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The Journal of Neuroscience, February 1, 2001, 21(3):983-998
Abnormal Morphological and Functional Organization of the
Hippocampus in a p35 Mutant Model of Cortical Dysplasia Associated with
Spontaneous Seizures
H. J.
Wenzel1,
C. A.
Robbins1,
L.-H.
Tsai3, and
P. A.
Schwartzkroin1, 2
Departments of 1 Neurological Surgery and
2 Physiology/Biophysics, University of Washington, Seattle,
Washington 98195, and 3 Howard Hughes Medical Institute and
Department of Pathology, Harvard Medical School, Boston,
Massachusetts 02115
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ABSTRACT |
Cortical dysplasia is a major cause of intractable epilepsy in
children. However, the precise mechanisms linking cortical malformations to epileptogenesis remain elusive. The neuronal-specific activator of cyclin-dependent kinase 5, p35, has been recognized as a
key factor in proper neuronal migration in the neocortex. Deletion of
p35 leads to severe neocortical lamination defects associated with
sporadic lethality and seizures. Here we demonstrate that
p35-deficient mice also exhibit dysplasia/ heterotopia of principal
neurons in the hippocampal formation, as well as spontaneous behavioral
and electrographic seizures. Morphological analyses using
immunocytochemistry, electron microscopy, and intracellular labeling
reveal a high degree of abnormality in dentate granule cells, including
heterotopic localization of granule cells in the molecular layer and
hilus, aberrant dendritic orientation, occurrence of basal dendrites,
and abnormal axon origination sites. Dentate granule cells of
p35-deficient mice also demonstrate aberrant mossy fiber sprouting.
Field potential laminar analysis through the dentate molecular layer
reflects the dispersion of granule cells and the structural
reorganization of this region. Similar patterns of cortical
disorganization have been linked to epileptogenesis in animal models of
chronic seizures and in human temporal lobe epilepsy. The p35-deficient
mouse may therefore offer an experimental system in which we can
dissect out the key morphological features that are causally related to epileptogenesis.
Key words:
epilepsy; dentate gyrus; granule cell dispersion; heterotopia; neuronal migration disorder; biocytin; EEG
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INTRODUCTION |
Cortical dysplasia, an abnormality
of neocortical development associated with disturbed neural migration
and/or aberrant neuronal-glial differentation, is recognized as one of
the major causes of pediatric epilepsy (Honavar and Meldrum, 1997 ).
Severe forms of cortical dysplasia are often associated with medically
intractable seizures, developmental delay, and neurological deficits
(Mischel et al., 1995). Misplaced cells ("heterotopia") are a
common feature of cortical dysplasia, which is thought to result from a
failure of migration from the site of neuronal genesis to the normal
destination of the cells in the cortical plate (Guerrini et al., 1999;
Smith et al., 1999; Walsh, 1999). However, mechanisms linking cortical dysplastic malformations to epileptogenesis are still to be elucidated.
Control of the spatial and temporal patterns of neuronal
differentiation and migration involves a multitude of factors (e.g., protein molecules, adhesion molecules, channels/receptors, and intracellular cytoskeletal proteins) (for review, see Rakic
and Caviness, 1995; Huttenlocher et al., 1995). Defects in
developmental processes have been implicated in a large group of
genetic disorders associated with cortical dysplasia/heterotopia
(Raymond et al., 1995; Barkovich et al., 1996), including early onset
epilepsies (Guerrini et al., 1999; Walsh, 1999). To better understand
the relationship between these structural malformations and the
development of an epileptic state, investigators have turned to animal
model systems, including such mouse mutants as reeler (Falconer, 1951; Caviness and Sidman, 1973; Caviness, 1976), Lis-1 (Reiner et al., 1993;
Hirotsune et al., 1998; Fleck et al., 2000), doublecortin (Des Portes
et al., 1998), and cyclin-dependent kinase 5 (cdk5) (Ohshima et al.,
1996; Gilmore et al., 1998) (for review, see Chevassus-au-Louis et al.,
1999; Walsh, 1999). Surprisingly, although all of these mutations give
rise to disruption of normal cortical development, none is associated
with the occurrence of chronic spontaneous seizures.
Recently, a mouse "knock-out" for p35, a neuronal activator
of cdk5 that plays an important role in brain development, has been
generated (Chae et al., 1997). The protein p35 is highly expressed in
postmitotic neurons but completely absent in proliferating neuronal
progenitor cells (Tsai et al., 1994; Delalle et al., 1997). Mice with
the p35 mutation lack p35/cdk5 kinase activity and exhibit severe
cortical lamination disruption similar to that seen in reeler (i.e., an
inversion of cortical lamination). In addition, these animals suffer
from sporadic adult lethality and spontaneous seizures and display a
diminished corpus callosum and abnormal tangential fiber fascicles
within the neocortex (Chae et al., 1997; Kwon and Tsai, 1998).
Although the morphological disruptions of the cerebral cortex in p35
knock-outs have been studied in detail (Chae et al., 1997; Kwon and
Tsai, 1998; Kwon et al., 1999), much less is known about malformations
in hippocampus, and little is understood about the relationship of
disturbed neuronal organization to neuronal activity and seizure
generation. Here we report that significant structural abnormalities
appear in the hippocampus (particularly in dentate gyrus) of
p35-deficient mice, similar to pathology observed in resected
hippocampi of patients with temporal lobe epilepsy (tLE) (Houser, 1990,
1999; El Bahh et al., 1999). Furthermore, a high proportion of p35
knock-out mice exhibits spontaneous behavioral and/or electrographic
seizures reminiscent of limbic seizures. These observations provide an
initial basis for relating epileptogenesis to dysplastic malformations
resulting from errors in brain development.
Preliminary data have been reported previously in abstract form (Wenzel
et al., 1998; Robbins et al., 1999).
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MATERIALS AND METHODS |
Animals
A complete description of the gene deletion protocol and intial
characterization of these mice has been published (Chae et al., 1997).
Morphological analysis of hippocampal neurons was performed in brains
of p35 knock-out mice (p35 /). The p35/ colony was
maintained via brother-sister matings, and wild-type controls (+/+)
were derived from the same stock as originally used to generate /
breeding pairs (129/Sv × C57BL/6 background). Most of the
analysis was performed on 2- to 5-month-old mice; animals at ages 2, 10, and 20 d and 14-15 months were included for comparison.
Various electrophysiological and morphological methods were used to
analyze seizure-related behaviors and related properties of dentate
granule cells. All animal care and use conformed to the NIH Guide
for Care and Use of Laboratory Animals and were approved by the
Animal Care Committee of the University of Washington.
Observations of seizure behavior and EEG monitoring
Threshold testing. Seizure susceptibility
was measured at 60 d postnatally in 15 p35 knock-out and 12 wild-type mice, using the volatile convulsant flurothyl
(2,2,2-trifluroethyl ether; Aldrich, Milwaukee, WI) according to well
established protocols (Samoriski and Applegate, 1997; Rho et al.,
1999). The mice were placed individually in a Plexiglas chamber, and
flurothyl was infused into the chamber at a rate of 20 µl/min.
