons
are present. For example, we identified cells expressing S100b
in a heterotopion suggesting the presence of layer IV cells
within the malformation (Fig. 11C).
Changes in Gene Expression Density in Heterotopia
Colorimetric analyses allowed us to examine possible changes
in transcriptional activity for those genes hybridized in brains
Page 10 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
Figure 11. Cellular constituents of dorsal cortical heterotopia. (A--F) Representative
photomicrographs taken from the ABA containing dorsal cortical heterotopia and
hybridized for genes enriched in upper or deep-layer cells. Hybridization data shown
on left and quantitative colorimetric scale shown on right. Hybridized gene listed next
to each micrograph taken from ABA.
containing malformations. Two possible changes in transcriptional activity were hypothesized. Thus, 1) genes with low
expression levels may be upregulated in cells found in
malformations or 2) genes with high expression levels may
be downregulated in cells found in malformations. Surprisingly,
we did not find any evidence of significant changes of
expression density in cells found within malformations.
Within the ABA, we identified malformations in both the
sagittal and coronal-sectioned tissue hybridized for the same
gene (Supplementary Table 1). In addition, as described above,
individual brains with multiple malformations were often
observed. This allowed for a unique test of the reproducibility
of our observations of a lack of change in the expression
density of cells in malformations. For example, Figure 12
contains photomicrographs of a midline and dorsal heterotopion found in coronal and sagittal-cut tissue hybridized for
the voltage-gated K+ channel gene, Kcnj4. As can be seen, cells
in the midline (A, lower panel) or dorsal cortical heterotopion
(B, lower panel) do not exhibit changes in expression density
compared with normotopic cortex. Similar results were
obtained for tissue hybridized for Cox4i1, Clcn2, Grm5, and
Plagl1 (data not shown). These data indicate no change in
expression density among the genes we identified present in
cells found in malformations.
Discussion
The results from the present report detail the cytoarchitecture
of neocortical malformations found in a number of inbred mice
commonly used in behavioral, anatomical, and physiological
studies. Molecular layer heterotopia was the most commonly
observed malformation and could be classified according to
spatial/areal distribution. Specifically, molecular layer heterotopia in dorsal cortex had very stereotyped cytoarchitecture
and are similar to that previously observed in autoimmune mice
(Sherman et al. 1985, 1987; Sherman, Morrison, et al. 1990).
These heterotopia where observed across a wide range of
rostro-caudal and medio-lateral positions and were often
observable without magnification as bumps on the surface of
the intact brain. Interestingly, heterotopia was never found in
lateral cortical areas (rhinal cortices) indicating that heterotopia formation may be area specific.
Molecular layer heterotopia was also found in cortical areas
along the IHF (e.g., cingulate cortex). In contrast to those
observed in dorsal cortex, midline heterotopia had diverse
cytoarchitecture. Cells were observed in the white mater as
well as in between the 2 hemispheres. Cells were often
surrounded by white matter forming ‘‘islands.’’ We do not know
of any previous reports of midline heterotopia in inbred mice
although similar malformations have been observed in mutant
and transgenic mice (discussed below).
Other malformations of cortical development were also
observed in a number of inbred strains. These malformations
displayed a diverse cytoarchitecture and could be found in
more spatially diverse locations including rhinal cortices. These
malformations included phenotypes with tissue folds, invaginations, and aberrant white matter patterning.
Development of Neocortical Malformations
Molecular layer heterotopia in dorsal cortex has been studied
in detail especially in autoimmune mice, which display a high
incidence for neocortical malformation (Sherman et al. 1987;
Figure 12. No change in expression density in cells found in heterotopia. Data from ABA
where multiple brains, each containing a heterotopia, were hybrid for the same gene,
Kcnj4. Left panels (A) from brain cut along coronal plane containing a midline heterotopia.
Right panels (B) from brain cut along sagittal plane containing a dorsal cortical
heterotopia. Magnified images found in middle (ISH) and bottom (colorimetric) panels.
Sherman, Morrison, et al. 1990). These studies have revealed
several structural deficits in the developing neocortex of mice
with heterotopia, which likely contribute to their formation.
First, radial fibers from radial glial cells are reduced in number
and abnormally organized in areas underlying heterotopia
(Sherman et al. 1992). Thus, migrating neurons in areas lacking
the structural (and molecular) cues provided by radial glia
might migrate past their intended destination and into layer I.
Disruption of radial fibers along developing midline cortical
areas may therefore contribute to the formation of midline
heterotopia. These data emphasize the important role that
radial fibers play in neuronal migration and the development of
neocortical lamina (reviewed in Nadarajah and Parnavelas 2002;
Nadarajah 2003).
