Download Ethanol Induces Heterotopias in Organotypic

Ethanol Induces Heterotopias in
Organotypic Cultures of Rat Cerebral Cortex
Sandra M. Mooney1, Julie A. Siegenthaler1 and
Michael W. Miller1,2
1Department
of Neuroscience and Physiology, SUNY Upstate
Medical University, Syracuse, NY 13210, USA and 2Research
Service, Veterans Affairs Medical Center, Syracuse, NY 13210,
USA
Abnormalities in the migration of cortical neurons to ectopic sites
can be caused by prenatal exposure to ethanol. In extreme cases,
cells migrate past the pial surface and form suprapial heterotopias or
‘warts’. We used organotypic slice cultures from 17-day-old rat
fetuses to examine structural and molecular changes that accompany wart formation. Cultures were exposed to ethanol (0, 200, 400
or 800 mg/dl) and maintained for 2–32 h. Fixed slices were sectioned
and immunolabeled with antibodies directed against calretinin,
reelin, nestin, GFAP, doublecortin, MAP-2 and NeuN. Ethanol
promoted the widespread infiltration of the marginal zone (MZ) with
neurons and the focal formation of warts. The appearance of warts
is time- and concentration-dependent. Heterotopias comprised
migrating neurons and were not detected in control slices. Warts
were associated with breaches in the array of Cajal–Retzius cells
and with translocation of reelin-immunoexpression from the MZ to
the outer limit of the wart. Ethanol also altered the morphology of the
radial glia. Thus, damage to the integrity of superficial cortex allows
neurons to infiltrate the MZ, and if the pial–subpial glial barrier is also
compromised these ectopic neurons can move beyond the normal
cerebral limit to form a wart.
Keywords: alcohol, Cajal–Retzius cells, doublecortin, ectopia, fetal alcohol
syndrome, migration, radial glia, reelin
Introduction
characterized by migration defects in the cortex and cerebellum (Hong et al., 2000).
Reeler mice cannot express reelin, a large glycoprotein
produced primarily by Cajal–Retzius cells in the MZ and deposited into the extracellular space (D’Arcangelo et al., 1995;
Hirotsune et al., 1995; Ogawa et al., 1995; Soda et al., 2003).
Reelin binds to at least two receptors, very low density lipoprotein receptor (VLDLR) and apolipoprotein-E receptor type
2 (ApoER2) (Hiesberger et al., 1999; Trommsdorff et al., 1999).
Reelin-receptor binding causes the phosphorylation of an
adaptor protein Disabled 1 (Dab1). Dab1 is attached to the
intracellular domain of VLDLR and ApoER2 (Rice et al., 1998;
Howell et al., 1999). Reelin also can bind to integrin α3,
integrin β1 and members of the cadherin-related neuronal
receptor, all of which are important for neuronal migration
(Sezaki et al., 1999; Dulabon et al., 2000).
Though controversial, reelin (i) may act as a chemoattractant, goading the young neurons to migrate into the
cortical plate, or (ii) it may serve as a signal for young neurons
to stop their migration and to detach from their glial guide
(Pearlman and Sheppard, 1996; Marin-Padilla, 1998; Dulabon et
al., 2000; Hack et al., 2002; Nishikawa et al., 2002; Tabata and
Nakajima, 2002; Luque et al., 2003). Reelin may also regulate
the function and identity of radial glia (Marín and Rubenstein,
2003).
Neuron–Radial Glia Interactions
The interaction between neurons and radial glia is important
for guiding young neurons through the complex matrix of the
intermediate zone (IZ) and cortical plate (CP). Breakdown in
the neuron–radial glia interaction can result in neurons being
distributed in an ectopic site(s). An example of such a disruption occurs in the murine mutant reeler. In the reeler mouse,
neuron-glia interactions are obstructed (Pinto-Lord et al., 1982)
and cells in the marginal zone (MZ), notably Cajal–Retzius
cells, are disorganized (Derer, 1985) and dysfunctional
(D’Arcangelo et al., 1995). A small collection of lissencephaly
cases in humans are linked to mutations in the reeler gene,
Errors in Neuronal Migration
Small alterations in neuronal migration can produce defects in
cortical organization. For example, exposure to moderate
amounts of ethanol in vivo can desynchronize the migration
process. The initiation of migration can be delayed and the rate
of migration retarded (Miller, 1993). The net result is that the
columnar organization of cortex can be disrupted so that cells
with particular (generational and/or connectional) phenotypes
can terminate their migration in ectopic sites (Miller, 1986,
1987, 1988b, 1993, 1997) and form abnormal synaptic relations (Miller, 1987; Al-Rabiai and Miller, 1989). Likely these
alterations underlie the mental retardation associated with fetal
alcohol exposure. Other neurological disorders, e.g. autism,
Asperger’s syndrome, dyslexia and Rett syndrome, exhibit
similar alterations in cortical columnar organization and
circuitry (Casanova et al., 2002a,b,c, 2003).
Disruptions of the orderly sequence of neuronal migration
can produce heterotopic clusters: intra- and supracortical
heterotopias. They are common among people with a variety
of neurological disorders (e.g. Dunn et al., 1986; Shaw, 1987).
These include epilepsy (Blom et al., 1984; Livingston and
Aicardi, 1990; Palmini et al., 1991), dyslexia (Galaburda and
Kemper, 1979; Galaburda et al., 1985; Humphreys et al.,
1990), fetal alcohol syndrome (Clarren and Smith, 1978;
Cerebral Cortex V 14 N 10 © Oxford University Press 2004; all rights reserved
Cerebral Cortex October 2004;14:1071–1080; doi:10.1093/cercor/bhh066
During normal migration, neurons with a particular time of
origin are distributed within a specific stratum of cortex (e.g.
Angevine and Sidman, 1961; Berry and Rogers, 1965; Rakic,
1974; Luskin and Shatz, 1985; Miller, 1985, 1988a). Earlygenerated neurons migrate to deep cortex and later generated
neurons take progressively more superficial positions. This
inside-to-outside development of normal cerebral cortex
depends upon interactions between young neurons and radial
glia. The neuron–glia association is key for establishing the
columnar organization that characterizes cerebral cortex.
Clarren et al., 1978; Peiffer et al., 1979; Konovalov et al.,
1997), Fukuyama muscular dystrophy disease (Fukuyama et al.,
1960), muscle–eye–brain disease (Santavuori et al., 1977;
1989), subcortical laminar heterotopia (also known as band
heterotopia or doublecortex; Gleeson et al., 1998; des Portes et
al., 1998a, b), Walker–Warburg syndrome (Williams et al.,
1984), X-linked lissencephaly (Dobyns et al., 1996; Ross et al.,
1997) and X-linked periventricular heterotopias (Fox et al.,
1998).