Latencies were measured from the beginning of infusion to the onset of
a first generalized seizure (clonic) and to a second generalized
seizure (tonic-clonic) that follows a refractory postictal period.
Shorter latencies reflect greater seizure susceptibility (i.e., lower threshold). Exposure to flurothyl was terminated at the onset of the
second seizure. Data were analyzed using the Student's t
test (between-group comparison) using the SigmaStat Program (Jandel Scientific).
Video/EEG monitoring. Using ketamine/xylazine anesthesia, we
surgically implanted chronic EEG electrodes in 12 mice between 3 and 5 months of age. Microscrews (stainless steel) served as epidural
recording electrodes, and a twisted pair of fine stainless steel wires
served as a depth electrode, which was stereotactically implanted into
the right hippocampus. Electrodes were attached to a microplug, then
cemented to the cranium with dental acrylic. A Telefactor Video/EEG
monitoring system (Telefactor, W. Conshohocken, PA) was used to record
simultaneously both behavior and EEG from freely moving animals. Each
animal was monitored for 40-50 hr over the course of 3-4 weeks.
Tissue preparation for light
microscopy/immunocytochemistry
General tissue preparation. Forty-five mice used for
light microscopic and immunocytochemical studies were anesthetized with Nembutal (100 mg/kg, i.p.), then perfused with isotonic saline with
heparin (500 U/ml saline), followed by a solution of 4%
paraformaldehyde (PFA) in 0.1 M sodium phosphate
buffer (PB), pH 7.4. For electron microscopy, a fixation solution
consisting of 2-4% PFA and 0.1-2% glutaraldehyde was used. The
brains were immediately removed and placed in the same fixative for 4 hr at 4° C. After post-fixation, the brains were rinsed in PB,
cryoprotected in 10% sucrose in 0.1 M PB for 1 hr, followed by 30% sucrose in 0.1 M PB for 24 hr at 4° C, then frozen on dry ice. Thirty micrometer transverse serial sections were cut on a sliding microtome equipped with a
freezing stage, then selected for further processing.
For the quantitative analysis of granule cells, brains were post-fixed
for 24 hr, then the hippocampi were isolated and cryoprotected. Before
sectioning, hippocampi were slightly extended along the septotemporal
axis, then frozen and mounted (oriented transversely to the
septotemporal axis) on the freezing stage of a sliding microtome.
Serial transverse sections were then cut at 40 µm, collected in PB,
and chosen for staining using an unbiased, systematic method (West et
al., 1991; Buckmaster and Dudek, 1997); starting from a random position
near the septal pole, every 10th section was sampled, yielding an
average of 12-15 samples per hippocampus. After they were mounted on
slides, sections were stained with cresyl violet. Photomicrographs were
taken on 35 mm slide film, scanned, and imported into Adobe Photoshop
(Adobe Systems, Mountain View, CA) to assemble the figures.
Timm's histochemistry. The Timm's method for staining
heavy metals was used for the detection of synaptic vesicular zinc
[particularly enriched in mossy fiber (MF) boutons]. After initial
fixation with 4% paraformaldehyde as described above, the brains were
transferred to a solution containing 3-4% glutaraldehyde, 0.1%
Na2S, and 0.136 mM
CaCl2 in 0.12 M Millonig's
PB, pH 7.3, for 48 hr at 4° C, followed by cryoprotection in 30%
sucrose. Frozen sections were cut at 30 µm and then mounted on
slides, air-dried, and transferred to a fresh developer solution
containing 30 ml gum Arabic (50%), 5 ml 2 M
citrate buffer, 15 ml hydroquinone (5.76%), and 250 µl silver
nitrate (0.73%) for 1 hr in the dark. Sections were counterstained with cresyl violet, dehydrated, cleared in toluene, and coverslipped.
Quantitative analysis of granule cells within the dentate
gyrus. The number of granule cells in the granule cell layer, as well as the number of heterotopic granule cells within the molecular layer, were estimated per entire dentate gyrus using the optical fractionator method (West et al., 1991). In Nissl-stained preparations, a granule cell was defined as a neuron with a small cell body and an
oval or round nucleus; the Nissl-stained cytoplasm is found at the
apical and basal portions of the cell body (Seress and Pokorny, 1981;
Wenzel et al., 1981; Seress, 1992). The granule cell and molecular
layers also contain the cell bodies of glial cells, basket cells, and
other interneurons (for review, see Freund and Buszaki, 1996). The
nuclei of glial cells were easily identified and excluded from the
counting. The nuclei of basket cells are similar in appearance to those
of granule cells, but the cell body of basket cells (and also other
interneurons) in these layers is larger and more intensely stained for
Nissl substance. On the basis of these criteria, nongranule cells could
be identified and excluded from the counting.
Cell counting was performed by an investigator who was blind to the
genotype of the animals. Total section thickness was used for dissector
height, and only "caps" located within counting frames were counted
(Buckmaster and Dudek, 1997); caps were defined as the nuclei of
granule cells that came into focus while focusing down through the
dissector height. Counting frames (10 × 10 µm) were distributed
systematically and randomly over the dentate granule cell layer,
according to the method described by West et al. (1991). Using a camera
lucida-like microscope/computer interface (Nikon Optiphot-2 Microscope
with a Cohu CCD Camera; Nikon, Tokyo, Japan) and an NIH Image 1.62 b4
morphometry software package, the numerical density of granule cells,
the volume of the granule cell and molecular layers, and the total
number of granule cells for a given hippocampus were estimated.
Adequacy of sampling was assessed by determining the intra-animal
coefficient of variation (CE) as well as the inter-animal coefficient
of variation (CV) for each measure (West and Gundersen,
1990).
Immunocytochemistry. Different sets of sections were
processed for immunocytochemistry (ICC), using a modification of the avidin-biotin complex (ABC)-peroxidase technique (Hsu et al., 1981).
Immunocytochemical procedures were performed as described previously
(Wenzel et al., 1997). Briefly, sections were rinsed in PB, followed by
0.1 M Tris-HCL buffer (TB), pH 7.4; endogenous peroxidases were then inactivated by treatment with 0.5-1%
H2O2 in TB for 2 hr.