Migration deficits where neurons fail to migrate, such as
periventricular nodular heterotopia or subcortical band heterotopia, are often associated with radial fiber disruption.
Interestingly, molecular layer heterotopia is also observed after
genetic or environmental perturbation of radial glia. Overmigration into the molecular layer may be due to focal changes
in radial glia structure or molecular signaling which dramatically affects migrating neurons. For example, changes in the
terminal tufts of radial glia (which ramify in the molecular
layer) may no longer provide structural and molecular ‘‘detachment’’ cues for migrating neurons. Changes in detachment
cues may therefore result in neurons in the molecular layer.
A number of transgenic mice exhibit overmigration phenotypes such as molecular layer heterotopia, including mice with
deletion of extracellular matrix proteins, glycoproteins, and
Cerebral Cortex Page 11 of 15
transcription factors. Among many knockout mice worth
mentioning, the Intergrin linked kinase (Ilk) deletion mice
show a dramatic overmigration phenotype including molecular
layer heterotopia in both dorsal and midline cortices
(Niewmierzycka et al. 2005). Interestingly, Ilk knockouts
exhibit disorganization of radial glia fibers and displacement
of Cajal-Retzuis cells (Niewmierzycka et al. 2005). It is possible
that one or more of these anatomical changes are also present
in the developing brain of inbred mice and underlie the
formation of heterotopia. Supporting the role of radial glia in
overmigration phenotypes, it was shown recently that molecular layer heterotopia are found in mouse knockouts that have
radial glial fibers that fail to attach to the pial/glial limitans
(Haubst et al. 2006). Such is the case in mice knockouts for
Laminin c1III4, a6 integrin, and perlecan. In these mutants,
overmigration phenotypes were observed despite no changes
in the proliferative properties of radial glia (Haubst et al. 2006).
In addition to reduced and disorganized radial fibers, mice
with heterotopia also demonstrate disruption of the glial
limiting membrane (GLM; Rosen et al. 1992). Serving as an
important boundary between the brain and the subarachnoid
space, the GLM as well as the basement membrane (BM) keep
neurons from migrating out of the neocortex. Breaches in the
GLM and BM might enable neurons to migrate past layer I.
Thus, neuronal ‘‘islands’’ along the IHF are likely formed by
disruption of the GLM/BM along the midline. Likewise, tissue
folds and neurons found above the pial surface, as was observed
in more complex malformations, might be formed by neurons
that migrate past breaches in the GLM/BM. Consistent with an
important role in neuronal migration played by the GLM/BM,
disruption of the GLM/BM by surgical puncture at P1, results in
the neocortical heterotopia in mice (Rosen et al. 1992).
Moreover, neocortical heterotopia is prevalent in the neocortex
of the dreher mutant mouse (Lmx1a–/–) which exhibits
abnormalities in both the GLM and in blood vessel organization
in layer I (Costa et al. 2001). Likewise, bumps on the surface of
the cortex as we show in the intact brain, were recently
described in Laminin a6 knockouts (Georges-Labouesse et al.
1998). Recently, a number of mouse knockouts with deletion of
important BM proteins demonstrate a neocortical phenotype
similar (although more severe) to the malformations we observed
(Halfter et al. 2002; Michele et al. 2002; Moore et al. 2002; Beggs
et al. 2003; Niewmierzycka et al. 2005; Hu et al. 2007).
Interestingly, heterotopic ‘‘islands’’ of cells in between cerebellar
folia have been observed in dystroglycan knockout mice (Li et al.
forthcoming). The presence of this heterotopia phenotype at the
IHF and at cerebellar folia may indicate a common occurrence of
heterotopia at sites of cortical apposition.
A number of knockouts display complex malformations
similar to those we observed. For example, large neocortical
‘‘sulci-like’’ folds/invaginations and/or aberrant patterning of
layer I have been observed after deletion of Cpp32 (Caspase-3;
Kuida et al. 1996), Abi2 (Grove et al. 2004), and CgkI
(Demyanenko et al. 2005). How perturbation of these different
molecules result in the malformations of cortical development
is unknown.
cytes) were identified as well as 2 major neuronal subtypes
found in the cortex (GABAergic and glutamatergic neurons).
These data are consistent with previous data using electrophysiological techniques to identify putative glutamatergic
neurons as well as immunohistochemical methods to detect
GABAergic neurons in heterotopia (Sherman, Stone, Rosen,
et al. 1990; Gabel and LoTurco 2001).