Recent studies suggest that heterotopias form following
damage to the pial membrane (PM; also known as pial basement membrane). Mice lacking integrins α3, α6 or β1 display
major defects in the cortical PM that lead to the formation of
ectopias (Georges-Labouesse et al., 1998; Graus-Porta et al.,
2001). During development, damage to the PM caused by physical trauma precedes the appearance of heterotopias (MarinPadilla, 1996). Loss or dysfunction of proteins that comprise
the PM, including laminin (Halfter et al., 2002), perlecan
(Costell et al., 1999) and chondroitin sulfate proteoglycans
(Blackshear et al., 1997), induce a breakdown of the PM
leading to over-migration of neurons and the formation of peripial heterotopias.
Developmental exposure to ethanol can produce errors in
migration in rodents (e.g. Miller, 1986, 1992, 1993). Such
defects have been identified throughout the nervous system,
e.g. neural crest cells (Rovasio and Battiato, 2002), brainstem
neurons (Zhou et al., 2001), and cerebellum (Kornguth et al.,
1979; Lewis, 1985; Liesi, 1997). The most thoroughly studied
area is the neocortex. Based on their time of origin, neurons
migrate to inappropriate sites and the kinetics of neuronal
migration is altered (Miller, 1986, 1988c, 1993, 1997; Miller et
al., 1990; Komatsu et al., 2001; Siegenthaler and Miller, 2004).
A common sequela is the formation of heterotopic cell clusters
within cortex and beyond the pial surface (Miller, 1986, 1987,
1988c, 1993; Gressens et al., 1992; Kotkoskie and Norton,
1988; Miller et al., 1990; Komatsu et al., 2001; Sakata-Haga et
al., 2002; Mooney and Miller, 2003). Suprapial heterotopias are
also referred to as ‘warts’. We hypothesize that these warts (i)
represent neurotoxic effects of ethanol on the termination of
neuronal migration and (ii) result from disruptions in reelin
expression in the MZ.
Materials and Methods
Culture Models
Organotypic cultures were prepared from rat fetuses on gestational
day (G) 17. To ensure uniformity, the day on which a sperm-positive
plug was first found was designated G1. Pregnant dams were anesthetized (90 mg/kg ketamine and 9 mg/kg xylazine), their uteri were
exteriorized and the fetuses removed. The fetal cranium was opened
with a mid-sagittal cut to expose the cerebral vesicles. Brains were
carefully removed and cut into 300 µm thick coronal slices using a
McIlwain Tissue Chopper (Mickle Lab. Eng., Gomshall, UK).
Tissue was rinsed in ice-cold stock medium [minimum essential
medium (GibcoBRL, Grand Island, NY), 24% glucose, 1.0% penicillin/
streptomycin and 25 mM potassium chloride] supplemented with 20%
fetal bovine serum. Samples were transferred onto a culture filter
(PICMORG50; Millipore, Bedford, MA) in a Petri dish containing fresh
supplemented stock medium. After a 3 h incubation (37°C, 6% CO2),
the medium was replaced with a serum-free stock medium containing
ethanol (0, 200, 400, or 800 mg/dl). This bridges the range of ethanol
exposures described in human and rat studies documenting heterotopias (Clarren et al., 1978; Kotkoskie and Norton, 1988; Komatsu et al.,
2001; Sakata-Haga et al., 2002). Moreover, bioassays show that exposures to ethanol needed to alter neural development in vivo are less
1072 Cortical Heterotopias Induced by Ethanol • Mooney et al.
than half that needed to replicate the alteration in vitro (e.g. cf. Miller
and Nowakowski, 1991; Jacobs and Miller, 2001; and cf. Miller, 1993;
Siegenthaler and Miller, 2004). The preparations were maintained in
the ethanol-containing medium for 2, 6, 8, 16, 24, or 32 h. Some preparations were incubated for 17 h in a medium containing 0.0040% 5bromo-2-deoxyuridine (BrdU) to label cells passing though the S-phase
of the cell cycle.
Anatomical Preparations
Tissues were fixed with 4.0% paraformaldehyde in 0.10 M phosphate
buffer (pH 7.4; PB) for 30 min at room temperature. Fixed samples
were washed in solutions of 10% and then 30% sucrose in 0.10 M PB
at 4°C. Tissue was rapidly frozen and cryosectioned at 15 µm.
The immunolabeling was begun by a pre-incubation step to quench
non-specific immunoreactivity. Sections were washed for 1 h in a solution of 5.0% non-fat milk and 1.0% bovine serum albumin in 0.10 M
phosphate buffered saline containing 0.10% Triton X-100 (mPBS).
Specific immunolabeling began by incubating the sections with a
specific primary antibody. Primary antibodies were directed against
calretinin (MAB1568, 1:500 in mPBS; Chemicon), reelin (SC-5578,
1:250 in mPBS; Santa Cruz), nestin (MAB353, 1:200 in mPBS;
Chemicon), vimentin (1:500 in mPBS; Sigma), glial fibrillary acidic
protein (GFAP; G-3893, 1:1000 in mPBS; Sigma), NeuN (MAB377,
1:1000 in mPBS; Chemicon), microtubule associated protein 2 (MAP2, 1:500 in mPBS; Sigma), and doublecortin (generously provided by
Christopher Walsh; 1:250 in mPBS).
Unbound primary antibody was removed by washing the sections
in mPBS. Tissue was then incubated in biotinylated secondary antimouse or anti-rabbit IgG (1:1000; Vector, Burlingame CA) for 1 h.
Sections were incubated in avidin–horseradish peroxidase conjugate
(Vector). Immunoreactivity was visualized by washing the sections
with 3,3′-diaminobenzidine (DAB; Vector) in the presence of
hydrogen peroxidase. Some secondary antibodies were bound to a
fluorescent tag. All immunolabeling steps were performed at room
temperature. Finally, sections were counterstained with cresyl
violet.
Cultures treated with BrdU were immunolabeled with an anti-BrdU
antibody (347580, 1:30 in mPBS; BD Biosciences). Sections were
treated for immunohistochemistry as described above with the addition of a pretreatment in 2 N HCl prior to application of the anti-BrdU
antibody.
Some sections were processed for terminal uridylated nick-end
labeling (TUNEL, S7100; Intergen) which identifies cells exhibiting
fragmented DNA, i.e. apoptotic cells (e.g. Jacobs and Miller, 2001).
Briefly, tissue was exposed to terminal deoxynucleotidyl transferase
TdT for 1 h at 37°C, rinsed in PBS and then incubated in a peroxidaseconjugated anti-digoxigenin. Immunolabeling was visualized by
washing the tissue in a solution of DAB in the presence of hydrogen
peroxide (see above). The sections were counterstained with methyl
green to assess nuclear appearance.
Results
Normal Cortical Structure
Five main strata were distinguished in the cerebral walls of
organotypic slice cultures (Fig. 1). The upper part of the cerebral wall contained the MZ and CP. The MZ was relatively acellular and the CP was cell-dense. The IZ was deep to the CP. It
was discriminable by linear arrays of cells. Two deepest zones,
the ventricular zone (VZ) and the subventricular zone (SZ)
were densely packed with cell bodies. The VZ was a pseudostratified columnar epithelium and the SZ contained more
haphazardly arranged cell bodies.
Heterotopias were not evident in control-treated cultures.
