Epilepsy in Dcx knockout mice associated with discrete lamination defects and enhanced excitability in the hippocampus.
ABSTRACT Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence, were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to further study how lamination defects underlie the genesis of epileptiform activities.
- SourceAvailable from: Bruce T Schaar[show abstract] [hide abstract]
ABSTRACT: Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.Neuron 07/1999; 23(2):247-56. · 15.77 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.Neuron 07/1999; 23(2):257-71. · 15.77 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.Cell 02/1998; 92(1):51-61. · 31.96 Impact Factor
Epilepsy in Dcx Knockout Mice Associated with Discrete
Lamination Defects and Enhanced Excitability in the
Marika Nosten-Bertrand1,2., Caroline Kappeler3,4., Ce ´line Dinocourt5., Ce ´cile Denis1,2¤, Johanne
Germain1,2, Franc ¸oise Phan Dinh Tuy3,4, Soraya Verstraeten1,2, Chantal Alvarez6,7, Christine Me ´tin6,7,
Jamel Chelly3,4, Bruno Giros1,2,8, Richard Miles5, Antoine Depaulis9, Fiona Francis3,4*
1INSERM, U513, Universite ´ Pierre et Marie Curie, Paris, France, 2UMPC Universite ´ Paris 06, Neurobiologie et Psychiatrie, Paris, France, 3Institut Cochin, Universite ´ Paris
Descartes, CNRS (UMR 8104), Paris, France, 4INSERM, U567, Paris, France, 5INSERM, U739, UPMC, CHU Pitie ´ Salpe ˆtrie `re, Paris, France, 6UPMC, Paris, France, 7INSERM,
U839, Institut du Fer a ` Moulin, Paris, France, 8Douglas Hospital Research Center, Department of Psychiatry, McGill University, Montreal, Quebec, Canada, 9Grenoble
Institute of Neurosciences, Inserm U836-UJF-CEA-CHU, Universite ´ Joseph Fourier, Grenoble, France
Patients with Doublecortin (DCX) mutations have severe cortical malformations associated with mental retardation and
epilepsy. Dcx knockout (KO) mice show no major isocortical abnormalities, but have discrete hippocampal defects. We
questioned the functional consequences of these defects and report here that Dcx KO mice are hyperactive and exhibit
spontaneous convulsive seizures. Changes in neuropeptide Y and calbindin expression, consistent with seizure occurrence,
were detected in a large proportion of KO animals, and convulsants, including kainate and pentylenetetrazole, also induced
seizures more readily in KO mice. We show that the dysplastic CA3 region in KO hippocampal slices generates sharp wave-
like activities and possesses a lower threshold for epileptiform events. Video-EEG monitoring also demonstrated that
spontaneous seizures were initiated in the hippocampus. Similarly, seizures in human patients mutated for DCX can show a
primary involvement of the temporal lobe. In conclusion, seizures in Dcx KO mice are likely to be due to abnormal synaptic
transmission involving heterotopic cells in the hippocampus and these mice may therefore provide a useful model to
further study how lamination defects underlie the genesis of epileptiform activities.
Citation: Nosten-Bertrand M, Kappeler C, Dinocourt C, Denis C, Germain J, et al. (2008) Epilepsy in Dcx Knockout Mice Associated with Discrete Lamination
Defects and Enhanced Excitability in the Hippocampus. PLoS ONE 3(6): e2473. doi:10.1371/journal.pone.0002473
Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan
Received February 11, 2008; Accepted May 8, 2008; Published June 25, 2008
Copyright: ? 2008 Nosten-Bertrand et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The contribution of the Re ´gion Ile-de-France to the Institut Cochin animal care facility is acknowledged. This work was supported by INSERM, the CNRS
and the Fe ´de ´ration pour la Recherche sur le Cerveau (awarded to FF). C. Dinocourt was supported by the EC (EPICURE-LSH-CT-2006-037315). C. Kappeler was
supported by a fellowship from the Ministe `re de l’Education Nationale, de la Recherche et de la Technologie, the Fondation pour la Recherche Me ´dicale (FRM) and
the ANR (A05183KS).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Celogos, 91058 Evry, France
. These authors contributed equally to this work.
Doublecortin (DCX) is a microtubule-associated protein [1,2]
which is critical for human cortical development. Type I lissenceph-
aly, due to DCX [3,4] or LIS1  mutations, is associated with severe
mental retardation and refractory epilepsy . This form of cortical
dysplasia is due most probably to neuronal migration abnormalities
during development . In type 1 lissencephaly patients the
neocortex has a smooth surface and generally consists of four
disorganized layers of neurons instead of the six highly organized
layerspresent ina normalbrain. Furthermore the hippocampus is
disorganized . The exact causes of epilepsyand mental retardation
in these cases remain to be determined. However, epileptic seizures
are likely to result from abnormal connectivity associated with the
aberrant positioning of cortical neurons [9,10].
Dcx KO mice have no obvious lamination defects in the isocortex
[11,12]. Indeed, radial migration of isocortical pyramidal cells
appears to occur correctly leading to normally organized cortical
layers. However, in the hippocampus of Dcx KO mice, the CA3
region ofthepyramidallayerisdisorganized and dividedintoat least
two distinct layers [8,11]. This is due to a slowed or arrested
migration of a proportion of hippocampal pyramidal neurons at late
embryonic stages . Cortical interneuron migration abnormalities
have also been identified with Dcx inactivation [12,13]. Thus
tangentially migrating neurons derived from the medial ganglionic
eminence of Dcx KO or RNAi-treated mice show abnormal
morphologies during migration in explant and slice cultures.
Interneuron numbers were also found to be reduced in the isocortex
of P0 animals . With limited pyramidal cell and interneuron
defects, Dcx KO mice may be a useful model to investigate the
pathological processes of cognitive impairment, seizure susceptibility
and epilepsy, independent of major isocortical lamination defects.
In the present study we show that Dcx KO animals are
hyperactive and prone to epileptic seizures. This latter phenotype
may be associated with changes in the expression of calbindin
(CB), neuropeptide Y (NPY) and calretinin in the hippocampus. In
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addition, KO mice show an increased susceptibility to convulsants
(pentylenetetrazole, PTZ and kainic acid, KA). Video-EEG
monitoring demonstrated that spontaneous electrographical and
behavioural seizures occur in the absence of convulsants. These
appear to be initiated in the hippocampus and to rapidly diffuse to
the cortex. Electrophysiological recordings in vitro revealed a lower
threshold for epileptiform discharges and a higher frequency of
spontaneous, sharp wave-like events in hippocampal slices from
Dcx KO animals. Thus, Dcx KO mice, without severe isocortical
disorganization, are susceptible to epilepsy most probably due to
abnormal cell positioning, connectivity and synaptic transmission
in the hippocampus.
Morphological Abnormalities in the Hippocampus of Dcx
The Dcx KO hippocampal CA3 region is divided into several
distinct pyramidal cell layers (Figure 1A, B). We questioned here
the innervation of this region by the dentate gyrus (DG), and its
association with interneurons, in order to better understand the
potential functional consequences of such defects.
Histological analyses of cresyl violet-stained sections of 5 KO and
3 wild type (WT) adult brains at age 4 months on the C57BL/6 (B6)
background, revealed a very similar organization of the DG
between the genotypes (Figure 1A–D). Our previous studies also
showed no major differences in the development of the DG at late
embryonic and neonatal stages . However, in KO hippocampi,
CA3 cells were abnormally dispersed in the CA3b and CA3c
regions, between the fimbria and the entrance to the DG, in at least
two distinct layers (Figure 1D, H), compared with the single diffuse
layer of WT hippocampi (Figure 1C). Such abnormalities were
observed at all levels in the hippocampus along the rostro-caudal
axis. CB-labeled mossy fibers, the axons of DG cells, appeared
correspondingly less well-organized in the CA3 region of KO
hippocampi(Figure1E,F,WT,n=9;KO,n=6).Mossy fibers may
therefore innervate abnormally dispersed CA3 pyramidal cells in a
spatially appropriate fashion in KO mice. In addition, interneuron-
like cells, identified by their morphology  using Golgi staining,
were also observed in close proximity to pyramidal cells in each of
the KO CA3 layers (Figure 1G–K, Figure S1). Altogether, these
data suggest that mossy fibers and interneurons adapt their
innervation patterns to target abnormally dispersed CA3 cells.