Our search for malformations in tissue assayed for layerspecific genes, revealed the origin of neurons found in midline
heterotopia. These data indicate that layer II neurons are found
in midline heterotopia in contrast to deep-layer neurons, which
were not observed. Thus, the presence of upper layer neurons
in heterotopia suggests that these malformations may result
from deficits in migration of late-born neurons. In contrast, we
found evidence that neurons from both upper as well deeper
layers may contribute to the cellular constituents of dorsal
heterotopia. These data are consistent with the presence of
layer V--like burst firing neurons in heterotopia (NZB mice;
Gabel and LoTurco 2001). Future, birth-dating experiments
will help determine the precise birth dates of neurons in brains
with heterotopia.
Cellular Constituents of Neocortical Malformations
We used the ABA to identify a number of cell-types present in
heterotopia according to cell-type specific gene expression.
These data demonstrate that both neurons and glia are present
in heterotopia. Glial subtypes (astrocytes and oligodendro-
Neocortical Malformations in Inbred Mice Used in Strain
Studies and the Production of Transgenic Mice
Inbred mouse strains are an important tool toward greater
understanding of the genetic and neural organization of
behavior. Strain surveys coupled with genetic analyses such
Page 12 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
Transcriptional Diversity and Density in Neocortical
Malformations
Quantification of gene expression density (colorimetric) of
tissue found in the ABA allowed for determination of transcriptional up- or downregulation by cells found within heterotopia
compared with normotopic areas. Interestingly, we found no
evidence of significant changes in expression density. In light of
the fact that our data included tissue assayed for ion channel and
neurotransmitter receptor genes, these results suggest that little,
if any, changes in the synaptic and electrophysiological features
of cells found in malformations. An important caveat to this
interpretation is that not all the tissue examined for important
transmitter receptor and ion channel genes contained malformations. Moreover, messenger RNA and/or protein level changes
may take place during fetal and/or early postnatal periods.
Supporting a model of little change in ion channel genes, recent
electrophysiological recordings did not find major differences in
the intrinsic firing properties of neurons found in heterotopia
(Gabel and LoTurco 2001).
It remains unclear to what extent altered synaptic connections (Jenner et al. 2002) contribute to the decreased seizure
thresholds in mice with heterotopia (Gabel and LoTurco 2002).
Thus, even in the absence of any changes in the expression of
ion channels/transmitter receptors, brains with heterotopia may
exhibit excitability changes due to alteration of the balance
between excitatory and inhibitory synaptic connections. Our
observations of changes in the pattern of myelinated fibers in
dorsal cortical heterotopia are in agreement with axonal
changes in heterotopia found after neurofilament immunocytochemistry (Sherman, Stone, Press, et al. 1990) and suggest
dramatic circuit and synaptic reorganization. Future studies
using anterograde or retrograde tracing techniques will help
elucidate circuit-level changes in the neocortex of inbred mice
with malformations of cortical development.
as quantitative trait loci (QTL) analysis are critical in the
identification of genes and molecules involved in numerous
behaviors and phenotypes. Recently, inbred strains have been
used to study the genetic components of a wide range of
behaviors such as food intake (Bachmanov et al. 2002), learning
(fear: Bolivar et al. 2001; motor: McFadyen et al. 2003; spatial:
Wahlsten et al. 2005), visual acuity (Wong and Brown 2006),
grooming (Kalueff and Tuohimaa 2005), sexual behaviors
(Burns-Cusato et al. 2004), and aggressive behaviors (Mineur
et al. 2003). How neocortical malformations contribute to
intrastrain variability as well as interstrain differences in these
studies is unknown. However, results from behavioral and
physiological studies in mice with molecular layer heterotopia
are clear, indicating changes in cognitive function (Hyde et al.
2000, 2001, 2002) and sensory processing (Frenkel et al. 2000;
Peiffer et al. 2001). These results emphasize the importance of
histological examination of neocortical tissue from inbred mice
used in strain surveys.
An important finding from the present report is the
observation of neocortical malformations in nearly all inbred
mouse strains examined, whereas outbred mice were normal.
Although the incidences of malformations varied according to
strain, these data point to malformations of cortical development as a characteristic feature of several inbred mice strains.
How the diverse genotypes of the inbred mice examined result
in similar malformation phenotypes is not known. Our data and
that published previously by others points to a large list of
inbred mice and mouse mutants with cortical malformations
including many not found in our analysis such as NZB, BXSB,
MRL, Snell dwarf mutant (dw/dw), and Dreher mutant (dr/dr;
Sherman, Morrison, et al. 1990; Sherman, Stone, Press, et al.
1990). Interestingly, many different knockout (KO) mice have
also been identified with neocortical heterotopia like that which
we describe. These results point to potential overlapping/
multigene regulation of neocortical malformation development.