The MZ was largely devoid of neuronal somata and no cells
broached the integrity of the pial surface.
Figure 1. Cytoarchitecture of the cerebral wall. (A) Five main strata are distinguished
in the cerebral wall of an organotypic slice culture: the marginal zone (MZ), cortical
plate (CP), intermediate zone (IZ), subventricular zone (SZ) and ventricular zone (VZ).
(B) These five regions are discriminable in ethanol-treated slices. (C) This ethanoltreated slice shows an ill-defined MZ characterized by increased cellularity. Scale bars
= 50 µm.
Ethanol-induced Warts
Appearance
Heterotopias were apparent in many ethanol-treated preparations. Within the cerebral wall, heterotopic cells were
common in the MZ (Fig. 1; Mooney and Miller, 2003). Often
the crowding in the MZ was so great that it was difficult to
distinguish the MZ from the CP. That said, these heterotopias
did not occupy the entire MZ. Wide expanses of the MZ
appeared normal, i.e. as in the untreated slices.
In instances where the pial or ventricular surface was
broached, cells were found beyond the normal boundaries in
the formation of warts (Fig. 2). Two types of warts were identified: peduncular and appositional. In peduncular warts, the
cells formed an enlarged mass that was connected by a stalk to
the cerebral wall. In appositional warts, a mass of cells lay
directly over a >200 µm wide swath of the cortical surface.
Thus, it had the appearance of adhering to the outer surface of
the cultured tissue.
Factors Defining Warts
The length of time a slice was treated with ethanol altered the
incidence and the morphology of the warts. Warts first
appeared in cultures treated with ethanol (400 mg/dl) for 8 h
and the frequency of warts increased over time (Fig. 3A). All of
the warts detected at 8 h were peduncular warts. With longer
exposure to ethanol, the frequency of appositional warts
increased.
The presence of warts was concentration-dependent (Fig.
3B). In slices treated with ethanol for 24 h, the incidence of
warts was 30% greater in slices treated with 800 mg/dl ethanol
than it was for slices treated with 400 mg/dl ethanol. Cultures
treated with low concentrations of ethanol (200 mg/dl) did not
exhibit warts after 24 h; however, such low concentrations of
ethanol eventually can induce warts to form. Small warts were
Figure 2. Wart morphology. (A) A peduncular wart is characterized by a large cluster
of cells attached to the cerebral wall by a ‘stalk’. (B) An appositional wart is a suprapial
aggregate of cells juxtaposed to the surface of the cerebral wall. Scale bars = 50 µm.
evident in slices treated with only 200 mg/dl ethanol for 72 h
(data not shown).
Ethanol concentration altered the morphology of the warts.
Following treatment with 800 mg/dl ethanol, the number of
slices that had appositional warts was 50% higher than slices
treated with 400 mg/dl ethanol. The number of slices showing
peduncular warts was the same for slices treated with
moderate (400 mg/dl) or high (800 mg/dl) concentrations of
ethanol. Interestingly, tissue treated with 800 mg/dl ethanol
for 24 h also showed warts on the ventricular surface (Fig. 4).
Composition of the CP and Wart
Few cells in the warts were TUNEL-positive or contained
condensed chromatin (Fig. 5). Thus, the majority of cells was
viable. No somata in the warts were GFAP-positive. In contrast,
virtually all cell bodies in the CP and in the warts were MAP-2-
Cerebral Cortex October 2004, V 14 N 10 1073
Figure 3. Factors influencing the appearance of warts. (A) The effect of the length of
exposure to ethanol (400 mg/dl) on the frequency of slices exhibiting a wart is
described. Slices (n = 54) taken from a dozen litters were treated with ethanol for 2,
8, 16, 24, or 32 h. (B) The effect of ethanol concentration on the frequency of warts is
shown. Bars depict the percentage of slices that have warts caused by treatment with
ethanol (0, 200, 400 and 800 mg/dl) for 24 h. The solid and open portions of the bars
represent the ratios of peduncular and appositional warts, respectively. n = 31 slices
from seven litters.
or NeuN-immunopositive. This suggests that the somata were
neuronal. Furthermore, most cell bodies in the superficial CP
and in suprapial and ventricular warts, were doublecortin-positive implying that they were actively migrating (Figs 4 and 6).
Together, these data argue that most cells in the superficial CP
and in the warts were migrating neurons.
Immunoexpression of calretinin, a calcium binding protein,
was localized to cells and neuropil in the MZ, presumably
Cajal–Retzius cells. Reelin-positive staining was also evident in
the MZ and the superficial CP. The MZ subjacent to a wart
exhibited a break in the layer of calretinin immunolabeling. In
many preparations, cells in the MZ converged towards this
break. Interestingly, calretinin and reelin immunolabeling was
seen at the outer margins of the wart, but reelin-positive
staining was noticeably absent underneath a wart.
BrdU-positive cells were seen in the proliferative VZ, never
in the CP or the wart, indicating that cells in the latter two
regions were post-mitotic (Fig. 7).
The developing cortex was rife with nestin- (Fig. 8) and
vimentin-immunoreactive elements. Coarse, parallel nestinpositive fibers, presumably bundles of radial glial fibers, were
evident through the IZ. These fibers were thinner in the CP. In
more superficial CP and in the MZ fibers became more
randomly oriented. On occasion, thin fibers could be seen
coursing through the MZ and into the suprapial wart.
Ventricular warts also contained nestin-positive fibers (data not
shown).
Discussion
Ethanol-induced Alterations
Ethanol induces structural malformations in superficial cortex.
(i) The density of cells in the MZ is increased. This increased
cellularity concurs with previous reports of ectopic cells in the
superficial cortex (e.g. Clarren et al., 1978; Sakata-Haga et al.,
2002; Mooney and Miller, 2003). These ectopic cells in the MZ
are NeuN-positive, i.e. they are neurons. (ii) Clusters of cells, or
1074 Cortical Heterotopias Induced by Ethanol • Mooney et al.
warts, are evident in the suprapial space. These are similar to
heterotopias described in vivo in humans (Clarren et al., 1978)
and rodents (Kotkoskie and Norton, 1988; Gressens et al.,
1992; Komatsu et al., 2001; Sakata-Haga et al., 2002). (iii) There
are abnormalities in the organization of nestin-positive radial
glia and calretinin-positive (Cajal–Retzius) neurons.
The changes detected in the slices treated with 400 mg/dl
ethanol are similar to those in rats with moderate (∼150 mg/dl;
Komatsu et al., 2001) or high (<300 mg/dl; Kotkoskie and
Norton, 1988) blood ethanol concentrations. This pattern of
requiring higher concentrations of ethanol in vitro to match
ethanol-induced changes in vivo is evident for specific developmental events. For example, 2½-fold more ethanol is needed
in vitro to mimic in vivo changes in cell proliferation (cf. Miller
and Nowakowski, 1991; Jacobs and Miller, 2001) and migration (cf. Miller, 1993; Siegenthaler and Miller, 2004).