Sections were treated with 3% bovine serum albumin (BSA) (Boehringer
Mannheim, Indianapolis, IN), 3% goat or horse serum (Sigma, St. Louis,
MO), and 0.25% Triton X-100 (TX) in 0.05 M TB, 0.15 M NaCl, pH 7.4 (TBS) for 1 hr to reduce nonspecific staining. Sets of alternating
sections were rinsed in TBS for 30 min and incubated for 24 hr at 4°C
in the various antisera and dilutions in TBS containing 1% goat or
horse serum, 2% BSA, and 0.25% TX: anti-neuron-specific nuclear
protein (NeuN) (Chemicon, Temecula, CA), 1:1000;
anti-microtubule-associated protein-2 (Boehringer Mannheim), 1:500;
anti-glial fibrillary acidic protein (GFAP) (Dako Corporation,
Carpinteria, CA) 1:4000; anti-glutamate decarboxylase 67 (GAD67)
(Chemicon, Temecula, CA), 1:100; anti-calretinin (Chemicon), 1:4000;
anti-parvalbumin (Chemicon), 1:4000; anti-somatostatin (SOM) (Peninsula
Laboratories, Belmont, CA), 1:5000; and anti-zinc transporter ZnT3
(provided by Dr. R. D. Palmiter, University of Washington,
Seattle, WA), 1:250. After rinses for 2 hr in TBS, sections were
incubated in biotinylated goat anti-rabbit IgG or horse anti-mouse IgG
(Vector Laboratories, Burlingame, CA), diluted 1:500 for 24 hr at
4°C, rinsed for 2 hr in TBS, and then incubated in ABC (Elite ABC
Kit, Vector Laboratories), diluted 1:500 in 1% goat or horse serum,
2% BSA, 0.25% TX, and TBS for 24 hr at 4° C. Sections were rinsed
thoroughly in TB, pH 7.6, and then incubated for 15 min in 0.025%
3,3'-diaminobenzidine (DAB; Sigma) in TB. After reaction for 5-10 min
in fresh DAB with 0.003%
H2O2, sections were rinsed
in TB, followed by PB. Specificity of the immunostaining was evaluated
by omitting primary antibodies from the regular staining sequence. ZnT3
immunoreactivity in synaptic vesicles of mossy fiber boutons was also
localized at the ultrastuctural level using a protocol described by
Wenzel et al. (1997).
Electrophysiology and intracellular labeling
with biocytin
Intracellular recording and labeling. Hippocampal
slices were prepared conventionally as described previously (Wenzel et
al., 2000) for in vitro experiments. Mice were anesthetized
with halothane and decapitated, and the brain was removed quickly,
cooled briefly in ice-cold oxygenated [95%
O2/5% CO2] artificial CSF
(ACSF) containing (in mM): 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 26 NaCO3, 2 CaCl2, 10 dextrose, and blocked to contain the
hippocampus. Using a vibroslicer, 400-µm-thick slices transverse to
the longitudinal axis of the hippocampus were cut into a bath of
oxygenated ACSF at 4°C. Sections were then transferred to a holding
chamber and allowed to equilibrate for at least 1 hr while submerged in
ACSF at room temperature. Slices were then individually transferred to
a standard interface recording chamber. In the chamber, slices rested
on a nylon mesh over a well that was perfused (1 ml/min) with warmed
(33-35°C), oxygenated ACSF. In addition, warmed, humidified air was
circulated above the slice. Slices remained undisturbed for at least 15 min in the chamber before recording began.
Intracellular electrodes made from borosilicate glass were pulled using
a horizontal puller (Sutter Instruments, San Rafael, CA) and filled
with 2% biocytin (Molecular Probes, Eugene, OR) dissolved in 1 M potassium acetate (80-230 M resistance, pH 7.4). Intracellular potentials were recorded using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Neurons within the granule cell
layer were impaled, and biocytin was iontophoretically injected with
700 msec duration and 0.5-1.0 nA hyperpolarizing current pulses
delivered every second for 10-30 min. After biocytin injection, the
electrode was withdrawn, and the slice was left in the chamber for at
least 30 min before removal for fixation.
Retrograde labeling with biocytin. Visualization of multiple
dentate granule cells was achieved through retrograde labeling via
iontophoresis of 4% biocytin (in 0.05 M Tris
HCl, pH 7.3) into extracellular space of hippocampal stratum
(s.) lucidum in CA3b subfield (Okazaki et al., 1995). The
microelectrode (5-10 µm) was placed ~200 µm below the surface of
the slice, then 600 nA positive current pulses (7 sec on/7 sec off)
were passed for 18-20 min. Slices remained incubating in the chamber
for an additional 3 hr, then were fixed in 4% paraformaldehyde and
further processed as described below.
Extracellular field recordings. Field EPSPs (fEPSPs) were
recorded in the dentate gyrus of both p35/ and wild-type mice with
extracellular electrodes (filled with ACSF, 5-10 M resistance) in
response to perforant path stimulation. A laminar profile of evoked
fEPSPs was constructed by recording sequentially from several sites
through the molecular layer, perpendicular to the granule cell layer
(superior blade of the dentate gyrus).
Tissue processing and morphological analysis of biocytin-labeled
granule cells (light and electron microscopy). After the intracellular recording procedure and iontophoretic injection of
biocytin, slices were removed from the recording chamber and immersion-fixed in a solution of 4% paraformaldehyde and 0.1-1% glutaraldehyde in 0.1 M sodium PB, pH 7.4, for
2-4 hr at 4°C. The slices were rinsed in 0.1 M
PB, then infiltrated for cryoprotection with 10% sucrose in 0.1 M PB for 1 hr, followed by 30% sucrose for 8-12
hr. Frozen sections were cut (60 µm) and further processed with a
histochemical procedure. Sections of a total of 99 slices with
biocytin-filled granule cells were subsequently processed for light and
electron microscopy, as follows.
Sections were rinsed in 0.1 M PB, pH 7.4, and then in 0.1 M TB, pH 7.4. Endogenous peroxidases were suppressed with
0.5-1% H2O2 in 0.1 M TB for 2 hr. Sections were pretreated with 2% BSA, 0.25-0.4% dimethylsulfoxide (DMSO) (Sigma), and 0.05 M
TBS, pH 7.4, for 1 hr to reduce nonspecific background staining and to permeabilize membranes. Sections were rinsed in 0.1 M TBS
for 30 min and then incubated in ABC (Elite ABC Kit, Vector), diluted 1:500 in 2% BSA, 0.25-0.4% DMSO, and 0.05 M TBS for
36-48 hr at 4°C. Sections were then rinsed throughly in 0.1 M TBS followed by 0.1 M TB, pH 7.6, and
preincubated in 0.025% DAB with 0.005% NiNH4SO4 added to increase
the density of stain for 15 min. Subsequently, the sections were
reacted with fresh
DAB/NiNH4SO4 solution
containing 0.01% H2O2 for
15-60 min. The reaction was stopped by rinses in 0.1 M TB.
Sections were further processed for electron microscopy (EM) using a
method that included post-fixation in 1% osmium tetroxide in 0.15 M PB, pH 7.4, for 1 hr at room temperature, alcohol
dehydration, and flat-embedding in Eponate 12 Resin (Ted Pella,
Redding, CA) between two aclar sheets. The biocytin-filled granule
cells (and their dendrites and axon arborizations) were visualized at
the light microscopical level and photographed before remounting and further sectioning for EM. Camera lucida drawings were made of each
biocytin-filled granule cell, and its dendrites and axon arborizations
were reconstructed by superimposing all sections of the hippocampal
slice. Serial thin sections from various slices containing different
portions of the biocytin-filled granule cells (e.g., mossy fiber axon
collaterals in the dentate inner molecular layer) were stained with
uranyl acetate and Reynold's lead citrate and examined on a Philips
410 electron microscope.