In the case of KO mice, is not clear why germline mutation or
deletion of a given gene results in such focal disruption of
neocortical lamination as is the case with molecular layer
heterotopia. This might be the reason why, despite numerous
descriptions of heterotopia in inbred/mutant/transgenic mice,
there does not exist adequate models of the genetic origin of
these neocortical malformations. Future studies, examining
other inbred strains and employing crosses of mice with high
and low incidence of heterotopia as well as QTL may provide
clues to the genetic component of neocortical malformations.
Similar to data relating to neocortical malformations, it is
well established that many different inbred mouse strains show
callosal dysgenesis/agenesis (Wahlsten et al. 2003). Moreover,
many different KO mice have been identified with callosal
dysgenesis/agenesis including those with deletion of transcription factors, extracellular matrix molecules, growth factors, and
intracellular signaling molecules (reviewed by Richards 2002).
Thus, similar acallosal phenotypes in inbred and KO mice point
to diverse genetic factors implicated in callosum development.
An interesting previous study is worth mentioning regarding
the genetic origin of the neocortical malformations that we
describe. For example, Sherman et al. (1992) reported that
when inbred mouse embryos belonging to a strain with high
incidence of heterotopia (NZB mice), are implanted into
a pregnant female of a strain with 0% incidence (outbred
mice), there is no change in the total incidence of malformations observed in the resulting litter. That is to say, the resulting
pups of that litter (100% NZB genotype; brains examined as
adults) have high incidence of heterotopia. Conversely, when
embryos of an outbred strain are implanted into a pregnant
NZB female, no animals in that resulting litter (100% outbred
genotype) are observed to contain malformations (brains also
examined as adults). Taken together, these data suggest that
neither the maternal (in utero) nor early postnatal environment
play a role in the etiology of malformations.
Mutant and transgenic mice have recently contributed
a great deal to the identification of genes and molecules
critical to brain development and plasticity. With the continued
utilization of engineered mouse models it is becoming increasingly important to characterize the intrinsic anatomy,
physiology, and behavior of the background strains from which
these models are created (Bothe et al. 2004). Specifically, KO
mice displaying phenotypes including neocortical heterotopia
should be carefully compared against observed incidences of
malformations in the background strain. Our results indicate
that inbred mouse strains often have rather severe malformation of cortical development. Thus, histological examination
can facilitate interpretation of results from studies with
transgenic as well as inbred mice.
Malformation of Cortical Development in Humans
Malformations of cortical development in humans are often
accompanied by developmental delay, cognitive deficits, and/or
epilepsy (Schwartzkroin and Walsh 2000; Schwartzkroin
et al. 2004). For example, post-mortem analysis of brains of
dyslexic patients revealed the presence of numerous molecular layer heterotopia in nearly every brain examined which
were identical to those heterotopia we observed in inbred
mice (Galaburda and Kemper 1979; Galaburda et al. 1985;
Humphreys et al. 1990). Molecular layer heterotopia has also
been observed in post-mortem epileptic patients (Eriksson
et al. 2001), implicating malformations of neocortical development with epilepsy. However, the role of heterotopia in
epilepsy and cognitive impairment has remained controversial
due to the presence of heterotopia in the general population
(Meencke and Janz 1984; Lyon and Gastaut 1985; Kasper et al.
1999). For example, in addition to finding heterotopia in the
brains of dyslexics, Galaburda and colleagues also found them
in a sample of brains of clinically normal individuals (Kaufmann
and Galaburda 1989). These authors identified heterotopia in
approximately 30% of brains of clinically normal individuals
which were identical in morphology and cytoarchitecture to
those found in brains from dyslexics. However, the total
number of heterotopia found in each affected brain (1--2
heterotopia) of clinically normal individuals was dramatically
reduced compared with that found in brains of dyslexics
(30--140 heterotopia; Kaufmann and Galaburda 1989).
It is not clear how to compare the incidences of malformations found in humans with those found in inbred mice or
other animal models. The reported occurrence of malformations in inbred mice varied considerably according to strain
and we did not find any malformations in brains from outbred
mice or SWR/J inbred mice. Thus, based on our data and
that reported from studies of brains of normal individuals,
one would conclude that humans display greater incidence of
heterotopia that that observed in outbred mice and SWR inbred
mice. The incidences of malformations in A/J, C57BL/6J,
C3H/HeJ, and CBA/J mice (Table 1) most closely match that
observed in brains of clinically normal humans (e.g., Kaufmann
Cerebral Cortex Page 13 of 15
and Galaburda 1989), whereas C57BL/10J mice had a very high
incidence of malformation (~80%) similar to that observed in
dyslexic humans (Galaburda and Kemper 1979; Galaburda et al.
1985; Humphreys et al. 1990).