Effects of Ethanol on Neuronal Migration
The various ethanol-induced changes in the structure of superficial cortex are consistent with data showing that ethanol
affects neuronal migration. (i) The infiltrated MZ and warts
contain doublecortin-positive cells. Doublecortin is expressed
by migrating neurons and, to a lesser extent, by differentiating
neurons that have recently completed their migrations (Francis
et al., 1999; Gleeson et al., 1999; Friocourt et al., 2003). (ii)
Physical and chemical guides for neuronal migration (radial
glia, Cajal–Retzius cells, and reelin) are affected by ethanol.
(iii) It appears that the warts are formed by cells that are
nearing the end of their migration. Warts appear within eight h
of ethanol treatment. The MZ is ∼20 µm wide. In the presence
of ethanol, cell migration in ethanol-treated cultures proceeds
at a mean rate of 3.5 µm/h (Siegenthaler and Miller, 2004).
Hence, it takes ∼6 h for a cell to traverse and move beyond the
MZ. (iv) Warts do not contain any BrdU-labeled cells. Thus, it is
not an ectopic site of cell proliferation and cells forming the
wart must have been generated elsewhere and migrated into
the wart.
Ethanol affects the active process of neuronal migration. It
alters the rate of migration and the expression of cell adhesion
proteins (CAPs) in cultured cortical preparations (Hirai et al.,
1999; Siegenthaler and Miller, 2004). Ethanol also induces CAP
expression by dissociated primary neural cells and neural
cancer cells (Miller and Luo, 2002a,b) and alters the actions of
CAPs (Charness et al., 1994; Ramanathan et al., 1996; Wilkemeyer and Charness, 1998; Bearer et al., 1999). The data from
these culture studies concur with findings from in vivo studies.
Prenatal exposure to ethanol delays the onset of neuronal
migration and retards the rate of migration (Miller, 1993),
alters the structure of radial glia (Miller and Robertson, 1993;
Miller, 2003) and affects CAP expression (Miñana et al., 2000).
The net result is that ethanol causes neurons to end their migration at ectopic sites throughout cortex (e.g. Miller, 1986,
1988c; Miller et al., 1990; Sakata-Haga et al., 2002; Mooney and
Miller, 2003). Interestingly, patterns of ectopic neurons are
also evident in mice with conditional knockouts of integrin β1
(Graus-Porta et al., 2001). These animals exhibit infiltration of
neurons into the MZ in regions where the extracellular matrix
is degraded.
Figure 4. Doublecortin labeling in a ventricular surface wart. (A) Doublecortin
immunolabeling in the cerebral wall is highest in the IZ. Reduced labeling is evident in
the SZ, CP and MZ, and none is detected in the VZ. (B) Treatment with a high
concentration of ethanol induces a wart at the ventricular surface. Most cells in this
wart are doublecortin-positive. Scale bars = 50 µm.
Figure 5. Cells in wart are viable and Neu-N positive. (A) Few cells in the cerebral wall
or the wart are TUNEL-positive (solid arrowheads) or have condensed chromatin (open
arrowheads). Thus, the majority of cells are viable. The inset is a high magnification
image depicting the two types of cells. (B) Most cells in the CP and the wart are NeuN immunolabeled (solid arrows). Only a few cells in the wart are Neu-N-negative (open
arrows). Scale bars = 50 µm for the low magnification images (A, B) and 10 µm for
the inset.
Figure 6. MZ is compromised beneath wart region. (A) Calretinin-positive somata
(crossed, solid arrows) and processes (solid arrows) are distributed in both the MZ and
subplate. The immunolabeling in the MZ is interrupted beneath the wart and calretininnegative cells (open arrows) are in the breach. (B) Reelin immunolabeling (open
arrows) is common in the MZ. Labeled cells tend to be aligned in the MZ. This array is
noticeably absent below the wart, however, reelin immunolabeling is evident toward
the limit of the wart (solid arrows). (C) Most cells in a suprapial wart express
doublecortin. Scale bars = 50 µm.
Cerebral Cortex October 2004, V 14 N 10 1075
Figure 7. Cumulative BrdU labeling. BrdU-labeled cells are confined to the proliferative
zones of the cerebral wall, the ventricular zone (VZ) and the subventricular zone
(SZ)(open arrows). The wart is devoid of BrdU-labeled cells. Scale bar = 50 µm.
Suprapial Warts
The migration defects underlying neuronal infiltration of the
MZ and wart formation are not artifacts of the preparation.
Instead, they must be caused by ethanol. After all, neither
structural abnormality occurs in control preparations and both
occur in ethanol-treated slices in a concentration-dependent
manner. Moreover, warts emanate from the middle of the slice,
that is, they are not growing from the cut surfaces of the slice.
Why then do warts form? Some insight comes from studies in
which the pia is mechanically damaged. Such damage results in
ectopic cells in the MZ and in disruption of the radial glial
fibers near the wound (Rosen et al., 1992; Marin-Padilla, 1996,
1999). In addition, mechanical insult leads to defects in cortical
lamination which are akin to those caused by ethanol (e.g.
Clarren et al., 1978; Miller, 1986, 1988c).
Warts in the present study are not caused by mechanical
damage to the pia or the MZ. The most compelling reason for
this is that warts are not present in control slices. Ethanolinduced warts are solid clusters of cells with definite borders,
e.g. as shown in the reelin and nestin immunostaining near the
edge of the wart (see Figures 6 and 8).
1076 Cortical Heterotopias Induced by Ethanol • Mooney et al.
Figure 8. Radial glial fibers. (A) Nestin-immunoreactive radial glial processes extend
into the superficial cerebral wall. These processes extend into an appositional wart
(black arrows). The breach of the MZ is identified by an asterisk. (B) The normal and
abnormal termination of radial glia are shown in a whole-mount preparation. End-feet
of radial glia (crossed arrows) are commonly located in the MZ. Radial glia enter and
ramify in the wart (white arrows). (C) A drawing of this wart shows the passage of
nestin-positive fibers from the MZ into the wart (black arrow) and the disorganized
array of fibers and glial end-feet in the wart. Scale bars = 50 µm.
Ventricular Zone Warts
In rare instances, warts are evident at the ventricular surface.
VZ warts, also known as periventricular heterotopias, are like
the suprapial warts in that they are bounded nodules of cells.
They contain doublecortin- and nestin-positive elements,
which implies that the cells in the VZ warts are actively
migrating along radial glia. This finding contrasts with the
conclusions of others (Santi and Golden, 2001) who state that
periventricular heterotopias form from a failure of cells to
migrate from the VZ and result from disrupted radial glia.
The VZ is the site of origin for most migrating neurons. VZ
cells are dynamic. The nuclei of these cells move pially and adpially as the cells pass through the stages of the cell cycle
(Sauer, 1935, 1936; Seymour and Berry, 1975; Tramontin et al.,
2003). Presumably, VZ warts form after the ventricular surface
is abrogated at a particular point possibly by a focal disruption
of tight junctions among adjacent VZ cells (Sakata-Haga et al.,
2002). Radial migration is bidirectional (e.g. Hatten, 1990,
2002; T.F. Haydar, personal communication). Although the
prevailing route is away from the ventricular surface, some
cells may begin their migration in an ad-pial direction and
pioneer the formation of a VZ wart if they pass through an
ethanol-induced breach in the ventricular surface.