Adult Dcx KO Mice Weigh Less than WT Mice and Are
At weaning (4 weeks), there were no differences in body weight
between KO (n=35) and WT (n=42) mice (respectively 11.260.42
and 11.760.41 g). However, at 4 months of age, the body weight of
Dcx KO mice (n=45) was significantly lower than WT littermates
(n=47), (respectively 28.960.4 and 30.860.4 g; ANOVA test,
P,0.005, F1,90=11.18). We also observed that exposure to the novel
environment of actimeter cages triggered a significantly higher level
of spontaneous horizontal and vertical activity in KO adult mice (5–6
months of age) compared with their WT littermates (WT n=31; KO
n=30; ANOVA test, Figure 2). This novelty-induced hyperactivity
was also observed in younger animals (data not shown).
Dcx KO Mice Exhibit Spontaneous Seizures and Show
Changes in Seizure Sensitive Markers
Occasional spontaneous seizures were observed in Dcx KO mice
(n=5), characterized by abnormal head movements, rearing, and
clonus of the forelimbs. No WT littermates showed similar behaviors.
Figure 1. Morphological Abnormalities in the Dcx KO Hippo-
campus. (A–D) Cresyl violet staining showed that dentate gyri were
indistinguishable between the genotypes, however, abnormally orga-
nized CA3 pyramidal cells in the KO hippocampus were observed (red
arrows in D). (E,F) Calbindin-labeling shows less well organized mossy
fibers in the KO section (F) compared to an equivalent WT section (E).
Red arrowheads indicate a more fragmented appearance of fibers in the
KO. Only strongly CB-labeled KO sections were used in this analysis. (G–
K) Golgi-Cox labeling shows the association of interneuron-like cells (red
arrows) with CA3 pyramidal cells, even those displaced in a separate
upper pyramidal layer in the KO (H). Pyramidal cells (blue, stained with
Nissl) were present in 3 approximate layers in this KO animal (2 are
observed here labeled 1, 2 in H). (I–K) The three cells shown in H are
shown in better focus individually in I–K. These cells show the typical
morphology of CA3 basket cell or axo-axonic-like interneurons ,
with fusiform cell bodies, and one or two dendrites originating from the
apical pole, which then branch proximally to give radially oriented
dendrites in the stratum radiatum. In addition, such cells have several
basal, spine-free dendrites, branched close to the cell body and
extended toward the alveus. Scale bars: A (for A, B), 500 mm; and C (for
C, D); E (for E, F); G (for G, H), 50 mm; I (for I, J, K), 44 mm. dg, dentate
gyrus; fi, fimbria; so, stratum oriens.
Epilepsy in Dcx Knockout Mice
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The expression of various markers has been shown to be altered
in the hippocampus after seizure activity induced by lesions, drug
treatment, direct electrical stimulation and gene inactivation [15–
22]. We therefore examined the expression of CB and NPY,
known to be modified in such models of recurrent seizures, in
untreated WT (n=13) and KO (n=16) mice of different ages
CB is present in subpopulations of interneurons, pyramidal and
DG cells in the isocortex and hippocampus and the occurrence of
seizures is often associated with a decrease in its expression,
particularly in the DG [20,23]. In the hippocampi of WT mice
(aged 3–18 months) we therefore observed CB expression in the
cell bodies and dendrites of DG cells, as well as in the mossy fibers
projecting to the CA3 region (Figure 1E,F; 3A,C). Sparse CB-
positive interneurons were observed in the stratum radiatum /
lacunosum moleculare region of the CA3 area. In 9/16 Dcx KO
animals tested (56.3 %), we observed a clearly reduced intensity of
CB staining in the dendrites of DG cells, their cell bodies and
mossy fibers, throughout the rostro-caudal axis of the hippocam-
pus, compared to labeling in WT mice (Figure 3A–D, Table 1).
By contrast, hippocampal NPY expression has been shown to be
upregulated after seizures in epileptic animals, possibly reflecting
an endogenous mechanism that opposes excessive hippocampal
excitability . In particular, a neoexpression of this neuropep-
tide in DG cell mossy fibers has been reported in epileptic animals
. We therefore examined this marker in adjacent slices to those
labeled for CB. A strong ectopic expression of NPY was observed
in mossy fibers stemming from the DG (Figure 3E–H) in the same
KO mice showing a reduced CB expression. Such a modification
was not observed in other Dcx KO mice with a normal expression
of CB, nor in WT mice.
In one mouse where spontaneous epileptic seizures were
previously observed and which was sacrificed at 11 months, a clear
reduction of CB expression and a dramatic increase in NPY
Figure 2. Dcx KO Mice are Hyperactive. Time-course of mean6SEM spontaneous horizontal (locomotion) and vertical (rearings) behavioural
activity in WT (n=31) and KO (n=30) mice. Adult mice (aged 5–6 months) were introduced individually in activity boxes and automatically recorded
over a 2 h period. An ANOVA test revealed significance between the 2 strains for horizontal activity, (genotype, F1,1416=14.48, P,0.0001; time,
F23,1416=80.82, P,0.0001 and genotype6time interaction, F23,1416=2.78, P,0.01); and for vertical activity, (genotype, F1,1416=102.71, P,0.0001;
time, F23,1416=76.41, P,0.0001 and genotype6time interaction, F23,1416=1.785, P,0.01).
Table 1. Modification in Calbindin and NPY Expression in Dcx KO Mice.
Number of mice testedGenotypeAge (months)
Number of mice with
modificationsCalbindin Neuropeptide Y
KO mice showing changes9 (56.3%)
A subset of Dcx KO mice show correlated calbindin and NPY expression changes potentially related to spontaneous seizures. WT: wild-type; KO: knockout (female and
male); +: slight modifications; ++: moderate modifications; +++: strong modifications; nd: not determined;*: mouse showing spontaneous epileptic seizures.
Epilepsy in Dcx Knockout Mice
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expression were detected (Table 1). In addition, KO mice as young
as 3 months were shown to have a changed expression of these
markers whereas none of the WT mice (n=13) showed these
changes, even at 15–18 months (n=2). These combined modifica-
tions of CB and NPY expression were therefore observed in 9 out of
16 KO mice (56.3 %) tested (Table 1). This proportion may
however be underestimated, since in other models such alterations
have been shown to occur with a delay after seizure onset, and the
duration of such changes is also unknown [20,23]. Altogether, the
combined results of our CB and NPY labelings, and the previous
association of these markers with epilepsy [20–23], suggest, albeit
indirectly, that a large proportion of Dcx KO mice may have
experienced recurrent spontaneous epileptic seizures.
Certain mice (9 KO, 4 WT) were also tested for calretinin
expression, which labels newly generated neurons of the
(Figure 3I, K). Four KO animals showed a moderate to extensive
increase in the number and intensity of labeled subgranular zone
neurons (Figure 3J, L), with 3/4 of these animals also showing
changes in NPY/CB (Table S1). Such modifications of calretinin
expression have previously been correlated with decreased CB
expression and epilepsy onset in other models . Three other
KO animals showed a minor disorganization of such cells (Figure
S2), often affecting the supragranular blade, as we previously
reported , with 2/3 animals also showing changes in NPY/
CB. The remaining 2 KO animals and the 4 WT mice tested
showed no obvious abnormalities in calretinin, CB or NPY. Thus
it appears that changes in calretinin expression can be variable and
are not always strictly correlated with changes in CB and NPY.
Indeed, the minor disorganization of cells observed in some
animals may represent a separate phenomenon, unrelated to the
increased number of cells observed in other animals. However,
Ruttimann et al.  showed a short transient change in
expression of calretinin, compared to longer term changes for
CB in epileptic GABA B1 -/- mutant mice and this may partly
explain the variability observed here.
Dcx KO Mice Are More Susceptible to Chemoconvulsants
than WT Mice
To further characterize seizure susceptibility in Dcx KO mice,
we first attempted to induce audiogenic seizures. WT (n=8) and
KO (n=8) animals, aged 28–33 days , were exposed to a 90
decibel sound for 90 sec in two tests separated by 48 h. Wild
running was observed in one WT animal, but no tonic or tonic-
clonic seizures occurred in either WT or KO animals (data not
shown). Similarly, the sensitivity to reflex-seizures induced by
tactile stimulation was explored in 2 month old WT (n=10) and
KO (n=10) animals. No behavioral arrest, myoclonic jerks or
clonic seizures were observed in either group (data not shown).