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RESULTS |
The gene-targeting strategy and generation of p35/ mice have
been reported previously (Chae et al., 1997). The initial histological study demonstrated a general disorganization of the forebrain in these
mice, with a major defect in the development of the normal lamination
pattern of the neocortex (Kwon and Tsai, 1998). In the present study,
histological examination of the neocortex of p35/ mice reconfirmed
this observation of severe defects in the lamination of neocortex,
associated with formation of aberrant fiber fascicles. Our observations
also revealed structural abnormalities within the hippocampus,
particularly associated with the appearance of dispersed granule cells
and abnormal mossy fiber localization. We have focused on these
hippocampal abnormalities and their functional correlations, with
respect to the occurrence of spontaneous seizures.
P35/ mutation is associated with low seizure threshold
and with occurrence of spontaneous electrographic and behavioral
seizures
Seizure susceptibility was compared in p35/ and wild-type mice
through flurothyl seizure testing. In all animals, exposure to
flurothyl elicited seizure activity. Statistical analysis of the
latencies to the flurothyl-induced seizures revealed that the p35/
mice had significantly shorter latencies than wild-type to both the
first (352.2 ± 1.6 vs 423.7 ± 12.0 sec, respectively) and
second seizures (590.6 ± 14.3 vs 688.4 ± 13.8 sec) (Fig.
1A). All statistical
data are presented as means ± SEM. These results represent a 16%
reduction in mean latency to the first seizure and a 14% reduction to
the second seizure in p35 / mice.
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Figure 1.
A, Flurothyl seizure threshold
testing demonstrated significantly shorter latencies, to both first and
second seizures, in p35 knock-out mice in comparison with control
(wild-type) mice. B, Representative EEG recording from a
p35/ mouse during a spontaneous tonic-clonic seizure. Ictal
episodes often began (top arrow) with a decrease in EEG
voltage, followed by fast spiking activity that increased in amplitude
and proceeded into a clonic pattern; seizure activity was followed by
an isoelectric postictal period (starting at bottom
arrow). The top two traces were recorded
epidurally from the left and right cortex, respectively; the
bottom trace was recorded with a depth electrode in the
right hippocampus.
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Video/EEG recording confirmed the occurrence of spontaneous seizures in
many p35/ mice. A typical example of EEG recorded from a p35/
mouse during a tonic-clonic seizure is shown in Figure
1B. Spontaneous epileptiform EEG activity was
verified in 75% of the p35/ mice (8 of 12 animals). Twenty-five
percent of p35/ animals exhibited spontaneous tonic-clonic
seizures with behavioral manifestations, whereas an additional 50%
displayed intermittent interictal electrographic activity. The
spontaneous seizures had durations ranging from 59 to 84 sec and were
followed by a prolonged isoelectric postictal period lasting 3-5 min.
Ten percent of the p35/ mice died during seizure. No epileptiform activity was observed either behaviorally or electrographically in
wild-type mice.
Most of the recorded spontaneous seizures were strong generalized
tonic-clonic episodes that appeared to occur simultaneously in cortex
and hippocampus. Seizures frequently began from a state of sleep or
quiet rest, with a sudden decrease in amplitude of the EEG, sometimes
preceded by a spike corresponding to the animal awakening; the mouse
then lifted and turned its head back and forth. As the seizure evolved,
both electrographic and behavioral activity reflected the tonic phase
of a seizure; spiking gradually increased in amplitude and frequency,
and the animal's head turned rigidly upward and back concurrent with
bilateral forelimb extension. This tonic phase was frequently prolonged
and usually progressed into a clonic EEG pattern. The animal's
behavior often progressed into vigorous bouncing, produced by rapid
bilateral hindlimb thrusting. The seizure ended with the onset of a
protracted isoelectric post-ictal period; behavioral inactivity was
seen when the cortical EEG flattened. Occasionally the hippocampus
would continue to spike for several seconds longer than neocortex.
Histological abnormalities are characteristic features of the
hippocampus in p35/ mice
Histological examination of the neocortex revealed striking
differences between wild-type and p35/ mice. Figure
2, A1 and A2, shows
the typical lamination of the wild-type neocortex with six layers
distinguished by neuronal morphology, cell density, and general
cytoarchitecture. This normally laminated cytoarchitecture is not
apparent in p35/ mice (Fig. 2B1,B2).
With the exception of lamina I containing a few Cajal-Retzius cells,
laminas II-VI represent an inversion of the normal neuronal
patterning; i.e., large pyramidal neurons normally localized in lamina
V are now present underneath lamina I, and small pyramidal cells are
distributed in the deep cortical zone [further details are described
by Chae et al. (1997)]. In addition, aberrant fiber fascicles course
through the neocortex, leading to further interruptions in the
cytoarchitecture.
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Figure 2.
A1, B1, Coronal
brain sections from wild-type (A1) and p35/
knock-out (B1) mice, stained with cresyl violet.
Neocortex (N), hippocampus
(H), and dentate gyrus (DG)
are indicated. A2, Higher magnification of neocortex and
hippocampus from a wild-type mouse shows normal neocortical laminae
(I-VI) and hippocampal subregions
CA1 and CA3 with s. oriens
(OR) and radiatum (RAD), pyramidal cell
layer (PCL), molecular layer
(ML), and dentate gyrus (DG) with hilus
(H), granule cell layer
(GCL), and molecular layer (ML).
B2, Coronal section of neocortex and hippocampus of a
p35/ mouse demonstrates heterotopic pyramidal neurons in CA1/CA3
(arrows) and dispersed dentate granule cells localized
within the inner molecular layer (ML;
arrows). Note the abnormal cortical lamination, the
presence of aberrant fiber fascicles within the neocortex
(arrowheads), and the absence of callosal fibers
crossing the midline (white arrow). Scale bars:
A1, B1, 1000 µm; A2,
B2, 300 µm.
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The most striking morphological finding in the hippocampal
formation of adult p35/ mice was a severe disarrangement of its neuronal architecture (Figs. 2, 3). The
principal cell layers (CA1-3 pyramidal and dentate granule cell
layers) are disorganized and show frequent interruptions and aberrantly
localized cell somata (Figs. 2B2,
3B1,B2,C1,C2). In
particular, pyramidal cells of the medial CA1 subfield and subiculum
tend to be dispersed into s. oriens, where they form a distinct cell
layer (Fig. 2B2). In the CA3 subfield, pyramidal
neurons are found outside the pyramidal cell layer, in both s. oriens
and s. lucidum/radiatum (Figs. 2B2, 3C1,D,E). To determine
the neuronal nature of these heterotopic cells, the neuron-specific
NeuN antibody was used. In hippocampal sections immunoreacted against
NeuN, a large proportion of the heterotopic cell population was
neuronal.