Ethanol-induced Damage to the Pial Surface
Basement and pial membranes at the ventricular and pial surfaces, respectively, constitute physical barriers between neural
tissue and surrounding environments. The PM provides a
contact point for radial glial thus forming the glial limitans
(Sievers et al., 1994). The basement membrane at the ventricular surface provides an attachment point for proliferating
progenitors undergoing interkinetic nuclear migration (Sauer,
1935, 1936; Seymour and Berry, 1975). Components of the
membranes — laminin, collagen and nidogen — are synthesized
and released by meningeal cells (Sievers et al., 1994).
Ethanol-induced alterations in the distribution of calretininand reelin-immunoexpression and inappropriate placement of
radial glia suggest that ethanol alters the PM and glial limitans.
This conclusion is supported by studies showing (i) that
ethanol-induced warts are linked to defects in the glial limitans
(Komatsu et al., 2001) and (ii) that ethanol causes the abnormal
degradation of laminin (Liesi, 1997). Moreover, mice expressing a mutant laminin exhibit disruptions in the extracellular
matrix of the PM, over-migration of cells into the MZ, and often
have warts (Halfter et al., 2002) like those in ethanol-treated
tissues. Interestingly, cellular infiltration of MZ occurs without
accompanying defects in the integrity of the glial limitans or in
laminin immunostaining (Sakata-Haga et al., 2002).
Role of Growth Factors
Growth factors regulate many processes in cortical development (Bibel and Barde, 2000; Huang and Reichardt, 2001).
Two neurotrophins involved in the migration of cortical
neurons are brain-derived neurotrophic factor (BDNF; Knusel
et al., 1994; Behar et al., 1997; Ringstedt et al., 1998; Polleux et
al., 2002) and neurotrophin-4 (NT-4; Behar et al., 1997;
Brunstrom et al., 1997; Polleux et al., 2002). Altered expression of these neurotrophins results in heterotopic MZ cells
(Brunstrom et al., 1997; Ringstedt et al., 1998). Apparently,
BDNF negatively regulates reelin expression (Ringstedt et al.,
1998) and intraventricular injection of NT-4 results in ectopic
cells in the suprapial space at the site of injection (Brunstrom
et al., 1997). The latter likely results from aberrant migration
and physical damage to the pia.
Another key regulator of neuronal migration is transforming
growth factor (TGF). Both TGFβ1 and TGFβ2 are expressed by
elements of the migratory system (migrating cells and radial
glia, respectively) in the developing cortex (Flanders et al.,
1991; Pelton et al., 1991; Miller, 2003). Exogenous TGFβ1
fosters cell migration (Siegenthaler and Miller, 2004). It
increases the rate of migration by 74% and promotes the
expression of CAPs (specifially, neural cell adhesion molecule
and isoforms of integrin) in a concentration-dependent
manner.
Wart Formation
We propose that wart formation is a three-step process.
Initially ethanol interferes with growth factor regulation. For
example, ethanol induces an increase in cortical BDNF expression (Heaton et al., 2003) and a decrease in cortical TGFβ1
content (Miller, 2003).
The second step is a change in the molecular microenvironment in the MZ and superficial CP. Increased BDNF reduces
reelin expression (Ringstedt et al., 1998). Hence, the inhibitory
effect of reelin is inhibited. This permits the infiltration of the
MZ by migrating neurons. Concurrently, ethanol alters TGFβ1promoted neuronal expression of CAPs (Siegenthaler and
Miller, 2004). The result is that the physical neuron–radial glia
interaction which normally ends in the superficial CP, is abnormally retained. This movement of neurons into the MZ is a
widespread phenomenon.
The final step is wart formation. This results from a compromise of the pial–subpial glial barrier. Focal damage allows
ectopic neurons in the MZ to move beyond the pial surface. At
first the warts have a peduncular appearance and then, as their
size increases, warts become appositional. Thus, the dynamics
of wart formation appear to involve a cascade of chemical and
then physical changes.
Notes
We thank Renee Mezza and Wendi Burnette for help in preparing the
tissue. This research was supported by grants from the National Institutes of Health (AA06916 and AA07568) and the Department of
Veterans Affairs.
Address correspondence to Michael W. Miller, Department of
Neuroscience and Physiology, SUNY Upstate Medical University,
750 East Adams Street, Syracuse NY 13210, USA. Email:
[email protected].
References
Al-Rabiai S, Miller MW (1989) Effect of prenatal exposure to ethanol on
the ultrastructure of layer V of mature rat somatosensory cortex. J
Neurocytol 18:711–729.
Angevine JB, Sidman RL (1961) Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature
192:766–768.
Bearer CF, Swick AR, ORiordan MA, Cheng G (1999) Ethanol inhibits
L1-mediated neurite outgrowth in postnatal rat cerebellar granule
neurons. J Biol Chem 274:13264–13270.
Behar TN, Dugich-Djordjevic MM, Li YX, Ma W, Somogyi R, Wen X,
Brown E, Scott C, McKay RD, Barker JL (1997) Neurotrophins
stimulate chemotaxis of embryonic cortical neurons. Eur J
Neurosci 9:2561–2570.
Berry M, Rogers AW (1965) The migration of neuroblasts in the developing cerebral cortex. J Anat 99:691–709.
Cerebral Cortex October 2004, V 14 N 10 1077
Bibel M, Barde Y-A (2000) Neurotrophins: key regulators of cell fate
and cell shape in the vertebrate nervous system. Gene Dev
14:2919–2937.
Blackshear PJ, Silver J, Nairn AC, Sulik KK, Squier MV, Stumpo DJ,
Tuttle JS (1997) Widespread neuronal ectopia associated with
secondary defects in cerebrocortical chondroitin sulfate proteoglycans and basal lamina in MARCKS-deficient mice. Exp Neurol
145:46–61.
Blom RJ, Vinuela F, Fox AJ, Blume WT, Girvin J, Kaufmann JC (1984)
Computed tomography in temporal lobe epilepsy. J Comput Assist
Tomogr 8:401–405.
Brunstrom JE, Gray-Swain MR, Osborne PA, Pearlman AL (1997)
Neuronal heterotopias in the developing cerebral cortex produced
by neurotrophin-4. Neuron 18:505–517.
Casanova MF, Buxhoeveden DP, Cohen M, Switala AE, Roy EL (2002a)
Minicolumnar pathology in dyslexia. Ann Neurol 52:108–110.
Casanova MF, Buxhoeveden DP, Switala AE, Roy E (2002b) Asperger’s
syndrome and cortical neuropathology. J Child Neurol 17:142–145.
Casanova MF, Buxhoeveden DP, Switala AE, Roy E (2002c) Minicolumnar pathology in autism. Neurology 58:428–432.