We then tested the response of KO animals to PTZ and KA.
Doses were adjusted from previous reports [25,26] and behavior
was subsequently assessed by two investigators blind to the
genotypes. Following PTZ injections (30 or 35 mg/kg, i.p.) in 3–6
Figure 3. Untreated KO Mice Show Changes in Seizure
Sensitive Marker Expression. (A–D) CB immunoreactivity in an
untreated WT mouse (A,C) and a KO mouse (B,D), sacrificed at 7 months
of age. In the hippocampus of the KO mouse, the expression of CB is
reduced in the dg cell layer and dendrites, and in mossy fibers (D).
Although the corpus callosum appears thinner in the KO mouse section,
this is not typical for Dcx KO mice on the C57BL/6 background (8). (E–H)
NPY immunoreactivity in an untreated WT mouse (E,G) and a KO mouse
(F,H), sacrificed at 7 months of age. High magnification of the
hippocampus shows NPY neoexpression in the mossy fibers of the dg
in the KO mouse (H). Ectopic NPY accumulates in the mossy fibers of
dentate granule cells probably because it is transported through the
fibers to the terminals, from which it can be released . No obvious
differences in the expression of NPY and CB were observed in other
brain structures, as previously reported by others . (I–L) Calretinin
immunoreactivity in an untreated WT mouse (I,K) and a KO mouse (J,L).
Note the disorganization and increase in expression and number of
calretinin-labeled cells in the subgranular zone (SGZ) in KO sections.
Analysis of 9 KO animals showed modifications in the SGZ in 7 of them
(see also Figure S2), although 3 of these animals showed only a minor
disorganization of the SGZ cells. The granular layer itself showed no
obvious differences between the genotypes. Cx: cortex; dg: dentate
gyrus; gc: granular cells; mf: mossy fibers; SGZ: subgranular zone. Scale
bars: A (for A, B, E, F), 1 mm; C (for C, D, G, H), 200 mm; I (for I, J),
300 mm; K (for K, L), 100 mm.
Epilepsy in Dcx Knockout Mice
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month old WT (n=10) and KO (n=10) animals, clonic or tonic
seizures were observed in 7 KO mice compared to only 1 WT
animal (exp. B and C, Table 2), suggesting an enhanced
susceptibility to PTZ-induced seizures in KO animals.
When PTZ (30 mg/kg, i.p.) was injected in mice equipped with
hippocampal and cortical electrodes (exp D, Table 2; n=3 WT and
n=7 KO), no behavioral seizures or EEG abnormalities were
observed in WT animals during 10 min following injection (Table
S2). In 2 KO mice, PTZ induced a clonic-tonic seizure within 3 min,
rapidly followed by death. In another KO mouse, hippocampal
spikes (4–5/min) occurred within 3 min and were followed by a
discharge of spikes and spike-and-waves associated with a clonic
seizure (latency=8.5 min; duration=24 sec). Myoclonic jerks were
observed in 2 further KO animals, although no changes were
observed in EEG activity. No overt changes of either behavior or
EEG activity were observed in the 2 remaining KO mice.
KA (20 mg/kg, i.p.) was injected in a different series of WT
(n=27) and littermate KO (n=27) animals, aged 3–6 months.
During the first hour following injection, 13 WT animals became
immobile or displayed stereotypic movements (behavioral scores
rated 1, 2 or 3, Table S3), whereas the 14 others showed
convulsions or severe seizures (scores 4, 5 or 6). In contrast, only 8
KO mice were rated 1–3 in this test compared to 19 KO animals
showing convulsions or severe seizures (Figure 4A, B; Table S3).
Furthermore, KO mice were scored significantly higher than WT
in the first 15 min following KA injection, as well as throughout
the 3h monitoring period (Figure 4B). In both groups, several mice
died in the week following the KA test (n=12 KO, 11 WT), with 4
KO compared to 0 WT mice dying in the first 40 min.
When KA (20 mg/kg, i.p.) was administered in animals
equipped with hippocampal and cortical electrodes (n=5 WT;
n=4 KO), discharges of spikes associated with behavioral arrest
were observed in the hippocampus and cortex in both groups.
These discharges were similar in number, latency and mean
duration during the first hour post-injection (Table 3). However,
all KO mice displayed forelimb clonic seizures associated with a
discharge of spikes and polyspikes within the first 60 min post-KA
whereas this was observed in only 2 WT mice. These convulsive
seizures were significantly more frequent, and with a shorter
latency and longer mean duration during the first hour post KA in
KO compared to WT mice (Table 3).
These data clearly show that Dcx KO mice are more prone to
seizures induced by convulsants, with both PTZ and KA inducing
convulsive seizures more readily in KO animals, than WT
Video-EEG Recordings Reveal Ictal Activity and the
Initiation of Spontaneous Seizures in the Hippocampus
Our data converge to suggest that Dcx KO mice are epileptic.
Indeed, spontaneous convulsive seizures were observed occasionally
in KO mice, expression of seizure-sensitive markers in the
hippocampus was abnormal and these animals showed an increased
sensitivity to chemo-convulsants. We performed video-EEG
monitoring in an attempt to detect spontaneous electrographical
and behavioural seizures in the absence of convulsant and to
characterize associated behaviors. Animals (6 WT, 5 KO, aged 5–6
months) were regularly recorded for periods of 12h during the day
time. EEG recordings showed occasional hippocampal spikes and
short periods of theta oscillations associated with exploratory
behavior in both WT and KO animals. Spectral analyses of the
EEG activity during rest did not reveal any significant differences
between the two lines (data not shown).
Spontaneous seizures were observed in 2 of 5 KO mice and a
total of 5 seizures were recorded (Figure 5, Video S1). These
seizures occurred when the animal was at rest or during slow wave
sleep and always started with a slight movement of the head
immediately followed by stereotyped sniffing associated with the
first spikes in the hippocampus. As the discharge developed and
the amplitude increased, the animal reared and displayed head
nodding and clonus of the forelimbs. The discharge lasted between
28 and 40 sec after which flattening of the EEG was observed
(Figure 5A). During this post-ictal EEG depression, forelimb
clonus could still be observed for about 1 min. Following this
phase the animals remained immobile for 2–3 min after which
they generally groomed. No running, jumping or tonic phases
were observed during these analyses. Examination of EEG
recordings at lower speed suggested that the seizures were initiated
in the hippocampus and then propagated to the cortex (Figure 5B).
This was confirmed by time-frequency analyses in the 2–48 Hz
range showing a higher power of the EEG signal recorded in the
hippocampus as compared to the cortex (Figure 5C). In addition,
estimation of the direction of information transfer  indicated
that the hippocampus mainly leads the cortex at least during the
first 20 sec of the seizures (Figure 5D).
These data hence better characterize and confirm electrograph-
ically the occurrence of spontaneous clonic seizures in the absence
of convulsant in Dcx KO mice and localize seizure onset to the
hippocampus, the site of visible morphological abnormalities.
Enhanced Excitability in the CA3 Region of Dcx KO Mice
In order to test for functional changes in hippocampal circuitry
which might explain the indications of epileptic activity,
hippocampal slices were prepared (n=11 WT, n=10 KO) and
extracellular signals recorded from the stratum pyramidale of the
CA3b and CA3c regions (Figure 6A). Electrodes were placed with
a separation of 500 mm to detect the activity of distinct CA3
neuron populations .