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Figure 3.
Transverse sections (cresyl violet) of the
hippocampus (A1) and dentate gyrus (A2)
of the wild-type mouse. B1, B2,
Transverse sections of the dorsal hippocampus of a p35/ mouse
demonstrate heterotopic CA3 pramidal cells (arrows) and
dispersed granule cells within the dentate inner and middle molecular
layer (IML and MML)
(arrows). C1, C2,
Transverse sections of the ventral hippocampus of a p35/ mouse show
extensive granule cell dispersion in the dentate gyrus
(arrows) and a gap within the CA3 pyramidal cell layer
(arrow). Higher magnification of the granule cell layer
(C2) shows displaced cells in both the molecular layer
and the hilus. D, E, Comparison of the
CA3 subfield of wild-type and p35/ mice, showing pyramidal neurons
heterotopically localized within s. oriens (OR) and
lucidum/radiatum (LU, RAD)
(arrows). Scale bars:
A1-C1, 400 µm;
A2-D2, 100 µm.
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Most p35/ mice also show a severe disorganization of the granule
cell layer, particularly affecting the superior blade. Granule cells
are dispersed and heterotopically localized into the molecular layer
and hilus, blurring the borders of the granule cell layer with adjacent
layers or forming separate cell clusters deep in the hilus (Fig.
3B2,C2,
4A). Although no
statistical comparison was performed, there appeared to be a strikingly
higher degree of granule cell dispersion into the molecular layer (and
particularly into the hilus) in the ventral than in the dorsal
hippocampus (Fig. 3, compare B1-2, C1-2). NeuN
ICC confirmed the neuronal nature of these cells, and their shape
suggested that most of these neurons are displaced dentate granule
cells (Fig. 4B).
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Figure 4.
Transverse sections of the dentate gyrus from
p35/ mice, showing cresyl violet staining (A)
and NeuN immunoreactivity (B). Most of the
dispersed cells within the inner molecular layer (IML)
are NeuN-positive. C, D, Timm's
histochemistry for zinc in mossy fiber boutons shows the normal
staining pattern in the dentate hilus (H)
of a wild-type mouse (C). In p35/ mice
(D), Timm stain can be seen in the hilus
(H), the granule cell layer
(GCL), and inner and middle molecular layers
(IML and MML), indicating the abnormal
distribution of mossy fibers associated with heterotopically dispersed
granule cells (arrows). E,
Immunocytochemistry for GFAP shows a relatively normal astrocytic
border between hilus and granule cell layer of the inferior blade
(A, arrows). In contrast, note the
absence of GFAP-positive astrocytes at the hilus/granule cell layer
border in the superior dentate blade.
F1-F3, Sections immunoreacted against
calretinin antibody show normal distribution of the calretinin-positive
fiber plexus (CRP) within the IML of the wild-type mouse
(inset F2) and, in p35/ mice (F1 and
inset F3), dispersed granule cells (dGC)
in the IML surrounded by the CRP. G, H,
Photomicrographs show parvalbumin immunoreactivity in dentate
interneurons (arrows) and the axonal plexus
(arrowheads) in the wild-type (G1,
inset G2) and p35/ mice (H1,
inset H2). There is dramatically less parvalbumin
immunoreactivity in the axonal plexus surrounding the granule cells in
p35/ mice (H2). Scale bars: A,
D, E, F1, 100 µm;
B, C, G1,
H1, 200 µm; F2-3, G2,
50 µm.
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To further characterize the dentate region of p35/ mice, Timm
staining was used to label zinc in synaptic vesicles of MF boutons. In
the wild-type (Fig. 4C), Timm-stained MF axons normally project from the granule cell layer throughout the hilus toward the CA3
pyramidal cell layer, forming a compact projection in s. lucidum;
in addition, MF axon bundles are also localized infrapyramidally and in
the pyramidal cell layer. Timm staining in p35/ mice shows a
similar pattern of MF projection in hilus and CA3 subfield (Fig.
4D). However, in contrast to the wild-type, numerous
Timm-stained MF boutons/axons are also present in the granule cell
layer and particularly within the inner molecular layer (Fig.
4D, arrows). Although there was
significant animal-to-animal variability in the degree of granule cell
dispersion and MF distribution in granule cell and molecular layers,
these abnormal patterns were typical of p35/ mice and not seen in
wild-type animals.
Analysis of granule cell dispersion
Quantitative cell counts
To assess the extent of granule cell dispersion in the
dentate gyrus of p35/ mice, we analyzed the following histological parameters from 2-month-old p35/ mice and age-matched wild-type mice: (1) numbers of granule cells and (2) volumes of granule cell and
molecular layers; from these measures, we calculated the numerical
density of granule cells. Each measure was estimated separately for the
granule cell and molecular layers. Because in p35/ mice the border
between granule cell layer (GCL) and inner molecular layer is
irregular, the distinction between granule cell and molecular layers
was artificially (but consistently) defined in each section. The data
(Table 1, Fig.
5A,C)
revealed small but nonsignificant differences between p35/ and
wild-type mice for the total number of granule cells per dentate (Fig.
5C) and the number of granule cells in the granule cell
layer (Fig. 5A). However, p35/ mice do show
significantly more granule cells in the molecular layer than
wild-type mice (Fig. 5B). Approximately 29.8% of all
granule cells in p35/ mice are localized within the molecular layer
(compared with 14.4% in the wild-type). Statistical comparison of the
mean volume of the dentate layers revealed a difference between
p35/ mice and wild-type mice: molecular layer and the total
value of the granule cell plus molecular layers of p35/ animals are
significant smaller (p < 0.016 and
p < 0.03, respectively), but the difference in the
granule cell layer is not significant (p < 0.44). However, the numerical density of granule cells in the
molecular layer, the dispersed granule cells, is significantly
higher in p35/ mice compared with the wild-type. The intra-animal
CE for the number of cells counted and points counted (see Materials
and Methods), as well as the between-subjects CV and
CE2/CV2
ratio, were in the range that indicated adequate sampling (West et al., 1991).
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Table 1.
Total number and numerical density of granule cells (GCs),
and volume of granule cell layer (GCL) and molecular layer (ML), in the
dentate gyrus of 2-month-old wild-type and p35/ mice
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Figure 5.
Graphs show the numbers of granule cells counted
in the dentate granule cell and molecular layers per hippocampus of the
wild-type and p35/ mice at 2 months of age. A, The
number of granule cells within the granule cell layer per entire
hippocampus. The granule cell number is slightly greater
(nonsignificantly) in wild-type mice than in p35/ animals.
B, The number of heterotopic granule cells within the
molecular layer. In p35/ mice, ~30% of the total number of
granule cells is heterotopically localized within the molecular layer,
compared with ~14% in wild-type mice (*p < 0.001). C, The total number of granule cells per
hippocampus. There is no significant difference in the total number of
granule cells between wild-type and p35/ mice. Although the number
of dispersed granule cells is significantly greater in p35/ mice
(+125% in the molecular layer), the absolute number of
displaced neurons is relatively small compared with the number of cells
in the granule cell layer. Thus, there is no statistical difference
when total numbers are compared.