Whether our results in mice will serve as a good model for
studies of malformations in humans with dyslexia and/or
epilepsy will depend on further investigation of the relationship between heterotopia and these diseases. Nevertheless, the
well-documented relationship between the presence of heterotopia in inbred mice and cognitive/sensory deficits (Frenkel
et al. 2000; Peiffer et al. 2001) and excitability changes (Gabel
and LoTurco 2002) serves as an important platform upon
which to further investigate the effects of malformations of
cortical development on cortical function.
Supplementary Material
Supplementary material
oxfordjournals.org/.
can
be
found
at:
http://www.cercor.
Funding
Howard Hughes Medical Institute (Undergraduate Science
Education Program) award to Queens College, CUNY
(#52005118) supported R.L.R.; and (NS058758-01A1) to J.C.B.
Notes
We thank Sandra Giraldo and Jason Abramowitz for help with histology
and Dr Ben Kest and Dr Richard Bodnar for the generous gift of inbred
mice. We thank Gad Klein, Dr Lisa Gabel, Dr Matthew Sarkisian, and
Dr Eric Richfield for helpful comments. Conflict of Interest : None
declared.
Address correspondence to Joshua C. Brumberg, PhD, Department of
Psychology, Queens College, CUNY, Flushing, NY 11367, USA. Email:
joshua.brumberg@qc.cuny.edu.
References
Ang ES Jr, Gluncic V, Duque A, Schafer ME, Rakic P. 2006. Prenatal
exposure to ultrasound waves impacts neuronal migration in mice.
Proc Natl Acad Sci USA. 103(34):12903--12910.
Angevine JB Jr., Sidman RL. 1961. Autoradiographic study of cell
migration during histogenesis of the cerebral cortex in the mouse.
Nature. 192:766--768.
Bachmanov AA, Reed DR, Beauchamp GK, Tordoff MG. 2002. Food
intake, water intake, and drinking spout side preference of 28
mouse strains. Behav Genet. 32(6):435--443.
Balogh S, Sherman G, Hyde L, Denenberg V. 1998. Effects of neocortical
ectopias upon the acquisition and retention of a non-spatial
reference memory task in BXSB mice. Dev Brain Res. 111:291--293.
Beggs HE, Schahin-Reed D, Zang K, Goebbels S, Nave KA, Gorski J,
Jones KR, Sretavan D, Reichardt LF. 2003. FAK deficiency in cells
contributing to the basal lamina results in cortical abnormalities
resembling congenital muscular dystrophies. Neuron. 40(3):501--514.
Bielas S, Higginbotham H, Koizumi H, Tanaka T, Gleeson JG. 2002.
Cortical neuronal migration mutants suggest separate but intersecting pathways. Annu Rev Cell Dev Biol. 20:593--618.
Boehm G, Sherman G, Hoplight B, Hyde L, Waters N, Bradway D,
Galaburda A, Denenberg V. 1996. Learning and memory in the
autoimmune BXSB mouse: effects of neocortical ectopias and
environmental enrichment. Brain Res. 726:11--22.
Bolivar VJ, Pooler O, Flaherty L. 2001. Inbred strain variation in
contextual and cued fear conditioning behavior. Mamm Genome.
12(8):651--656.
Bothe GWM, Bolivar VJ, Vedder MJ, Geistfeld JG. 2004. Genetic and
behavioral differences among five inbred mouse strains commonly
used in the production of transgenic and knockout mice. Genes
Brain Behav. 3(3):149--157.
Page 14 of 15 Neocortical Malformations in Inbred Mice
d
Ramos et al.
Burns-Cusato M, Scordalakes EM, Rissman EF. 2004. Of mice and
missing data: what we know (and need to learn) about male sexual
behavior. Physiol Behav. 83(2):217--232.
Caviness VS Jr., Sidman RL. 1973. Time of origin of corresponding cell
classes in the cerebral cortex of normal and reeler mutant mice: an
autoradiographic analysis. J Comp Neurol. 148:141--151.
Clark MG, Sherman GF, Bimonte HA, Fitch RH. 2000. Perceptual
auditory gap detection deficits in male BXSB mice with cerebrocortical ectopias. Neuroreport. 11(4):693--696.
Costa C, Harding B, Copp AJ. 2001. Neuronal migration defects in the
Dreher (Lmx1a) mutant mouse: role of disorders of the glial limiting
membrane. Cereb Cortex. 11(6):498--505.
Demyanenko GP, Halberstadt AI, Pryzwansky KB, Werner C, Hofmann F,
Maness PF. 2005. Abnormal neocortical development in mice lacking
cGMP-dependent protein kinase I.. Brain Res Dev Brain Res. 160(1):1--8.