Casanova MF, Buxhoeveden D, Switala A, Roy E (2003) Rett syndrome
as a minicolumnopathy. Clin Neuropath 22:163–168.
Charness ME, Safran RM, Perides G (1994) Ethanol inhibits neural
cell–cell adhesion. J Biol Chem 269:9304–9309.
Clarren SK, Smith DW (1978) The fetal alcohol syndrome. N Engl J Med
298:1063–1067.
Clarren SK, Alvord EC Jr, Sumi SM, Streissguth AP, Smith DW (1978)
Brain malformations related to prenatal exposure to ethanol. J
Pediatr 92:64–67.
Costell M, Gustafsson E, Aszodi A, Morgelin M, Bloch W, Hunziker E,
Addicks K, Timpl R, Fassler R (1999) Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol
147:1109–1122.
D’Arcangelo G, Miao GG, Chen S-C, Soares HD, Morgan JI, Curran T
(1995) A protein related to extracellular matrix proteins deleted in
the mouse mutant reeler. Nature 374:719–723.
Derer P (1985) Comparative localization of Cajal–Retzius cells in the
neocortex of normal and reeler mutant mice fetuses. Neurosci Lett
54:1–6.
des Portes V, Francis F, Pinard JM, Desguerre I, Moutard ML, Snoeck I,
Meiners LC, Capron F, Cusmai R, Ricci S, Motte J, Echenne B,
Ponsot G, Dulac O, Chelly J, Beldjord C (1998a) Doublecortin is
the major gene causing X-linked subcortical laminar heterotopia
(SCLH). Hum Mol Genet 7:1063–1070.
des Portes V, Pinard JM, Billuart P, Vinet MC, Koulakoff A, Carrie A,
Gelot A, Dupuis E, Motte J, Berwald-Netter Y, Catala M, Kahn A,
Beldjord C, Chelly J (1998b) A novel CNS gene required for
neuronal migration and involved in X-linked subcortical laminar
heterotopia and lissencephaly syndrome. Cell 92:51–61.
Dobyns WB, Andermann E, Andermann F, Czapansky-Beilman D,
Dubeau F, Dulac O, Guerrini R, Hirsch B, Ledbetter DH, Lee NS,
Motte J, Pinard JM, Radtke RA, Ross ME, Tampieri D, Walsh CA,
Truwit CL (1996) X-linked malformations of neuronal migration.
Neurology 47:331–339.
Dulabon L, Olson EC, Taglienti MG, Eisenhuth S, McGrath B, Walsh
CA, Kreidberg JA, Anton ES (2000) Reelin binds α3 β1 integrin and
inhibits neuronal migration. Neuron 27:33–44.
Dunn V, Mock T, Bell WE, Smith W (1986) Detection of heterotopic
gray matter in children by magnetic resonance imaging. Magn
Reson Imaging 4:33–39.
Flanders KC, Lüdecke G, Engels S, Cissel DS, Roberts AB, Kondaiah P,
Lafyatis R, Sporn MB, Unsicker K (1991) Localization and actions
of transforming growth factor-βs in the embryonic nervous system.
Development 113:183–191.
Fox JW, Lamperti ED, Eksioglu YZ, Hong SE, Feng Y, Graham DA,
Scheffer IE, Dobyns WB, Hirsch BA, Radtke RA, Berkovic SF,
Huttenlocher PR, Walsh CA (1998) Mutations in filamin 1 prevent
migration of cerebral cortical neurons in human periventricular
heterotopia. Neuron 21:1315–1325.
Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, Vinet MC,
Friocourt G, McDonnell N, Reiner O, Kahn A, McConnell SK,
1078 Cortical Heterotopias Induced by Ethanol • Mooney et al.
Berwald-Netter Y, Denoulet P, Chelly J (1999) Doublecortin is a
developmentally regulated, microtubule-associated protein
expressed in migrating and differentiating neurons. Neuron
23:247–256.
Friocourt G, Koulakoff A, Chafey P, Boucher D, Fauchereau F, Chelly
J, Francis F (2003) Doublecortin functions at the extremities of
growing neuronal processes. Cereb Cortex 13:620–626.
Fukuyama Y, Kawazura M, Haruna H (1960) A peculiar form of
congenital progressive muscular dystrophy. Report of 15 cases.
Paediatr Univ Tokyo 4:5–8.
Galaburda AM, Kemper TL (1979) Cytoarchitectonic abnormalities in
developmental dyslexia: a case study. Ann Neurol 6:94–100.
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 8:983–986.
Gleeson JG, Allen KM, Fox JW, Lamperti ED, Berkovic S, Scheffer I,
Cooper EC, Dobyns WB, Minnerath SR, Ross ME, Walsh CA (1998)
Doublecortin, a brain-specific gene mutated in human X-linked
lissencephaly and double cortex syndrome, encodes a putative
signaling protein. Cell 92:63–72.
Gleeson JG, Lin PT, Flanagan LA, Walsh CA (1999) Doublecortin is a
microtubule-associated protein and is expressed widely by
migrating neurons. Neuron 23:257–271.
Graus-Porta D, Blaess S, Senften M, Littlewood-Evans A, Damsky C,
Huang Z, Orban P, Klein R, Schittny JC, Muller U (2001) Beta1class integrins regulate the development of laminae and folia in the
cerebral and cerebellar cortex. Neuron 31:367–379.
Gressens P, Lammens M, Picard JJ, Evrard P (1992) Ethanol-induced
disturbances of gliogenesis and neuronogenesis in the developing
murine brain: an in vitro and in vivo immunohistochemical and
ultrastructural study. Alcohol Alcohol 27:219–226.
Hack I, Bancila M, Loulier K, Carroll P, Cremer H (2002) Reelin is a
detachment signal in tangential chain-migration during postnatal
neurogenesis. Nat Neurosci 5:939–945.
Halfter W, Sucai D, Yip Y, Willem M, Mayer U (2002) A critical function of the pial basement membrane in cortical histogenesis. J
Neurosci 22:6029–6040.
Hatten ME (1990) Riding the glial monorail: a common mechanism for
glial-guided neuronal migration in different regions of the developing mammalian brain. TINS 13:179–184.
Hatten ME (2002) New directions in neuronal migration. Science
297:1660–1663.
Heaton MB, Paiva M, Madorsky I, Shaw G (2003) Ethanol effects on
neonatal rat cortex: comparative analyses of neurotrophic factors,
apoptosis-related proteins, and oxidative processes during vulnerable and resistant periods. Dev Brain Res 145:249–262.
Hiesberger T, Trommsdorff M, Howell BW, Goffinet A, Mumby M,
Cooper JA, Herz J (1999) Direct binding of reelin to VLDL receptor
and ApoE receptor 2 induces tyrosine phosphorylation of disabled1 and modulates tau phosphorylation. Neuron 24:481–489.
Hirai K, Yoshioka H, Kihara M, Hasegawa K, Sawada T, Fushiki S
(1999) Effects of ethanol on neuronal migration and neural cell
adhesion molecules in the embryonic rat cerebral cortex: a tissue
culture study. Dev Brain Res 118:205–210.