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Field potential analysis
A laminar profile of synaptic responses, recorded throughout the
granule cell and molecular layers of the superior blade of the dentate
gyrus, revealed clear differences between p35/ and wild-type mice
(Fig. 6). The amplitude of the evoked
fEPSPs was measured at a fixed latency (Fig. 6A) and
plotted as a function of location along a model granule soma-dendrite
axis (Fig. 6C). Consistent with the morphological findings
of a dispersed granule cell layer in p35/ animals, the fEPSP
positivity of p35/ dentate evoked by perforant path stimulation was
much broader in slices from p35/ mice (Fig. 6B).
This positivity is thought to reflect a "somal" current source
[see Sutula et al. (1998) for similar analysis of "sprouted"
dentate], suggesting that the heterotopic granule cells are activated
by incoming perforant path fibers.
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Figure 6.
A, Field EPSPs evoked by
stimulation in the dentate molecular layer were recorded from sites in
the superior blade of the dentate gyrus in wild-type and p35/ mice.
B, The amplitude of these EPSPs was measured at a fixed
latency [peak of EPSP recorded in the granule cell layer
(GCL)] and plotted as a function of recording location
along an axis perpendicular to the GCL (wild-type,  ; p35/,  ).
C, Schematic drawing showing location of normally placed
granule cells (solid ovals) and dispersed granule cells
(dotted ovals). IML, Inner molecular
layer; MML, middle molecular layer; OML,
outer molecular layer.
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Immunocytochemistry of astrocytes and interneurons reveals
cell-specific differences between p35/ and wild-type
mice
ICC techniques were used to examine the expression of several
markers specific for astrocytes and inhibitory interneurons (Fig.
4E-H). Using an antibody against
GFAP (Fig. 4E), we found no obvious differences in
distribution and density of GFAP-positive cells within the hippocampus
proper containing heterotopic neurons. However, in the dentate gyrus, a
characteristic population of astrocytes that is normally localized
along the hilar-granule cell border was absent in p35/ mice;
furthermore, occasional aberrant astrocyte somata were seen between the
granule cells.
The distribution of hippocampal inhibitory interneurons in p35/
hippocampus was examined using an antibody against GAD67 (synthesizing
enzyme of the inhibitory neurotransmitter GABA) to stain all GABAergic
neurons. In addition, we used a set of antibodies calretinin,
parvalbumin, and somatostatinthat were previously associated with
various GABAergic subpopulations of interneurons. The pattern of
GAD67-positive neuron distribution in p35/ and wild-type mice was
similar to that seen in other ICC and in in situ
hybridization studies on rodents, including mice (data not shown) (for
review, see Houser and Esclapez, 1994; Freund and Buszaki,
1996; Fukuda et al., 1997). In both p35/ and wild-type mice, GAD
immunoreactive neurons were found in the hippocampal pyramidal
cell layer, s. oriens and s. radiatum/moleculare. In the dentate,
GAD-positive cells were seen in the dentate hilus, in the granule cell
layer, and scattered in the molecular layer. GAD67 reveals consistent
"punctate" (presumed axon terminals) immunostaining surrounding
somata of principal cells, including the dispersed granule cells in the
dentate molecular layer (data not shown). This expanded region of
GABA-containing terminals surrounding granule cells constitutes the
major difference in the pattern of GAD67 immunoreactivity distribution
between p35/ and wild-type animals.
Immunostaining for SOM revealed a large number of neurons in all
subfields of the hippocampus and dentate gyrus, with laminar distribution consistent with previous studies (data not shown) (for
review, see Buckmaster et al., 1994; Freund and Buszaki, 1996). In
agreement with these previous studies on mouse, we found only light
labeling of SOM-positive axons in the outer two-thirds of the dentate
molecular layer (compared with a dense axon and terminal plexus in
rats). There were no apparent differences in the patterns of
distribution and intensity of SOM staining between p35/ and
wild-type mice.
Immunostaining for calretinin identifies another subpopulation of
interneurons in all hippocampal subfields and strata (for review, see
Freund and Buszaki, 1996). In addition, in the hilus of the ventral
dentate gyrus of wild-type and p35/ animals, calretinin
immunoreactivity labeled the somata and processes of mossy cells (data
not shown) as reported previously by Liu et al. (1996). Axon terminals
of these mossy cells form a dense supragranular-IR band in the inner
molecular layer in both dorsal (Fig. 4F) and ventral
dentate gyrus. In p35/ mice, this supragranular calretinin-positive band appeared more prominent than in wild-type mice and overlapped with
the dispersed granule cells in this layer.
In the hippocampus and dentate gyrus, parvalbumin ICC shows parvalbumin
positivity in interneuron somata, dendrites, and axon terminals.
Parvalbumin-containing cell bodies are mainly localized in the
pyramidal cell layer and s. oriens of the hippocampus, in the granule
cell layer and hilus of the dentate gyrus of wild-type mice (Fig.
4G), and only occasionally in other layers (e.g., s. lucidum
of the CA3 region). The dendrites of parvalbumin-positive neurons arise
parallel to the granule cell dendrites and exhibit prominent
varicosities spanning all layers of the molecular layer. In both
p35/ and wild-type mice, the axons and terminals immunoreactive for
parvalbumin form a dense axon plexus within the cell layers, surrounding pyramidal and granule cells, including the dispersed granule cells within the dentate molecular layer of p35/ mice. Although no systematic cell counting was performed, the number of
parvalbumin-containing neurons appears to be reduced in p35/ mice
as compared with the wild-type; furthermore, the intensity of the
parvalbumin staining of the axon plexus and terminals in the dentate
granule cell layer was weaker in p35/ mice (Fig. 4G,H).
Electron microscopy of mossy fiber connections
Electron microscopic examination of the granule cells in
p35/ mice localized in granule cell and molecular layers showed ultrastructural characteristics similar to the wild-type and those described previously for dentate granule cells (Laatsch and Cowan, 1966; Ribak and Anderson, 1980; Wenzel et al., 1981; Seress, 1992). Despite the variable granule cell localization (Fig.
7A) and dendritic orientation
in p35/ mice (see next section), dendrites from both
genotypes are covered with spines with a broad range of shape, size,
and length. Dendrites and dendritic spines form simple and multiple
asymmetric synaptic contacts with axon terminals. In p35/ dentate,
MFs form a widely distributed axon plexus around granule cell somata
and proximal dendrites, in both the granule cell and molecular layers;
these axons are associated with numerous terminals (i.e., MF boutons)
that vary considerably in size and shape (Fig.
5B-E). The identity of terminals as MF boutons
is confirmed by their ultrastructural features (packed with clear round
synaptic vesicles, occasional dense-core vesicles, a few coated
vesicles, and numerous mitochondria) and EM localization of ZnT3
IR of synaptic vesicle membranes in these terminals (identical to those seen in hilus and CA3 s. lucidum). The MF boutons, identified by ZnT3 immunoreactivity in both granule cell (Figs.