Denenberg VH, Sherman GF, Schrott L, Rosen GD, Galaburda AM. 1991.
Spatial learning, discrimination learning, paw preference and neocortical
ectopias in two autoimmune strains of mice. Brain Res. 562:98--104.
Eriksson SH, Rydenhag B, Uvebrant P, Malmgren K, Nordborg C. 2001.
Widespread microdysgenesis in therapy-resistant epilepsy—a case
report on post-mortem findings. Acta Neuropathol. 103:74--77.
Frenkel M, Sherman GF, Bashan KA, Galaburda AM, LoTurco JJ. 2000.
Neocortical ectopias are associated with attenuated neurophysiological responses to rapidly changing auditory stimuli. Neuroreport.
11(3):575--579.
Gabel LA, LoTurco JJ. 2001. Electrophysiological and morphological
characterization of neurons within neocortical ectopias. J Neurophysiol. 85(2):495--505.
Gabel LA, LoTurco JJ. 2002. Layer I ectopias and increased excitability
in murine neocortex. J Neurophysiol. 87(5):2471--2479.
Galaburda AM. 2005. Dyslexia—a molecular disorder of neuronal
migration: the 2004 Norman Geschwind Memorial Lecture. Ann
Dyslexia. 55(2):151--165.
Galaburda AM, Kemper TL. 1979. Cytoarchitectonic abnormalities in
developmental dyslexia: a case study. Ann Neurol. 6(2):94--100.
Galaburda AM, LoTurco J, Ramus F, Fitch RH, Rosen GD. 2006. From
genes to behavior in developmental dyslexia. Nat Neurosci.
9(10):1213--1217.
Galaburda AM, Sherman GF, Rosen GD, Aboitiz F, Geschwind N. 1985.
Developmental dyslexia: four consecutive patients with cortical
anomalies. Ann Neurol. 18:222--233.
Georges-Labouesse E, Mark M, Messaddeq N, Gansmuller A. 1998.
Essential role of alpha 6 integrins in cortical and retinal lamination.
Curr Biol. 8(17):983--986.
Gleeson JG, Walsh CA. 2000. Neuronal migration disorders: from
genetic diseases to developmental mechanisms. Trends Neurosci.
23(8):352--359.
Grove M, Demyanenko G, Echarri A, Zipfel PA, Quiroz ME,
Rodriguiz RM, Playford M, Martensen SA, Robinson MR,
Wetsel WC, et al. 2004. ABI2-deficient mice exhibit defective cell
migration, aberrant dendritic spine morphogenesis, and deficits in
learning and memory. Mol Cell Biol. 24(24):10905--10922.
Gupta A, Tsai LH, Wynshaw-Boris A. 2002. Life is a journey: a genetic
look at neocortical development. Nat Rev Genet. 3(5):342--355.
Halfter W, Dong S, Yip YP, Willem M, Mayer U. 2002. A critical function
of the pial basement membrane in cortical histogenesis. J Neurosci.
22(14):6029--6040.
Hevner RF, Daza RA, Rubenstein JL, Stunnenberg H, Olavarria JF, Englund C.
2003. Beyond laminar fate: toward a molecular classification of cortical
projection/pyramidal neurons. Dev Neurosci. 25(2--4):139--151.
Hu H, Yang Y, Eade A, Xiong Y, Qi Y. 2007. Breaches of the pial
basement membrane and disappearance of the glia limitans during
development underlie the cortical lamination defect in the mouse
model of muscle-eye-brain disease. J Comp Neurol. 501(1):168--183.
Haubst N, Georges-Labouesse E, De Arcangelis A, Mayer U, Götz M.
2006. Basement membrane attachment is dispensable for radial glial
cell fate and for proliferation, but affects positioning of neuronal
subtypes. Development. 133(16):3245--3254.
Humphreys P, Kaufmann WE, Galaburda AM. 1990. Developmental
dyslexia in women: neuropathological findings in three patients.
Ann Neurol. 28(6):727--738.
Hyde LA, Sherman GF, Hoplight BJ, Denenberg VH. 2000. Working
memory deficits in BXSB mice with neocortical ectopias. Physiol
Behav. 70(1--2):1--5.
Hyde LA, Hoplight BJ, Harding S, Sherman GF, Mobraaten LE, Denenberg VH.
2001. Effects of ectopias and their cortical location on several measures
of learning in BXSB mice. Dev Psychobiol. 39(4):286--300.
Hyde LA, Stavnezer AJ, Bimonte HA, Sherman GF, Denenberg VH. 2002.
Spatial and nonspatial Morris maze learning: impaired behavioral
flexibility in mice with ectopias located in the prefrontal cortex.
Behav Brain Res. 133(2):247--259.