Table 2. Increased Susceptibility of Dcx KO Mice to PTZ-
A: 50 mg/kgWT810215
B: 35 mg/kgWT430011
C: 30 mg/kgWT660000
D: 30 mg/kg WT330000
EEG (table 3) KO722032
Sum ofWT 13120011
B–D KO 1725463
0, no response; 1, myoclonic jerks; 2, clonic seizures associated with trembling
and chewing; 3, tonic-clonic seizures associated with extension of fore- and
hind limbs with animals falling on its side. Death, which could occur without
behavioral seizures, was noted independently of the seizure score. We observed
here that whereas the convulsive dose 50 s (CD50s) of 59.13 mg/kg was
previously reported in C57BL/6J mice, PTZ at 50 mg/kg i.p. induced death in a
large proportion of our KO and WT mice within less than 5 min following
injection (C57BL/6N background, exp A). Results obtained for the EEG
experiment (D) are shown separately to the data obtained at the same
concentration for the behavioral assessment alone (C), to take into account the
implantation of the electrodes and the different sites where these experiments
Epilepsy in Dcx Knockout Mice
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Spontaneous recurring population oscillations similar to sharp
waves observed in vitro  were generated in the CA3b and CA3c
regions from both WT and KO slices. This sharp wave activity is
similar to EEG activity generated in sleeping and resting animals
under normal physiological conditions. Each wave was evident as
a positive-going field potential with a sharp rise and a slower decay
(Figure 6C). Synchronous sharp wave-like events were however,
found to occur more frequently in Dcx KO slices (n=10,
1.9160.34 Hz) than WT slices (n=11, 0.4860.15 Hz, Students
t-test, P,0.001, Figure 6B and C). These results therefore show an
enhanced synchronous population activity in the Dcx KO CA3
region. Such activity might reflect an enhanced cellular excitability
or recurrent synaptic connectivity, both of which should favor
hippocampal epileptiform activity.
Dcx KO Mice Show a Lower Threshold for Epileptiform
Activity Induced by Bicuculline
Bicuculline, a GABAAreceptor antagonist, induces interictal-
like epileptiform events , characterized by high frequency
oscillations (150–300 Hz) superimposed on an underlying negative
field potential shift (Figure 6D). We compared the threshold dose
for the induction of these events in WT and Dcx KO hippocampal
slices. Slices were exposed sequentially to a series of concentrations
of bicuculline in the range 2–8 mM. Interictal bursts (Figure 6D)
were initiated in the CA3 region on exposure to 4 mM bicuculline
in 87.5 % of KO slices (n=14/16), and in the remaining 12.5 % at
8 mM bicuculline (n=2/16, Figure 6E). In contrast, only 41% of
WT slices reached bursting threshold with 4 mM bicuculline
(n=7/17), with a further 53% (n=9/17) at the higher
concentration of 8 mM. Thus, with the exception of one WT slice
bursting at 2 mM, KO slices showed a lower threshold for
interictal activity than WT slices. The frequency of interictal-like
epileptiformevents did not
(0.2460.03 Hz) and WT (0.1960.03 Hz) slices.
These data show that KO hippocampal slices have an enhanced
excitability and lower threshold for the induction of epileptiform
events, compared to slices from WT littermates. Combined with
the anatomical data, behavioral and EEG analyses, these results
strongly support the dysplastic hippocampus as the site of original
epileptogenic events in Dcx KO animals.
differsignificantly in KO
Figure 4. Dcx KO Mice are More Susceptible to KA-induced Seizures. (A) In the first 60 min after KA injection, a significant increase in the
progression of seizure-related behavior was observed in KO mice (white circles), compared to WT (black squares). ANOVA: genotype, F1,1565=37.65,
P,0.0001; time, F11,565=12.5, P,0.0001 and genotype6time interaction, F11,565=1.4, P.0.05. KO mice were scored significantly higher at 15 min
post-injection than WT mice and the increased severity of their reaction to KA continued throughout the initial 1 h monitoring period. *** differs from
control at P,0.0001. (B) Bar histograms show that, during the three hour monitoring period, more KO mice show rearing and falling (score 4) or
progression to severe tonic, clonic seizures (score 6) compared to WT mice. Inversely, more WT mice show less severe behavior (1, immobility). Thus,
KO mice are significantly more susceptible to KA-induced seizures than WT mice. Seizures were rated according to a previously defined scale : 1:
immobility; 2: forelimb and/or tail extension; 3: repetitive movements, head bobbing; 4: rearing and falling; 5: continuous rearing and falling: 6: severe
Epilepsy in Dcx Knockout Mice
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We previously showed that Dcx KO animals have a normally
laminated isocortex, but they exhibit interneuron migration
defects and a disorganized hippocampus. In the present study,
we set out to determine the consequences of these defects. We
show that KO animals are hyperactive, and exhibit signs of
chronic epilepsy, abnormalities that were not previously identified
in another Dcx KO model .
Hyperactivity was observed in Dcx KO mice during exploration
of novel environments. It is not yet clear if this phenotype is linked
to the epilepsy exhibited by these mice, to potential cognitive
deficits, or to other factors unrelated to the hippocampus. Indeed,
hyperactivity and additional sensorimotor abnormalities have been
linked to hippocampal dysfunction . Hyperactivity was thus
identified following hippocampal lesions in C57BL/6 mice ,
and in mouse models of mental retardation associated with
hippocampal-dependent cognitive defects [33,34]. Tuba1a mutant
mice, another lissencephaly model, are also hyperactive and
exhibit prominent hippocampal lamination defects . On the
other hand, hyperactivity has also been linked to isocortical
interneuron abnormalities . It will thus be important to further
explore whether this particular phenotype is directly related to
hippocampal lamination defects and potential cognitive deficits in
Dcx KO animals, or alternatively, to more subtle isocortical
We demonstrate here that Dcx KO mice develop epilepsy, with
seizures initiated in the hippocampus. Indeed, we observed both
behavioral features of limbic seizures, and facilitated seizure
induction by KA and PTZ, which have both been shown to target
limbic structures [37,38]. In contrast, seizures were not triggered
by audiogenic or tactile stimuli, known instead to involve
brainstem structures . The primary involvement of the
hippocampus is supported by the higher frequency of spontaneous
synchronous activities observed in the Dcx KO CA3 region in vitro,
combined with a lower threshold for interictal-like activities in the
presence of bicuculline, suggesting enhanced neuronal excitability
or recurrent synaptic connectivity of CA3 pyramidal cells. In
addition, our EEG recordings of spontaneous seizures, along with
time-frequency and information transfer analyses suggest that the
hippocampus is the primary epileptogenic focus in Dcx KO
animals. These converging data strongly suggest that discrete
hippocampal dysplasia gives rise to functional abnormalities which
render these animals more prone to seizures.
DCX mutations in human cause a spectrum of abnormalities
ranging from type I lissencephaly to subcortical laminar
heterotopia (SCLH), which are associated with mental retardation
and epilepsy, the latter frequently refractory to treatment . In
type I lissencephaly, EEG recordings, show high amplitude and
high frequency discharges, due to the neocortical disorganization
and smooth brain surface, where neurons are organized in similar
orientations, leading to an amplified signal . In SCLH, at the
other end of the spectrum, the EEG pattern and clinical symptoms
can be correlated with the thickness of the heterotopic band.
Patients with thin bands show a later onset of seizures which
originate mostly in the temporal lobe as shown by scalp EEG .
In some cases the discharges evolve into complex partial seizures
. On the other hand, patients with thick bands usually start
epilepsy early in infancy with mostly generalized seizures, and
interictal multifocal abnormalities are generally observed on scalp
EEG . However, in these latter cases it is possible that the
multifocal abnormalities mask an initiation of seizures in the
hippocampus. Indeed, in some patients stereotactic EEG record-
ings using depth electrodes have indicated a regional or focal
seizure onset which can involve the temporal lobe . Thus the
epilepsy observed in Dcx KO mice, with seizures initiated in the
hippocampus and propagated to the cortex, is somewhat
reminiscent of that observed in SCLH patients. However, it is
also possible that the hippocampus is not the only structure
generating seizures in such patients, and the human disorder is
obviously more complex than the mouse model. To our
knowledge, there are no clinical reports of isolated hippocampal
lamination defects in other human patients, although such subtle
abnormalities may be difficult to identify.
Other mouse models associating a susceptibility to epilepsy with
neuronal lamination defects, such as p35, Lis1 and reeler mutant
mice, show severe hippocampal pyramidal cell defects affecting
both the CA1 and CA3 regions [45–47]. Notably p35 and Lis1 KO
mice exhibit spontaneous tonic-clonic seizures, whilst reeler mice
have a reduced threshold for electroshock-induced seizures . A
defect in the hippocampal pyramidal cell layer is thus a common
pathological feature of these mice. However, the DG and the
isocortex are also abnormally organized, making it difficult to
determine the primary causes of the epilepsy. In this respect, the
Dcx KO mouse is unique, exhibiting subtle modifications of the
hippocampal circuitry, associated with the susceptibility to
epilepsy. No signs of hippocampal sclerosis (e.g. cell loss in hilar,
CA1 or CA3 areas) or granule cell dispersion  were obvious in
Dcx KO mice and the DG appears to form normally .