Hirotsune S, Takahara T, Sasaki N, Hirose K, Yoshiki A, Ohashi T,
Kusakabe M, Murakami Y, Muramatsu M, Watanabe S, Nakao K,
Katsuki M, Hayashizaki Y (1995) The reeler gene encodes a protein
with an EGF-like motif expressed by pioneer neurons. Nature
Genet 10:77–83.
Hong SE, Shugart YY, Huang DT, Shahwan SA, Grant PE, Hourihane
JO, Martin ND, Walsh CA (2000) Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human
RELN mutations. Nature Genet 26:93–96.
Howell B, Herrick T, Cooper J (1999) Reelin-induced tyrosine phosphorylation of disabled 1 during neural positioning. Genes Dev
13:643–648.
Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677–736.
Humphreys P, Kaufmann WE, Galaburda AM (1990) Developmental
dyslexia in women: neuropathological findings in three patients.
Ann Neurol 28:727–738.
Jacobs JS, Miller MW (2001) Proliferation and death of cultured fetal
neocortical neurons: effects of ethanol on the dynamics of cell
growth. J Neurocytol 30:391–401.
Knusel B, Rabin SJ, Hefti F, Kaplan DR (1994) Regulated neurotrophin
receptor responsiveness during neuronal migration and early
differentiation. J Neurosci 14:1542–1554.
Komatsu S, Sakata-Haga H, Sawada K, Hisano S, Fukui Y (2001)
Prenatal exposure to ethanol induces leptomeningeal heterotopia
in the cerebral cortex of the rat fetus. Acta Neuropathol 101:22–26.
Konovalov HV, Kovetsky NS, Bobryshev YV, Ashwell KW (1997)
Disorders of brain development in the progeny of mothers who
used alcohol during pregnancy. Early Hum Dev 48:153–166.
Kornguth SE, Rutledge JJ, Sunderland E, Siegel F, Carlson I, Smollens J,
Juhl U, Young B (1979) Impeded cerebellar development and
reduced serum thyroxine levels associated with fetal alcohol intoxication. Brain Res 177:347–360.
Kotkoskie LA, Norton S (1988) Prenatal brain malformations following
acute ethanol exposure in the rat. Alcohol Clin Exp Res
12:831–836.
Lewis PD (1985) Neuropathological effects of alcohol on the developing nervous system. Alcohol Alcohol 20:195–200.
Liesi P (1997) Ethanol-exposed central neurons fail to migrate and
undergo apoptosis. J Neurosci Res 48:439–448.
Livingston JH, Aicardi J (1990) Unusual MRI appearance of diffuse
subcortical heterotopia or ‘double cortex’ in two children. J
Neurol Neurosurg Psychol 53:617–620.
Luque JM, Morante-Oria J, Fairen A (2003) Localization of ApoER2,
VLDLR and Dab1 in radial glia: groundwork for a new model of
reelin action during cortical development. Dev Brain Res
140:195–203.
Luskin MB, Shatz CJ (1985) Neurogenesis of the cat’s primary visual
cortex. J Comp Neurol 242:611–631.
Marín O, Rubenstein JLR (2003) Cell migration in the forebrain. Annu
Rev Neurosci 26:441–483.
Marin-Padilla M (1996) Developmental neuropathology and impact of
perinatal brain damage: I. hemorrhagic lesions of neocortex. J
Neuropath Exp Neurol 55:758–773.
Marin-Padilla M (1998) Cajal–Retzius cells and the development of the
neocortex. Trends Neurosci 21:64–71.
Marin-Padilla M (1999) Developmental neuropathology and impact of
perinatal brain damage. III. Gray matter lesions of the neocortex. J
Neuropath Exp Neurol 58:407–429.
Miller MW (1985) Co-generation of projection and local circuit
neurons in neocortex. Dev Brain Res 23:187–192.
Miller MW (1986) Fetal alcohol effects on the generation and migration of cerebral cortical neurons. Science 233:1308–1311.
Miller MW (1987) Effect of prenatal exposure to ethanol on the distribution and time of origin of corticospinal neurons in the rat. J
Comp Neurol 257:372–382.
Miller MW (1988a) Development of projection and local circuit
neurons in cerebral cortex, In: Cerebral cortex. Vol. 7. Development and maturation of cerebral cortex (Peters A, Jones EG, eds) ,
pp. 133–175. New York: Plenum.
Miller MW (1988b) Maturation of rat visual cortex: IV. The generation,
migration, morphogenesis, and connectivity of atypically oriented
pyramidal neurons. J Comp Neurol 274:387–405.
Miller MW (1988c) Effect of prenatal exposure to ethanol on the development of cerebral cortex: I. Neuronal generation. Alcohol Clin
Exp Res 12:440–449.
Miller MW (1992) Development of the central nervous system: effects
of alcohol and opiates. New York: Wiley-Liss.
Miller MW (1993) Migration of cortical neurons is altered by gestational exposure to ethanol. Alcohol Clin Exp Res 17:304–314.
Miller MW (1997) Effects of prenatal exposure to ethanol on callosal
projection neurons in rat somatosensory cortex. Brain Res
766:121–128.
Miller MW (2003) Expression of transforming growth factor-β in developing rat cerebral cortex: effects of prenatal exposure to ethanol.
J Comp Neurol 460:410–424.
Miller MW, Luo J (2002a) Effects of ethanol and basic fibroblast
growth factor on the transforming growth factor beta1 regulated
proliferation of cortical astrocytes and C6 astrocytoma cells.
Alcohol Clin Exp Res 26:671–676.
Miller MW, Luo J (2002b) Effects of ethanol and transforming growth
factor β (TGFβ) on neuronal proliferation and nCAM expression.
Alcohol Clin Exp Res 26:1281–1285.
Miller MW, Nowakowski RS (1991) Effect of prenatal exposure to
ethanol on the cell cycle kinetics and growth fraction in the proliferative zones of fetal rat cerebral cortex. Alcohol Clin Exp Res
15:229–232.
Miller MW, Robertson S (1993) Prenatal exposure to ethanol alters the
postnatal development and transformation of radial glia to astrocytes in rat somatosensory cortex. J Comp Neurol 337:253–266.
Miller MW, Chiaia NL, Rhoades RW (1990) Intracellular recording and
labeling study of corticospinal neurons in the rat somatosensory
cortex: effect of prenatal exposure to ethanol. J Comp Neurol
296:1–15.
Miñana R, Climent E, Barettino D, Segui JM, Renau-Piqueras J, Guerri C
(2000) Alcohol exposure alters the expression pattern of neural
cell adhesion molecules during brain development. J Neurochem
75:954–964.
Mooney SM, Miller MW (2003) Ethanol-induced neuronal death in
organotypic cultures of rat cerebral cortex. Dev Brain Res
147:135–141.
Nishikawa S, Goto S, Hamasaki T, Yamada K, Ushio Y (2002) Involvement of reelin and Cajal–Retzius cells in the developmental formation of vertical columnar structures in the cerebral cortex:
evidence from the study of mouse presubicular cortex. Cereb
Cortex 12:1024–1030.