Jenner A, Galaburda A, Sherman G. 2002. Connectivity of ectopic
neurons in the molecular layer of the somatosensory cortex in
autoimmune mice. Cereb Cortex. 10:1005--1013.
Kaufmann WE, Galaburda AM. 1989. Cerebrocortical microdysgenesis
in neurologically normal subjects: a histopathologic study. Neurology. 39(2 Pt 1):238--244.
Kalueff AV, Tuohimaa P. 2005. Contrasting grooming phenotypes in
three mouse strains markedly different in anxiety and activity
(129S1, BALB/c and NMRI). Behav Brain Res. 160(1):1--10.
Kasper BS, Stefan H, Buchfelder M, Paulus W. 1999. Temporal lobe
microdysgenesis in epilepsy versus control brains. J Neuropathol
Exp Neurol. 58:22--28.
Kuida K, Zheng TS, Na S, Kuan C, Yang D, Karasuyama H, Rakic P,
Flavell RA. 1996. Decreased poptosis in the brain and premature
lethality in CPP32-deficient mice. Nature. 384(6607):368--372.
Lein ES, et al. 2007. Genome-wide atlas of gene expression in the adult
mouse brain. Nature. 445(7124):168--176.
Li X, Zhang P, Yang Y, Xiong Y, Qi Y, Hu H. forthcoming. Differentiation
and developmental origin of cerebellar granule neuron ectopia in
POMGnT1 knockout mice. Neuroscience. doi: 10.1016/j.neuroscience.
2007.06.041.
Ling EA, Paterson JA, Privat A, Mori S, Leblond CP. 1973. Investigation of
glial cells in semithin sections. I. Identification of glial cells in the
brains of young rats. J Comp Neurol. 149:43--71.
Lyon G, Gastaut H. 1985. Considerations on the significance attributed
to unusual cerebral histological findings recently described in eight
patients with primary generalized epilepsy. Epilepsia. 26:365--367.
McFadyen MP, Kusek G, Bolivar VJ, Flaherty L. 2003. Differences among
eight inbred strains of mice in motor ability and motor learning on
a rotorod. Genes Brain Behav. 2(4):214--219.
Meencke HJ, Janz D. 1984. Neuropathological findings in primary
generalized epilepsy: a study of eight cases. Epilepsia. 25:8--21.
Michele DE, Barresi R, Kanagawa M, Saito F, Cohn RD, Satz JS, Dollar J,
Nishino I, Kelley RI, Somer H, et al. 2002. Post-translational
disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature. 418(6896):417--422.
Mineur YS, Prasol DJ, Belzung C, Crusio WE. 2003. Agonistic behavior
and unpredictable chronic mild stress in mice. Behav Genet. 33(5):
513--519.
Molnar Z, Cheung AF. 2006. Towards the classification of subpopulations
of layer V pyramidal projection neurons. Neurosci Res. 55(2):105--115.
Moore SA, Saito F, Chen J, Michele DE, Henry MD, Messing A, Cohn RD,
Ross-Barta SE, Westra S, Williamson RA, et al. 2002. Deletion of brain
dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature. 418(6896):422--425.
Nadarajah B. 2003. Radial glia and somal translocation of radial neurons
in the developing cerebral cortex. Glia. 43(1):33--36.
Nadarajah B, Parnavelas JG. 2002. Modes of neuronal migration in the
developing cerebral cortex. Nat Rev Neurosci. 3(6):423--432.
Niewmierzycka A, Mills J, St-Arnaud R, Dedhar S, Reichardt LF. 2005.
Integrin-linked kinase deletion from mouse cortex results in
cortical lamination defects resembling cobblestone lissencephaly. J
Neurosci. 25(30):7022--7031.
Olson EC, Walsh CA. 2002. Smooth, rough and upside-down neocortical
development. Curr Opin Genet Dev. 12(3):320--327.
Peiffer AM, Dunleavy CK, Frenkel M, Gabel LA, LoTurco JJ, Rosen GD,
Fitch RH. 2001. Impaired detection of variable duration embedded
tones in ectopic NZB/BINJ mice. Neuroreport. 12(13):2875--2879.
Rakic P. 1974. Neurons in rhesus monkey visual cortex: systematic
relation between time of origin and eventual disposition. Science.
183(123):425--427.
Ramos RL, Bai J, LoTurco JJ. 2006. Heterotopia formation in rat but not
mouse neocortex after RNA interference knockdown of DCX.
Cereb Cortex. 16(9):1323--1331.
Richards LJ. 2002. Axonal pathfinding mechanisms at the cortical
midline and in the development of the corpus callosum. Braz J Med
Biol Res. 35(12):1431--1439.