Therefore, with stable abnormalities in lamination largely
restricted to the hippocampus proper, in particular the CA3
region, Dcx KO mice provide a pertinent model to further
investigate the cellular mechanisms involved in seizure initiation.
Although controversial, seizure activity may influence dentate
granule cell neurogenesis [50–53]. Our data, showing in certain
animals an increase in both the number of calretinin-positive
neurons and in the intensity of their labeling, point to increased
neurogenesis in the dentate gyrus of certain Dcx KO mice. Since
these newly generated neurons were often also disorganized, they
could in addition contribute to network hyperexcitability and
possibly promote subsequent seizures . Further studies
assessing neurogenesis in Dcx KO animals and its relationship to
epileptic seizures are therefore warranted.
We show here that epileptiform activity is likely to be initiated in
the CA3 region at or near the pyramidal cell defects. Interestingly
CB-labeled mossy fibers are disorganized, suggesting that ectopic
pyramidal cells still receive mossy fiber innervation. In addition,
interneuron-like cells were identified in close proximity to
Table 3. Summary of EEG Results within 1 Hour Following KA
Latency of first non convulsive discharge (min)26.0064.9524.0068.76
Number of non convulsive discharges2.8060.972.0060.91
Mean duration of non convulsive discharge (sec) 22.0265.7825.96613.19
Latency of first convulsive seizure (min) 106.00633.92 28.5068.88=
Number of convulsive seizures0.8060.583.5061.04*
Mean duration of convulsive seizure (sec) 19.53612.1246.3366.43
Mean6SEM of latency, number and mean duration of non-convulsive
discharges and clonic convulsive seizures recorded by hippocampal and cortical
EEG in WT and Dcx KO mice during the first hour following the injection of KA
(20 mg/kg, i.p.). Mann-Whitney tests,*z=2.01, P,0.05. Latency: =, P=0.08.
Epilepsy in Dcx Knockout Mice
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dispersed, as well as correctly localized CA3 cells. These data
therefore suggest that, following the malpositioning of neurons due
to disrupted neuronal migration, spatially specific synaptic
projections could still form contacts with ectopic cells. However,
it remains to be determined whether such connections are formed
appropriately and how synaptic transmission differs from that in
WT animals. These questions need to be addressed to better
understand the mechanisms underlying seizure initiation.
In summary, we describe here the first functional consequences
associated with discrete hippocampal lamination defects. Impor-
tantly, we demonstrate that Dcx KO mice have an epileptic
phenotype and suggest that disrupted neuronal migration results in
enhanced seizure susceptibility similar to the human condition. A
further characterization of the consequences of the lamination
defects and possible interneuron abnormalities will help to better
characterize the pathogenic mechanisms at play. The Dcx KO
Figure 5. Spontaneous Clonic Seizures in Dcx KO Mice. (A) Typical example of hippocampal and cortical EEG recordings of a spontaneous
seizure observed in a Dcx KO mouse associated with rearing, head nodding and forelimb clonus. (B) EEG recording at lower speed of the same seizure
suggesting initiation in the hippocampus. Hipp: right hippocampus; Cx L: left cortex; Cx R: right cortex. (C) Averaged (n=5 spontaneous seizures)
time–frequency chart of signal power 5 sec before and 20 sec after the onset of the seizure in the hippocampus (Hipp) and the cortex (Cx). Hz, hertz.
(D) Difference of GPDC (averaged over 5 seizures) between hippocampal and cortical recordings. A positive value indicates a direction of information
transfer from hippocampus towards cortex. Red areas indicate a significant GPDC difference (p,0.005). Statistics were obtained using surrogate data
in which phase relationships were destroyed by phase randomization of frequency spectra.
Epilepsy in Dcx Knockout Mice
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model will thus permit further studies of how abnormal neuron
position leads to possible changes in cell excitability or connectivity
resulting in epileptic synchrony. Such studies should shed light on
the pathophysiology of cortical migration disorders associated with
refractory epilepsy, and may in the future allow the development
of novel therapies.
Materials and Methods
Dcx mutant mice were maintained on the C57BL/6N (B6)
background after more than 10 generations of backcrosses and
were produced and genotyped as previously described .
Figure 6. Enhanced Excitability and Lower Threshold for Epileptical Events in Dcx KO Hippocampal Slices. (A) Position of the two
tungsten electrodes placed in the stratum pyramidale of the CA3b and CA3c regions. (B) Bar graph shows a higher frequency of the synchronously
generated sharp wave-like events in KO slices than in WT (1.9160.34 Hz, KO, n=10 vs. 0.4860.15 Hz, WT, n=11, Students’s t-test, P,0.001). Error
bars indicate SEM. (C) Extracellular recordings of synchronously generated sharp wave-like activities in the CA3b and CA3c regions from WT (left) and
KO (center) animals. * indicates synchronous events, which are seen in both CA3b and c regions. Right panel shows 1 synchronous sharp wave-like
activity in KO slices. (D) Interictal bursts initiated in the CA3 region on exposure to bicuculline at 4 mM in KO hippocampal slices. (E) Bar graph shows
the percentage of slices that reached bursting threshold in bicuculline from 2 to 8 mM. KO mice are predisposed to epileptiform activity with
moderate bicuculline application.
Epilepsy in Dcx Knockout Mice
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Experiments were conducted during the light phase of a 12 h
light/dark schedule, with lights on at 07:30 a.m. All behavioral
and pharmacological experiments were performed on 1–6 months
old males. The studies were performed in accordance with the
European Communities Council Directive (86/809/EEC) regard-
ing the care and use of animals for experimental procedures and
approved by local ethical committees.
Immunohistochemistry and Golgi-Cox staining
Immunohistochemistry was performed as described previously
. Markers used with immunoperoxidase detection were anti-
calretinin (1:10000, rabbit; Swant laboratories, Switzerland); anti-
NPY (1:10000, rabbit; Sigma, St. Louis, MO) and anti-CB
(1:20000, rabbit; Swant laboratories, Switzerland).
Golgi-Cox staining was performed as described by Gibb and
Kolb . WT and KO adult mice (6 months) were anesthetized
with pentobarbital (Ceva Sante ´ Animale; 0.15 ml/ 10g body
weight) and perfuzed intracardially with 9% (w/v) saline buffer, and
brains were placed in Golgi-Cox solution  at room temperature
in the dark for 10 days. Brains were then placed in 30% (w/v)
sucrose for 5 days at room temperature in the dark, prior to
sectioning in 6% (w/v) sucrose using a vibratome (200 mm, VT
1000S, Leica, Germany). Sections were incubated in the dark firstly
in NH4OH for 30 min, then after rinsing, in Kodak fixative for
30 min, and then were counter-stained with toluidine blue.
Horizontal (locomotion) and vertical (rearing) activities of naive
animals in novel environments were measured individually in
plexiglass cages (20615625 cm), with automatic monitoring of
photocell beam breaks every 5 min for 2 consecutive hours
(Imetronic, France). For horizontal activity, consecutive beam
breaks were recorded from two separate photocell beams. The
number of beam breaks in this case can be correlated with the
walking distance (cm) and provides a direct index of locomotion.
Behavioral assessment of seizure activity
susceptibility in both WT and Dcx KO animals was assessed
according to Todorova . The handling stimulus involved
lifting each mouse by the tail for 30 s, and moving it to a new cage.
Each animal was observed for 30 s on 5 consecutive days.
Audiogenic seizures (AS).
dependent , all animals were tested prior to weaning, (at 28-33
days) in a plexiglass cage (45645 cm) covered by a grid. After
15 sec of habituation, animals were exposed to a computer-
generated sound of 13 kHz at 90 decibels for 90 sec. Mice that did
not display a seizure response were re-exposed 48h later and tested
for sensitization-dependent audiogenic seizures. The intensity of
the response pattern was rated simultaneously by two observers
blind to the genotypes: 0, no response; 1, wild running; 2, clonic
seizures and/or tonic flexion and extension; 3, death.