7D,E) and molecular layers (Figs.
7B,C), form asymmetric synapses
predominantly with multiple simple dendritic spines (Fig.
7B); some MF boutons also form synapses with complex spines
and with dendrites of presumed granule cells (Figs.
7B,E). Occasionally, MF boutons
were found making synapses with the soma of heterotopic granule cells
and aspiny dendrites of interneurons (data not shown). The EM
observation on ZnT3-positive MF boutons abnormally localized in the
molecular layer (Fig. 7C) is consistent with the Timm
staining pattern of p35/ dentate and was not observed in wild-type
mice. However, there is some Timm labeling (seen at the light
microscopic level) within the granule cell layer of wild-type mice,
suggesting that even in normal dentate there may be some MF synapses in
this region.
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Figure 7.
Electron microscopy of mossy fiber boutons
(MFB) heterotopically localized within the granule cell
layer (GCL) and inner molecular layer
(IML) of p35/ mice. MFBs are identified with ZnT3
immunocytochemistry (black DAB-nickel reaction product in
C and E). A, Low-power
magnification of the dentate gyrus (superior blade) with heterotopic
GCs within the IML. B, In the IML (indicated by
top box and arrow in
A), an MFB forms asymmetric synapses with spines
(S) of granule cells. C, A
ZnT3-positive MFB makes an asymmetric synapse with a simple spine.
D, E, In the granule cell layer
(indicated by bottom box and arrow), a
nonlabeled (D) and ZnT3-positive
(E) MFB make asymmetric synapses with spines
(S) and dendrites (D) of
presumed granule cells. Scale bars: B-E,
0.25 µm.
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Intracellular labeling with biocytin reveals abnormal
patterns of granule cell localization, axonal and dendritic morphology,
and mossy fiber reorganization
Light microscopic examination of individual granule cells obtained
after in vitro injection of biocytin into the s. lucidum of
hippocampal slices, or intracellular labeling with biocytin, revealed a
high degree of granule cell abnormality in p35/ dentate with
respect to the localization of granule cell somata and in the
orientation of their dendritic fields and axon arbors (Figs. 8, 9).
Granule cell somata of wild-type mice have oval or round-shaped cell
bodies, typically giving rise to a spiny apical dendritic tree that
arborizes within the molecular layer; the mossy fiber axon arises from
the hilar side of the soma and runs toward the CA3 subfield. As shown
with Golgi and recent intracellular labeling studies (Spigelman et al.,
1998; Ribak et al., 2000), our examination of granule cells of
wild-type mice revealed only exceptional abnormalities. In contrast, as
shown in Figure 8, granule cells of p35/ mice are often displaced,
with cell bodies located either in the inner molecular zone (close to
the GCL) or in clusters within the hilus. Normal-appearing granule
cells in p35/ dentate show the expected monopolar-oriented
dendritic branching pattern within the molecular layer (Fig.
8A1,B, cell 1). However, many
granule cells exhibit a bipolar dendritic arborization (Fig. 8, cells
2, 3, 5; Fig. 9B1-2,C1-2). The spiny dendrites of some
p35/ granule cells arose from the hilar side of the cell body and
then turned 180° to form "recurrent" basal dendrites ascending
through the molecular layer (Fig. 8, cell 2; Fig.
9C); some dendrites originating from the hilar pole of the
cell body also extend into the subgranular hilar region (Fig.
8B, cell 5; Fig.
9D1-2). Granule cells heterotopically localized
within the hilus gave rise to one or two primary dendrites that ran
through the hilus toward the granule cell layer to branch within both
granule cell and molecular layers (Fig. 8, cell 4; Fig.
9B).
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Figure 8.
Photomicrographs (A) and camera
lucida drawings (B) of biocytin-filled granule
cells from a p35/ mouse. Granule cells and mossy fibers were
visualized after terminal uptake and retrograde transport of biocytin,
after in vitro injection of biocytin into the s. lucidum
of a hippocampal slice. A1, Biocytin-labeled granule
cell bodies (1-5) are present in the
granule cell layer (GCL), inner molecular layer
(IML), and hilus (H).
A2, Photomicrograph of a biocytin-labeled granule cell
(5) with basal dendrites (arrows)
in an adjacent section of the same slice. A3, Higher
magnification of the same granule cell showing a spiny dendrite
(arrows) and the mossy fiber axon
(arrowhead). B, Drawings of the
biocytin-labeled granule cells (GCs) shown in A,
demonstrating abnormal morphology with respect to location and
orientation of the cell body, and dendrites and axon arborization:
neuron 1, normal-appearing GC; neuron 2,
inverted GC with recurrent basal dendrites and an axon
(MF, arrowhead) originating from the
basal dendrite; neuron 3, GC with dendrites emerging
from the apical and hilar pole of the soma, and axon
(MF, arrowhead) arising from a dendrite;
neurons 4, heterotopic GCs within the hilus
(H), with dendrites extending into the
molecular layer; neuron 5, GC with basal dendrites
branching and extending into the hilus. Dashed line
indicates border between "normal" GCL and IML.
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Figure 9.
Photomicrographs
(A1-D1) and camera lucida drawings
(A2-D2) of single biocytin-filled
granule cells in wild-type (A1, A2) and
p35/ mice (B1-D2). Hippocampal
subregions CA3, dentate hilus
(H), granule cell layer
(GCL), and inner (IML), middle
(MML), and outer (OML) molecular layer
are indicated. A1, A2, Photomicrograph
and drawing show the regular dendritic pattern and axonal arborization
of a dentate granule cell (GC) within the GCL from a
wild-type mouse; an arrowhead indicates mossy fiber axon
(MF). B1, B2,
Heterotopically localized GC in the dentate hilus that gives rise to
two apical dendrites that extend into the GCL/IML and branch within the
ML. Note the extensive axon arborization (MF) within the hilus.
C1, C2, GC with abnormal dendritic and
axonal morphology. Dendrites emerge from the apical and hilar pole of
the GC body and extend into the ML. A recurrent hilar axon collateral
(arrows) arborizes within the GCL, indicating MF
sprouting (reorganization). D1, D2,
Biocytin-filled GC with abnormal apical dendritic arborization and
formation of basal dendrites (D, arrows)
extending into the hilus.
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Abnormally localized granule cells, as well as morphologically abnormal
granule cells localized within the granule cell layer, usually
displayed an axon that arose from the cell body and ran toward the CA3
s. lucidum, forming typical small "en passant" boutons and large MF
boutons with filipodial extensions (Fig. 9B2,C2).
These observations suggest that both heterotopic and normally
positioned granule cells contribute to the normal MF projection in p35
/ mice. However, many of these displaced cells also showed an
unusually extensive MF arborization within the hilus (Fig.