Ogawa M, Miyata T, Nakajima K, Yagyu K, Seike M, Ikenaka K,
Yamamoto H, Mikoshiba K (1995) The reeler gene-associated
antigen on Cajal–Retzius neurons is a crucial molecule for laminar
organization of cortical neurons. Neuron 14:899–912.
Palmini A, Andermann F, Olivier A, Tampieri D, Robitaille Y,
Andermann E, Wright G (1991) Focal neuronal migration disorders
and intractable partial epilepsy: a study of 30 patients. Ann Neurol
30:741–749.
Pearlman AL, Sheppard AM (1996) Extracellular matrix in early
cortical development. Prog Brain Res 108:117–134.
Peiffer J, Majewski F, Fischbach H, Bierich JR, Volk B (1979) Alcohol
embryo- and fetopathy. Neuropathology of 3 children and 3
fetuses. J Neurol Sci 41:125–137.
Pelton RW, Saxena B, Jones M, Moses HL, Gold LI (1991) Immunohistochemical localization of TGFβ1, TGFβ2, and TGFβ3 in the mouse
embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 115:1091–1105.
Pinto-Lord MC, Evrard P, Caviness VS Jr (1982) Obstructed neuronal
migration along radial glial fibers in the neocortex of the reeler
mouse: a Golgi-EM analysis. Brain Res 256:379–393.
Polleux F, Whitford KL, Dijkhuizen PA, Vitalis T, Ghosh A (2002)
Control of cortical interneuron migration by neurotrophins and
PI3-kinase signaling. Development 129:3147–3160.
Rakic P (1974) Neurons in rhesus monkey visual cortex: systematic
relation between time of origin and eventual disposition. Science
183:425–427.
Ramanathan R, Wilkemeyer MF, Mittal B, Perides G, Charness ME
(1996) Alcohol inhibits cell–cell adhesion mediated by human L1.
J Cell Biol 133:381–390.
Rice D, Sheldon M, D’Arcangelo G, Nakajima K, Goldowitz D, Curran
T (1998) Disabled-1 acts downstream of Reelin in a signaling
pathway that controls laminar organization in the mammalian
brain. Development 125:3719–3729.
Ringstedt T, Linnarsson S, Wagner J, Lendahl U, Kokaia Z, Arenas E,
Ernfors P, Ibanez CF (1998) BDNF regulates reelin expression and
Cajal–Retzius cell development in the cerebral cortex. Neuron
21:305–315.
Cerebral Cortex October 2004, V 14 N 10 1079
Rosen GD, Sherman GE, Richman JM, Stone LV, Galaburda AM (1992)
Induction of molecular layer ectopias by puncture wounds in
newborn rats and mice. Dev Brain Res 67:285–291.
Ross ME, Allen KM, Srivastava AK, Featherstone T, Gleeson JG, Hirsch
B, Harding BN, Andermann E, Abdullah R, Berg M, CzapanskyBielman D, Flanders DJ, Guerrini R, Motte J, Mira AP, Scheffer I,
Berkovic S, Scaravilli F, King RA, Ledbetter DH, Schlessinger D,
Dobyns WB, Walsh CA (1997) Linkage and physical mapping of Xlinked lissencephaly/SBH (XLIS): a gene causing neuronal migration defects in human brain. Hum Mol Genet 6:555–562.
Rovasio RA, Battiato NL (2002) Ethanol induces morphological and
dynamic changes on in vivo and in vitro neural crest cells. Alcohol
Clin Exp Res 26:1286–1298.
Sakata-Haga H, Sawada K, Hisano S, Fukui Y (2002) Administration
schedule for an ethanol-containing diet in pregnancy affects types
of offspring brain malformations. Acta Neuropathol 104:305–312.
Santavuori P, Leisti J, Kruus S (1977) Muscle, eye and brain disease: a
new syndrome. Neuropaediatrie 8(Suppl.):553–558.
Santavuori P, Somer H, Sainio K, Rapola J, Kruus S, Nikitin T, Ketonen
L, Leisti J (1989) Muscle–eye–brain disease (MEB). Brain Dev
11:147–153.
Santi MR, Golden JA (2001) Periventricular heterotopia may result
from radial glial fiber disruption. J Neuropathol Exp Neurol
60:856–862.
Sauer FC (1935) Mitosis in the neural tube. J Comp Neurol 62:377–405.
Sauer FC (1936) The interkinetic migration of embryonic epithelial
nuclei. J Morphol 60:1–11.
Seymour RM, Berry M (1975) Scanning and transmission electron
microscope studies of interkinetic nuclear migration in the cerebral vesicles of the rat. J Comp Neurol 160:105–126.
Sezaki K, Ogawa M, Yagi T (1999) Proteins of the CNR family are
multiple receptors of reelin. Cell 99:635–647.
1080 Cortical Heterotopias Induced by Ethanol • Mooney et al.
Shaw CM (1987) Correlates of mental retardation and structural
changes of the brain. Brain Dev 9:1–8.
Siegenthaler JA, Miller MW (2004) Transforming growth factor β1
modulates cell migration in rat cortex: effects of ethanol. Cereb
Cortex 14:791–802.
Sievers J, Phlemann F, Gude S, Berry M (1994) Meningeal cells
organize the superficial glial limitans of the cerebellum and
produce both the interstitial matrix and basement membrane. J
Neurocytol 23:135–149.
Soda T, Nakashima R, Watanabe D, Nakajima K, Pastan I, Nakanishi S
(2003) Segregation and coactivation of developing neocortical
layer 1 neurons. J Neurosci 23:6272–6279.
Tabata H, Nakajima K (2002) Neurons tend to stop migration and
differentiate along the cortical internal plexiform zones in the
reelin signal-deficient mice. J Neurosci Res 69:723–730.
Tramontin AD, Garcia-Verdugo JM, Lim DA, Alvarez-Buylla A (2003)
Postnatal development of radial glia and the ventricular zone (VZ):
a continuum of the neural stem cell compartment. Cereb Cortex
13:580–587.
Trommsdorff M, Gotthardt M, Hiesberger T, Shelton J, Stockinger W,
Nimpf J, Hammer R, Richardson J, Herz J (1999) Reeler/disabledlike disruption of neuronal migration in knockout mouse lacking
the VLDL receptor and ApoE receptor 2. Cell 97:689–701.
Wilkemeyer MF, Charness ME (1998) Characterization of ethanolsensitive and insensitive fibroblast cell lines expressing human L1.
J Neurochem 71:2382–2391.
Williams RS, Swisher CN, Jennings M, Ambler M, Caviness VS Jr (1984)
Cerebro-ocular
dysgenesis
(Walker–Warburg
syndrome):
neuropathologic and etiologic analysis. Neurology 34:1531–1541.
Zhou FC, Sari Y, Zhang JK, Goodlett CR, Li T-K (2001) Prenatal alcohol
exposure retards the migration and development of serotonin
neurons in fetal C57BL mice. Dev Brain Res 126:147–155.