Since AS susceptibility is age
Chemically induced seizures
Pentylenetetrazole (PTZ, 30–50 mg/kg, Sigma, St
Louis, USA) and kainate (KA, 15–20 mg/kg, OPIKA-1TM, Ocean
Produce International) were dissolved in 0.9% NaCl on the day of
test and were injected intraperitoneally (i.p.) in a volume of 0.1 ml
Induction and scoring of seizures.
convulsions were induced after an initial 2h period of habituation
to the test cage, by i.p. injection of either PTZ, at the doses of 30,
35 and 50 mg/kg, or KA (15 or 20 mg/kg i.p.). The animals were
immediately placed back into the test cages and their behavior and
seizure-related activities were observed by two experimenters blind
to the genotypes.
Following PTZ injections, scoring of seizures  was
performed every minute for 10 min after injection as follows: 0,
no response; 1, myoclonic jerks; 2, clonic seizures associated with
trembling and chewing; 3, tonic-clonic seizures associated with
extension of fore- and hind limbs with animals falling on its side.
Death, which could occur without behavioral seizures, was noted
independently of the seizure score. In our PTZ experiments, we
observed that whereas the convulsive dose 50 s (CD50s) of
59.13 mg/kg was previously reported in C57BL/6J mice, PTZ at
50mg/kg i.p. induced death in a large proportion of our KO and
WT mice within less than 5 min following injection (C57BL/6N
background, exp A, Table 2).
For KA, mice were monitored for 30 seconds every 5 minutes
during the first hour after the injection and every 20 minutes
during the following 2 hours. Seizures were rated according to a
previously defined scale  1, immobility; 2, forelimb and/or tail
extension; 3, repetitive movements, head bobbing; 4, rearing and
falling; 5, continuous rearing and falling; 6, severe tonic-clonic
seizures, death (after seizures).
Video- EEG recordings.
Mice were anaesthetized (chloral
hydrate, 0.4 g/kg,i.p.) and implanted with cortical and
hippocampal electrodes as previously described . After at
least 2 weeks of recovery, video-EEG activity was recorded using a
digital acquisition computer-based system (Coherence, Deltamed,
France, sampling rate 256 Hz) while the mice were freely moving
in a Faraday cage, as in previous studies . When mice were
examined for spontaneous seizures, their EEG activity was
recorded concomitantly with their behavior (synchronized video)
for up to 12 hours during the day, on 6–7 different days during a
period of 6 weeks. To test convulsants, the animals were first
recorded for 5 or 10 min before the PTZ or KA injection,
respectively, and then for 10 min (PTZ) or 3 hours (KA)
A time-frequency analysis of the spontaneous seizures in 2 KO
mice was performed using an in-house developed toolbox of
Statistical Parametric Mapping 5 software (www.fil.ion.ucl.ac.uk/
spm, Wellcome Department of Imaging Neuroscience, University
College London, UK) for dynamical analysis of intracerebral
EEG. For each discharge, the amplitude (square-root of power) of
oscillatory activity between 1 and 48 Hz, from 5 sec before the
onset and up to 20 sec thereafter, was obtained using a standard
time-frequency analysis based on Morlet wavelet transform .
For each frequency, the amplitude was computed on a 7 period
length sliding time-window, providing an effective frequency
specific time resolution. Time-frequency sampling of the time-
frequency plane was 4 ms/2 Hz. The time-frequency plane was
averaged over the 5 seizures.
To determine whether spontaneous seizures were initiated in
the hippocampus or in the cortex, estimation of the direction of
information transfer was performed using the generalized partial
directed coherence (GPDC) test . The GPDC was computed
using the BioSig toolbox (http://biosig.sourceforge.net/) on a
sliding time window (time width: 3 s; time step: 100 ms). The
model order of autoregressive models used in GPDC was defined
according to the Schwarz’s Bayesian criterion.
Upon completion of the experiments, all mice were injected
with a lethal dose of pentobarbital (Nembutal, 100 mg/kg, i. p.).
Brains were frozen, and cut into 20-mm sections using a cryostat
(CM 3050S, Leica, Germany). Histological analysis to determine
electrode position was performed following cresyl violet staining.
Slices were cut at 380 mm from WT and
KO mice (3–4 months) using a vibratome (HM 650V, Microm). 6
Epilepsy in Dcx Knockout Mice
PLoS ONE | www.plosone.org10 June 2008 | Volume 3 | Issue 6 | e2473
KO and 4 WT mice were used for the sharp wave experiments
and 5 KO and 4 WT animals for the bicuculline experiments.
Mice were anesthetized with 80 mg/kg of ketamine HCl/
Xylazine HCl solution (Sigma, St. Louis, MO). The brain was
removed andchilled inice-cold,
cerebrospinal fluid (ACSF) of the following composition (in
mM): 250 sucrose, 1 KCl, 26 NaHCO3, 10 D-glucose, 1 CaCl2,
10 MgCl2. Transverse slices were prepared from the level
approximately two-thirds of the way down the septo-temporal
arc and placed in an interface recording chamber . Slices were
perfused with an oxygenated ACSF containing (in mM): 124
NaCl, 4 KCl, 26 NaHCO3, 10 D-glucose, 2 CaCl2, 2 MgCl2at
35uC, while their upper surface was exposed to a humidified 95%
O2/ 5% CO2(v/v) atmosphere. Slices were exposed sequentially
for 20 min to a series of concentrations of bicuculline (Tocris,
Ellisville, USA) in range of 2–8 mM.
Multi-unit activities were recorded with extracellular electrodes
made from tungsten wire of 50 mm diameter (Phymep, Paris,
France). Up to 3 electrodes were mounted on holders controlled
by separate manipulators. Differences in potential between each
tungsten electrode and a reference Ag-AgCl electrode were
measured using a 4-channel amplifier (AM system, model 1700,
Carlsborg WA, USA). Extracellular signals were amplified 1000 x
and filtered with pass band between 1 Hz and 10 kHz. Signals
were digitized at 10–20 kHz using a 12-bit, 16 channel analog-to-
digital converter (Digidata 1200A, Axon Instruments), and
visualized on a PC using the program Axoscope (Axon
The data were analyzed by unpaired
Student’s t-tests, and repeated measures of analysis of variance
(ANOVA) to assess the interaction between genotypes (between
factor) and time (within factor). When variables did not follow a
normal distribution, statistical analyses were carried out using the
nonparametric Mann-Whitney rank sum test. Only significant
statistical tests are reported in the text, with the significance
established at a P-value,0.05. Error bars represent s.e.m. The
data presented in Figure 4B are raw data (descriptive statistics).
Expression in Dcx KO Mice.
Found at: doi:10.1371/journal.pone.0002473.s001 (0.04 MB
Modification in Calbindin NPY and Calretinin
Found at: doi:10.1371/journal.pone.0002473.s002 (0.03 MB
Summary of EEG results within 10 min following
1 hour observation period).
Found at: doi:10.1371/journal.pone.0002473.s003 (0.03 MB
Susceptibility of Dcx KO and WT mice to KA (during
Fig 1H, compared to a CA3 pyramidal cell in the same
hippocampal section. (A–E) The Golgi-Cox stainings were
performed in 40 mm brain slices and the three soma of
interneuron-like cells are not all in focus in the same plane. This
focal series helps show that each cell shows the typical morphology
of a CA3 basket cell or axo-axonic-like interneuron , with
fusiform cell bodies located within or adjacent to the pyramidal
cell layer, and one or two dendrites originating from the apical
A focal z series of interneuron-like cells shown in
pole, which then branch proximally to give radially oriented
dendrites in the stratum radiatum. In addition, such cells have
several basal dendrites branched close to the cell body and
extended toward the alveus. Spines are rarely present on these few
branches. Despite this recognisable morphology, we cannot
specifically say what type of interneurons these are, because of
the absence of a labelled axon arbor. (F, G) CA3 pyramidal cells
(arrow in F and same cell shown in G) differ from this because they
have one prominent apical dendrite emerging from a triangular
soma, and this is radially oriented in the stratum radiatum where it
is branched into large diameter segments, and in the stratum
lacunosum moleculare where they emit several branches. Basal
dendrites are also numerous in the stratum oriens. In addition the
dendritic tree is typically densely covered with spines. The
proximal apical dendrite has large complex spines (the thorny
excrescence, arrowhead G) which form complexes with the large
mossy fiber terminal. Scale bar E (for A-E); F, 75 mm; G, 37.5 mm.