Rosen GD, Sherman GF, Richman JM, Stone LV, Galaburda AM. 1992.
Induction of molecular layer ectopias by puncture wounds in
newborn rats and mice. Brain Res Dev Brain Res. 67(2):285--291.
Satorre J, Cano J, Reinoso-Suárez F. 1986. Quantitative cellular changes
during postnatal development of the rat dorsal lateral geniculate
nucleus. Anat Embryol. 174:321--327.
Schmidt SL, Lent R. 1987. Effects of prenatal irradiation on the
development of cerebral cortex and corpus callosum of the mouse.
J Comp Neurol. 264(2):193--204.
Schmitz C, Hof PR. 2005. Design-based stereology in neuroscience.
Neuroscience. 130(4):813--831.
Schwartzkroin PA, Roper SN, Wenzel HJ. 2004. Cortical dysplasia and
epilepsy: animal models. Adv Exp Med Biol. 548:145--174.
Schwartzkroin PA, Walsh CA. 2000. Cortical malformations and
epilepsy. Ment Retard Dev Disabil Res Rev. 6(4):268--280.
Seecharan DJ, Kulkarni AL, Lu L, Rosen GD, Williams RW. 2003. Genetic
control of interconnected neuronal populations in the mouse
primary visual system. J Neurosci. 23(35):11178--11188.
Sherman GF, Galaburda AM, Behan PO, Rosen GD. 1987. Neuroanatomical
anomalies in autoimmune mice. Acta Neuropathol (Berl). 74(3):239--242.
Sherman GF, Galaburda AM, Geschwind N. 1985. Cortical anomalies in
brains of New Zealand mice: a neuropathologic model of dyslexia?
Proc Natl Acad Sci USA. 82(23):8072--8074.
Sherman GF, Holmes LB. 1999. Cerebrocortical microdysgenesis is
enhanced in C57BL/6J mice exposed in utero to acetazolamide.
Teratology. 60(3):137--142.
Sherman GF, Morrison L, Rosen GD, Behan PO, Galaburda AM. 1990. Brain
abnormalities in immune defective mice. Brain Res. 532(1--2):25--33.
Sherman GF, Rosen GD, Stone LV, Press DM, Galaburda AM. 1992. The
organization of radial glial fibers in spontaneous neocortical
ectopias of newborn New Zealand black mice. Brain Res Dev Brain
Res. 67(2):279--283.
Sherman GF, Stone JS, Press DM, Rosen GD, Galaburda AM. 1990. Abnormal
architecture and connections disclosed by neurofilament staining in
the cerebral cortex of autoimmune mice. Brain Res. 529(1--2):202--207.
Sherman GF, Stone JS, Rosen GD, Galaburda AM. 1990. Neocortical VIP
neurons are increased in the hemisphere containing focal cerebrocortical microdysgenesis in New Zealand Black mice. Brain Res.
532(1--2):232--236.
Wahlsten D, Bachmanov A, Finn DA, Crabbe JC. 2006. Stability of inbred
mouse strain differences in behavior and brain size between laboratories
and across decades. Proc Natl Acad Sci USA. 103(44):16364--16369.
Wahlsten D, Colbourne F, Pleus R. 2003. A robust, efficient and flexible
method for staining myelinated axons in blocks of brain tissue. J
Neurosci Methods. 123(2):207--214.
Wahlsten D, Cooper SF, Crabbe JC. 2005. Different rankings of inbred
mouse strains on the Morris maze and a refined 4-arm water escape
task. Behav Brain Res. 165(1):36--51.
Wahlsten D, Metten P, Crabbe JC. 2003. Survey of 21 inbred mouse
strains in two laboratories reveals that BTBR T/+ tf/tf has severely
reduced hippocampal commissure and absent corpus callosum.
Brain Res. 971(1):47--54.
Williams RW, Rakic P. 1988a. Elimination of neurons from the rhesus
monkey’s lateral geniculate nucleus during development. J Comp
Neurol. 272:424--436.
Williams RW, Rakic P. 1988b. Three-dimensional counting: an accurate
and direct method to estimate numbers of cells in sectioned
material. J Comp Neurol. 278:344--352.
Wonders CP, Anderson SA. 2006. The origin and specification of cortical
interneurons. Nat Rev Neurosci. 7(9):687--696.
Wong AA, Brown RE. 2006. Visual detection, pattern discrimination and
visual acuity in 14 strains of mice. Genes Brain Behav. 5(5):389--403.
Zheng QY, Johnson KR, Erway LC. 1999. Assessment of hearing in
80 inbred strains of mice by ABR threshold analyses. Hear Res.
130(1--2):94--107.
Cerebral Cortex Page 15 of 15