9B2). The axons of some granule cells arose from anomalous locations, e.g., the lateral side of the cell body, the apical dendrite, or the initial portion of a recurrent basal dendrite. Figures
8 (A1 and B, cells 2 and 3)
and 9 (C2) indicate some examples of abnormal axon origin.
Finally, granule cells of p35/ mice, either localized within the
granule cell layer or displaced into the molecular layer, show
recurrent MF sprouting into the granule cell layer and/or inner
molecular layer, indicative of MF reorganization. Figure
9C1-2 shows an example of a "bipolar" granule cell
forming not only recurrent basal dendrites but also an aberrant MF axon collateral that forms a plexus within the granule cell layer.
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DISCUSSION |
These results expand the previous description by Tsai and her
colleagues (Chae et al., 1997; Kwon and Tsai, 1998) showing that normal
development of hippocampus and dentate gyrus involves p35-mediated
processes. We have, in addition, found that (1) hippocampal pyramidal
neurons of the CA1 and CA3 subfields and granule cells of the dentate
gyrus are heterotopically localized, (2) a significant proportion of
p35/ mice exhibit spontaneous behavioral and electrographic seizure, and (3) heterotopic granule cells exhibit features (e.g., basal dendrites, abnormal axon origin, and aberrant mossy fiber collaterals and synapses) often observed in epileptic tissue.
The findings of Kwon and Tsai (1998) in p35 knock-out mice, together
with similar cortical migration defects in cdk5-deficient mice (Ohshima
et al., 1996; Gilmore et al., 1998) suggest a critical signal
transmission function of p35/cdk5 kinase for the migration of cortical
neurons (or glia) to their proper destinations. In contrast, Reelin, a
glycoprotein secreted by the Cajal-Retzius cells in the marginal zone,
is thought to act as a "stop signal" for migrating cortical plate
neurons at the boundary of the marginal zone (Sheppard and Pearlman,
1997; Kwon and Tsai, 1998), thus explaining the abnormal presence of
neurons in this zone in the reeler mutant (Frotscher, 1998).
Developmental processes in the hippocampus are similar to those in the
neocortex, except for the long-lasting neurogenesis of granule cells in
the dentate gyrus and their "outside-in" gradient of positioning
(Cowan et al., 1980; Soriano et al., 1994). The abnormal positioning of dentate granule cells within the hilus and molecular layer and of
hippocampal pyramidal neurons in adjacent layers (and absence of a
specific astrocyte population) indicates a defect in the migration of
later generated cells. It is yet not clear, however, whether the
heterotopic granule cell positioning in the hilus results from a
migration defect or whether these granule cells might be generated at a
later time point, from progenitor cells within the hilus (Altman and
Bayer, 1990; Parent et al., 1997), and do not migrate into the granule
cell layer (Schlessinger et al., 1975; Soriano et al., 1994).
The occasional appearance of heterotopic granule cells (displaced
granule cells) in the dentate hilus and molecular layer has been
observed in wild-type mice and rats (Ramon y Cajal, 1968; Amaral, 1978;
Seress and Pokorny, 1981; Marti-Subirana et al., 1986). Granule cell
dispersion in the dentate gyrus, similar to that seen in human
epileptic hippocampi (Houser 1990; Lurton et al., 1997; El Bahh et
al., 1999), has also been observed in animal models of epilepsy,
e.g., after kainic acid injection into the dorsal hippocampus
(Cavalheiro et al., 1982; Suzuki et al., 1995; Bouilleret et al.,
1999), and in the pilocarpine model (Mello et al., 1992). Two
mechanisms have been proposed to explain the genesis of these
heterotopic granule cells: (1) a disorder of the migration, occurring
early in development (before any seizure event) and perhaps
contributing to epileptogenesis (Scharfman et al., 2000), and (2) a
consequence of repetitive seizures (as seen after kainic acid or
pilocarpine administration), perhaps reflecting enhanced neurogenesis
(Mello et al., 1992; Bouilleret et al., 1999). It remains unclear
whether the appearance of heterotopic granule cells is related to
granule cell loss in the epileptic hippocampus (Babb et al., 1984;
Mello et al., 1992; Mathern et al., 1994; Suzuki et al., 1995).
Granule cell dispersion has not been observed when cell loss is minimal
(Lurton et al., 1997), but both appear to be directly related to mossy
fiber sprouting into the molecular layer (Cavazos and Sutula, 1990;
Babb et al., 1991; Houser, 1999). The present study, using Timm
staining and intracellular labeling with biocytin to vizualize mossy
fiber boutons, demonstrates that heterotopic granule cells generate (and receive) aberrant mossy fibers, in both granule cell and molecular layers. In addition, heterotopic granule cells show basal
dendrites (entering the hilus) and axons originating from the apical
pole of the soma or the apical dendrite. These findings represent
abnormal phenomena associated with epilepsy both in human hippocampus
(Franck et al., 1995) and in experimental animal models (Spigelman et
al., 1998; Ribak et al., 2000). The behavioral observations of the
present study substantiate the enhanced susceptibility of p35/ mice
to seizures. Simultaneous video/EEG recordings confirmed spontaneous
epileptiform activity, including both intermittent interictal discharge
and spontaneous generalized seizures. Although no fatalities were
observed during recorded seizures, the severity of observed seizures,
together with occasional sudden (unexplained) death of some animals,
suggests that the mortality rate of ~10% [see also Chae et al.
(1997)] might be attributable to fatal seizures. Previous experiments
comparing PTZ-induced seizures in p35/ and wild-type mice showed
that generalized convulsions were only fatal in p35/ mice (Chae et
al., 1997).
Early in postnatal development (before postnatal day 10), before any
spontaneous seizure, p35/ mice exhibit heterotopic granule cells,
indicating that this abnormality is not a result of seizure activity
and suggesting a causal relationship between structural abnormality and
occurrence of spontaneous seizures. Other studies investigating animal
models of cortical dysplasia with experimentally induced disruption of
cortical development also suggest a causal link between cortical
malformations and seizure activity (Chevassus-au-Louis et al., 1999;
Walsh, 1999; Fleck et al., 2000; Chen et al., 2000). Such animal models
include migrational malformations induced by freezing (Dvorak and Feit, 1977; Dvorak et al., 1978; Rosen et al., 1992, 1996; Jacobs et al.,
1996, Hablitz and DeFazio, 1998), x-irradiation (Roper et al., 1995,
1997; Hicks et al., 1959), and chemical treatment (e.g., methylazoxymethanol: Singh, 1977; Cattaneo et al., 1995; De Feo et al., 1995; Baraban and Schwartzkroin, 1996; Germano and Sperber, 1997; Chevassus-au-Louis et al., 1998, 1999; Baraban et al., 2000). Animals with abnormalities paralleling human genetic neuronal migration disorders also show enhanced seizure-sensitivity: e.g., type
1 lissencephaly (Lis1 mutant) (Reiner et al., 1993; Hirotsune et al., 1998; Fleck et al., 2000), and band heterotopia (tish mu |
TOP
ABSTRACT
INTRODUCTION