Found at: doi:10.1371/journal.pone.0002473.s004 (17.83 MB
subgranular zone of the dentate gyrus in Dcx KO animals. (A)
Two KO animals were observed to have a greatly augmented
number of disorganized calretinin-positive cells, as shown here for
one animal. Both animals also showed changes in NPY and CB.
(B, C) Two further KO animals showed increased and/or
disorganized calretinin-positive cells. The animal in B also showed
changed NPY and CB. (D, E) Certain KO animals (n=3) showed
subtly disorganized calretinin-positive cells which were however
not visibly increased number. The animal shown in D did not
show changes in NPY and CB, whereas two further animals (one
of which is shown in E) did. (F) Two KO animals, one of which is
shown here, showed no overt changes in calretinin-positive cells,
and no changes in NPY and CB. (G) A WT section is shown for
comparison. 4 WT animals were analyzed. Scale bar G (for A–G),
Found at: doi:10.1371/journal.pone.0002473.s005 (5.83 MB TIF)
Changes observed in calretinin -positive cells in the
equipes/equipe9/index.html). A spontaneous seizure is exhibited
by a Dcx KO mice in channel 2. Initially the mouse is resting. The
first behavioral signs of the seizure are agitation of the head,
followed by rearing and clonus of the forelimbs. This is followed by
immobility for a short period.
Found at: doi:10.1371/journal.pone.0002473.s006 (2.36 MB
Video-EEG of a Dcx KO mouse suffering a
We are grateful to H. Bernard, Y. Saillour and N. Fallet for technical
assistance, to O. David for performing EEG signal analysis, to N. Bahi-
Buisson for helpful discussions concerning human patients and S. Marty for
discussions and gifts of antibodies. We thank L. Hillard for animal care at
Inserm U513 and the animal care facilities at the Institut Cochin and the
Conceived and designed the experiments: RM MN FF AD. Performed the
experiments: CM MN FF CK CDinocourt CDenis JG SV CA FP AD.
Analyzed the data: RM CM MN FF CK CDinocourt CDenis JG SV CA
FP AD. Contributed reagents/materials/analysis tools: MN FF CK. Wrote
the paper: RM MN FF CK CDinocourt AD. Other: Head of the
laboratory: BG RM AD JC. Animal breeding and genotyping: MN.
Epilepsy in Dcx Knockout Mice
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1. Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, et al. (1999)
Doublecortin is a developmentally regulated, microtubule-associated protein
expressed in migrating and differentiating neurons. Neuron 23: 247–256.
2. 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.
3. des Portes V, Pinard JM, Billuart P, Vinet MC, Koulakoff A, et al. (1998) A
novel CNS gene required for neuronal migration and involved in X-linked
subcortical laminar heterotopia and lissencephaly syndrome. Cell 92: 51–61.
4. Gleeson JG, Allen KM, Fox JW, Lamperti ED, Berkovic S, et al. (1998)
Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly
and double cortex syndrome, encodes a putative signaling protein. Cell 92:
5. Reiner O, Carrozzo R, Shen Y, Wehnert M, Faustinella F, et al. (1993) Isolation
of a Miller-Dieker lissencephaly gene containing G protein beta-subunit-like
repeats. Nature 364: 717–721.
6. Guerrini R, Marini C (2006) Genetic malformations of cortical development.
Exp Brain Res. 173(2): 322–33.
7. Harding B (1996) Gray Matter Heterotopia. In Guerrini R, Andermann F,
Canapicchi R, Roger J, Zilfkin B, Pfanner P, eds (1996) Dysplasias of cerebral
Cortex and Epilepsy. Philadelphia-New York: Lippincott-Raven. pp 81–88.
8. Kappeler C, Dhenain M, Phan Dinh Tuy F, Saillour Y, Marty S, et al. (2007)
Magnetic resonance imaging and histological studies of corpus callosal and
hippocampal abnormalities linked to doublecortin deficiency. J. Comp. Neur.
500 (2): 239–254.
9. Chevassus-au-Louis N, Represa A (1999) The right neuron at the wrong place:
biology of heterotopic neurons in cortical neuronal migration disorders, with
special reference to associated pathologies. Cell Mol Life Sci. 55: 1206–1215.
10. Schwartzkroin PA, Roper SN, Wenzel HJ (2004) Cortical dysplasia and epilepsy:
animal models. Adv Exp Med Biol. 548: 145–74.
11. Corbo JC, Deuel TA, Long JM, LaPorte P, Tsai EA, et al. (2002) Doublecortin
is required in mice for lamination of the hippocampus but not the neocortex. J.
Neurosci. 22: 7548–7557.
12. Kappeler C, Saillour Y, Baudoin JP, Tuy FP, Alvarez C, et al. (2006) Branching
and nucleokinesis defects in migrating interneurons derived from doublecortin
knockout mice. Hum. Mol. Gen. 15: 1387–1400.
13. Friocourt G, Liu JS, Antypa M, Rakic S, Walsh CA, et al. (2007) Both
doublecortin and doublecortin-like kinase play a role in cortical interneuron
migration. J Neurosci. 27(14): 3875–83.
14. Freund TF, Buzsa ´ki G (1996) Interneurons of the hippocampus. Hippocampus.
15. White JD, Gall CM (1987) Differential regulation of neuropeptide and proto-
oncogene mRNA content in the hippocampus following recurrent seizures.
Brain Res. 427(1): 21–9.
16. Bellmann R, Widmann R, Olenik C, Meyer DK, Maas D, et al. (1991)
Enhanced rate of expression and biosynthesis of neuropeptide Y after kainic
acid-induced seizures. J Neurochem. 56(2): 525–30.
17. Pitka ¨nen A, Beal MF, Sirvio ¨ J, Swartz KJ, Ma ¨nnisto ¨ PT, et al. (1989)
Somatostatin, neuropeptide Y, GABA and cholinergic enzymes in brain of
pentylenetetrazol-kindled rats. Neuropeptides. 14(3): 197–207.
18. Bendotti C, Vezzani A, Serafini R, Servadio A, Rivolta R, et al. (1991) Increased
preproneuropeptide Y mRNA in the rat hippocampus during the development
of hippocampal kindling: comparison with the expression of preprosomatostatin
mRNA. Neurosci Lett. 132(2): 175–8.
19. Chafetz RS, Nahm WK, Noebels JL (1995) Aberrant expression of neuropeptide
Y in hippocampal mossy fibers in the absence of local cell injury following the
onset of spike-wave synchronization. Brain Res Mol Brain Res. 31(1–2): 111–21.
20. Ruttimann E, Vacher C-M, Gassmann M, Kaupmann K, Van der Putten H, et
al. (2004) Altered hippocampal expression of calbindin-D-28k and calretinin in
GABAB(1)-deficient mice. Biochem. Pharmacol. 68: 1613–1620.
21. Reibel S, Nadi S, Benmaamar R, Larmet Y, Carnahan J, et al. (2001)
Neuropeptide Y and epilepsy: varying effects according to seizure type and
receptor activation. Peptides 22(3): 529–39.
22. Vezzani A, Sperk G, Colmers WF (1999) Neuropeptide Y: emerging evidence
for a functional role in seizure modulation. Trends Neurosci. 22(1): 25–30.
23. Yang Q, Wang S, Hamberger A, Celio MR, Haglid KG (1997) Delayed
decrease of calbindin immunoreactivity in the granule cell-mossy fibers after
kainic acid-induced seizures. Brain Res. Bull. 43: 551–559.
24. Neumann PE, Collins RL (1991) Genetic dissection of susceptibility to
audiogenic seizures in inbred mice. Proc. Natl. Acad. Sci. U S A. 88(12):
25. Yoshihara Y, Onodera H, Iinuma K, Itoyama Y (2003) Abnormal kainic acid
receptor density and reduced seizure susceptibility in dystrophin-deficient mdx
mice. Neuroscience 117(2): 391–5.
26. De Sarro G, Ibbadu GF, Marra R, Rotiroti D, Loiacono A, et al. (2004) Seizure
susceptibility to various convulsant stimuli in dystrophin-deficient mdx mice.
Neurosci Res. 50: 37–44.
27. Baccala LA, Takahashi DY, Sameshima K (2006) Computer Intensive Testing for
the Influence Between Time Series, Eds. B Schelter, M Winterhalder, J Timmer:
Handbook of Time Series Analysis-Recent Theoretical Developments and
Applications, Wiley, pp 413.
28. Cohen I, Miles R. Contributions of intrinsic and synaptic activities to the
generation of neuronal discharges in in vitro hippocampus. J Physiol. 524 Pt 2:
29. Kubota D, Colgin LL, Casale M, Brucher FA, Lynch G (2003) Endogenous
waves in hippocampal slices. J Neurophysiol. 89: 81–9.
30. Campbell AM, Holmes O (1984) Bicuculline epileptogenesis in the rat. Brain
Res. 323(2): 239–46.
31. Bast T, Feldon J (2003) Hippocampal modulation of sensorimotor processes.
Prog. Neurobiol. 70: 319–345.
32. Goddyn H, Leo S, Meert T, D’Hooge R (2006) Differences in behavioural test
battery performance between mice with hippocampal and cerebellar lesions.
Behav Brain Res. 173(1): 138–47.
33. Bakker CE, the Dutch-Belgian Fragile X Consortium. (1994) Fmr1 knockout
mice: a model to study fragile X mental retardation. Cell 78: 23–33.
34. Khelfaoui M, Denis C, van Galen E, de Bock F, Schmitt A, et al. (2007) Loss of
X-linked mental retardation gene oligophrenin1 in mice impairs spatial memory
and leads to ventricular enlargement and dendritic spine immaturity. J Neurosci.
35. Keays DA, Tian G, Poirier K, Huang GJ, Siebold C, et al. (2007) Mutations in
alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in
humans. Cell 128(1): 45–57.
36. Pillai-Nair N, Panicker AK, Rodriguiz RM, Gilmore KL, Demyanenko GP, et
al. (2005) Neural cell adhesion molecule-secreting transgenic mice display
abnormalities in GABAergic interneurons and alterations in behavior. J
Neurosci. 25(18): 4659–71.
37. Ben-Ari Y, Tremblay E, Riche D, Ghilini G, Naquet R (1981) Electrographic,
clinical and pathological alterations following systemic administration of kainic
acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose
method with special reference to the pathology of epilepsy. Neuroscience 6:
38. Andre ´ V, Pineau N, Motte JE, Marescaux C, Nehlig A (1998) Mapping of
neuronal networks underlying generalized seizures induced by increasing doses
of pentylenetetrazol in the immature and adult rat: a c-Fos immunohistochem-
ical study. Eur J Neurosci. 10(6): 2094–106.
39. Browning RA (1985) Role of the brain-stem reticular formation in tonic-clonic
seizures: lesion and pharmacological studies. Fed Proc. 44(8): 2425–31.
40. De Rijk-van Andel Arts WFM, de Weerd AW (1992) EEG and evoked potentials
in a series of 21 patients with lissencephaly type I. Neuropediatrics 23: 4–9.
41. Barkovic AJ, Guerrini R, Battaglia G, Kalifa G, N’Guyen T, et al. (1994) Band
heterotopia: correlation of outcome with magnetic resonance imaging
parameters. Ann Neurol. 36(4): 609–17.
42. Grant AC, Rho JM (2002) Ictal EEG patterns in band heterotopia. Epilepsia
43. Janzen L, Sherman E, Langfitt J, Berg M, Connolly M (2004) Preserved episodic
memory in subcortical band heterotopia. Epilepsia 45 (5): 555–558.
44. Bernasconi A, Martinez V, Rosa-Neto P, D’Agostino D, Bernasconi N, et al.
(2001) Surgical resection for intractable epilepsy in ‘‘double cortex’’ syndrome
yields inadequate results. Epilepsia 42(9): 1124–9.
45. Deller T, Drakew A, Heimrich B, Forster E, Tielsch A, et al. (1999) The
hippocampus of the reeler mutant mouse: fiber segregation in area CA1 depends
on the position of the postsynaptic target cells. Exp Neurol. 156: 254–267.
46. Fleck MW, Hirotsune S, Gambello MJ, Phillips-Tansey E, Suares G, et al. (2000)
Hippocampal abnormalities and enhanced excitability in a murine model of
human lissencephaly. J. Neurosci. 20: 2439–2450.
47. Wenzel HJ, Robbins CA, Tsai L-H, Schwartzkroin PA (2001) Abnormal
morphological and functional organization of the hippocampus in a p35 mutant
model of cortical dysplasia associated with spontaneous seizures. J. Neurosci. 21:
48. Patrylo PR, Browning RA, Cranick S (2006) Reeler homozygous mice exhibit
enhanced susceptibility to epileptiform activity. Epilepsia 47(2): 257–66.
49. Riban V, Bouilleret V, Pham-Le BT, Fritschy JM, Marescaux C, et al. (2002)
Evolution of hippocampal epileptic activity during the development of
hippocampal sclerosis in a mouse model of temporal lobe epilepsy. Neuroscience
50. Heinrich C, Nitta N, Flubacher A, Muller M, Fahrner A, et al. (2006) Reelin
deficiency and displacement of mature neurons, but not neurogenesis, underlie
the formation of granule cell dispersion in the epileptic hippocampus. J.
Neurosci. 26(17): 4701–13.
51. Bengzon J, Kokaia Z, Elme ´r E, Nanobashvili A, Kokaia M, et al. (1997)
Apoptosis and proliferation of dentate gyrus neurons after single and intermittent
limbic seizures. Proc Natl Acad Sci U S A. 94(19): 10432–7.
52. Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, et al. (1997)
Dentate granule cell neurogenesis is increased by seizures and contributes to
aberrant network reorganization in the adult rat hippocampus. J Neurosci.
53. Gray WP, Sundstrom LE (1998) Kainic acid increases the proliferation of
granule cell progenitors in the dentate gyrus of the adult rat. Brain Res. 790(1–
54. Parent JM, Jessberger S, Gage FH, Gong C (2007) Is neurogenesis reparative
after status epilepticus? Epilepsia 48 Suppl 8: 69–71.
Epilepsy in Dcx Knockout Mice
PLoS ONE | www.plosone.org 12June 2008 | Volume 3 | Issue 6 | e2473
55. Gibb R, Kolb B (1998) A method for vibratome sectioning of Golgi-Cox stained
whole rat brain. J Neurosci Methods 79(1): 1–4.
56. Glaser EM, Van der Loos H (1981) Analysis of thick brain sections by obverse-
reverse computer microscopy: application of a new, high clarity Golgi-Nissl
stain. J Neurosci Methods 4(2): 117–25.
57. Todorova MT, Burwell TJ, Seyfried TN (1999) Environmental risk factors for
multifactorial epilepsy in EL mice. Epilepsia 40: 1697–1707.
58. Yuhas Y, Shulman L, Weizman A, Kaminsky E, Vanichkin A, et al. (1999)
Involvement of tumor necrosis factor alpha and interleukin-1beta in enhance-
ment of pentylenetetrazole-induced seizures caused by Shigella dysenteriae.
Infect Immun. 67(3): 1455–60.
59. Schauwecker PE, Steward O (1997) Genetic determinants of susceptibility to
excitotoxic cell death: implications for gene targeting approaches. Proc. Natl.
Acad. Sci. U S A 94: 4103–4108.
60. Le Van Quyen M, Foucher J, Lachaux J, Rodriguez E, Lutz A, et al. (2001)
Comparison of the Hilbert transform and wavelet methods for the analysis of
neuronal synchrony. J. Neurosci. Methods 111: 83–98.
61. McCarthy JB, Walker M, Pierce J, Camp P, White JD (1998) Biosynthesis and
metabolism of native and oxidized neuropeptide Y in the hippocampal mossy
fiber system. J Neurochem. 70(5): 1950–63.
Epilepsy in Dcx Knockout Mice
PLoS ONE | www.plosone.org13 June 2008 | Volume 3 | Issue 6 | e2473