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Volume 16, Number 12,
Issue of June 15, 1996
pp. 3827-3836
Copyright ©1996 Society for Neuroscience
Null Mutation of c-fos Impairs Structural and
Functional Plasticities in the Kindling Model of Epilepsy
Yoshinori Watanabe1,
Randall S. Johnson2,
Linda S. Butler1,
Devin
K. Binder3,
Bruce M. Spiegelman2,
Virginia E. Papaioannou4, and
James O. McNamara1, 3, 5
1 Department of Medicine (Neurology), Epilepsy Research
Laboratory, Duke University Medical Center, Durham, North Carolina
27710, 2 Dana-Farber Cancer Institute, Departments of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115, 3 Departments of
Neurobiology and Pharmacology, Duke University Medical Center, Durham,
North Carolina 27710, 4 Department of Genetics and
Development, College of Physicians and Surgeons, Columbia University,
New York, New York 10032, and 5 Veterans Affairs
Medical Center, Durham, North Carolina 27705
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
It has been suggested that expression of the immediate early gene
c-fos links fleeting changes in neuronal activity to lasting
modifications of neuronal structure and function in the mammalian
nervous system. To test this idea, we examined behavioral and
electrophysiological indices of kindling development and
kindling-induced sprouting of hippocampal granule cell axons in
wild-type (+/+), heterozygous (+/ ), and homozygous (/) mice
carrying a null mutation of c-fos. The rate of kindling
development was significantly attenuated in / compared with +/+
mice, as evidenced by both electrophysiological and behavioral
measures. Kindling-induced granule cell axon sprouting as measured by
the Timm stain was also attenuated in homozygous null mutants compared
with +/+ mice, with an intermediate effect in +/ mice. The impairment
of kindling-induced axonal sprouting in the null mutants could not be
attributed to either detectable loss of dentate hilar neurons or
reduced activation of the dentate granule cells by seizures. Instead,
our data are consistent with the hypothesis that the null mutation of
c-fos attenuates a pathological activity-determined
functional plasticity (kindling development) as well as a structural
plasticity (mossy fiber reorganization). We favor the hypothesis that
this ``fos-less phenotype'' is attributable to impaired
seizure-induced transcriptional activation of one or more
growth-related genes.
Key words:
c-fos;
immediate early genes;
kindling;
axonal
reorganization;
epilepsy;
plasticity
INTRODUCTION
Alterations in neuronal activity produce a lasting
reorganization of synaptic connections in the mammalian nervous system
(Hubel and Wiesel, 1965 ). Patterned activity is critical to the
formation of normal synaptic connections in the developing nervous
system (Cline and Constantine-Paton, 1990; Shatz, 1990), and excess
neuronal activity also contributes to the formation of abnormal
synaptic connections in the mature nervous system (Sutula et al., 1988;
Represa et al., 1993). Kindling is a model of epilepsy in which
fleeting changes of neuronal activity in the form of brief focal
seizures lead to lifelong structural and functional reorganization of
the mammalian brain. Kindling is induced most conveniently by focal
application of a low-intensity electrical stimulus that initially
evokes a brief, localized electrical seizure without behavioral change;
however, after repeated applications it evokes prolonged, widespread
electrical seizures accompanied by intense behavioral seizures (Goddard
et al., 1969; McNamara et al., 1993). Once established, this enhanced
sensitivity to electrical stimulation is lifelong. Kindling is
accompanied by lasting synaptic reorganization exemplified by the
aberrant synapses formed by mossy fiber axons of the hippocampal
dentate granule cells (Sutula et al., 1988; Represa et al., 1993); the
activity-dependence of this reorganization was demonstrated by its
preferential induction by high-frequency compared to low-frequency
stimulation of neurons that activate the granule cells (Sutula et al.,
1988).
Immediate early genes (IEGs) such as c-fos provide an
attractive mechanism by which fleeting changes of neuronal activity may
produce lifelong structural and functional changes via regulation of
gene expression. Circumstantial evidence suggests that c-fos
in particular is part of a chain of molecular events that culminate in
outgrowth of dentate granule cell axons in seizure models. Seizures are
sufficient to induce the transcriptional activation of c-fos
and other IEGs (Morgan and Curran, 1991; Kiessling and Gass, 1993;
Labiner et al., 1993) in the dentate granule cells, followed shortly
thereafter by expression of genes encoding neurotrophic factors [e.g.,
nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),
basic fibroblast growth factor (bFGF)] (Gall and Isackson, 1989;
Ernfors et al., 1991; Gall, 1993; Gall et al., 1994), neurotrophic
factor receptors (e.g., Trk B, FGFR-1) (Bengzon et al., 1993; Bugra et
al., 1994), and axonal growth-associated proteins such as GAP-43
(Bendotti et al., 1993; Meberg et al., 1993). Because neurotrophic
factors exert morphoregulatory effects on hippocampal neurons (Walicke
et al., 1986; Mattson et al., 1989; Ip et al., 1993; Patel and
McNamara, 1995), seizure-induced expression of these genes may underlie
the morphological rearrangements of granule cell axons. The presence of
AP-1 sites in the promoter region of several of these growth-related
genes (e.g., bFGF and GAP-43) (Shibata et al., 1991; Nedivi et al.,
1992) suggests that they may be IEG target genes contributing to the
lasting synaptic reorganization of the granule cells in kindling.
The development of a null mutation of the c-fos
proto-oncogene in transgenic mice by gene targeting (Johnson et al.,
1992; Wang et al., 1992) provides an experimental preparation in which
the role of c-fos in the structural and functional
plasticities of the kindling model can be assessed. The goal of these
experiments was to examine behavioral and electrophysiological indices
of kindling development and the kindling-induced sprouting of the mossy
fiber axons of the dentate granule cells in wild-type (+/+),
heterozygous (+/), and homozygous (/) mice carrying a null
mutation for c-fos. We show that indeed the development of
both kindling and kindling-induced mossy fiber sprouting are impaired
in c-fos / mice.
MATERIALS AND METHODS
Kindling and surgical procedures. Animals used in
this study (n = 63) were progeny of a cross between C57BL/6J
and 129/SvJ mice that were homozygous wild-type (+/+), heterozygous
(+/), or homozygous mutant (/) for the targeted c-fos
allele (Johnson et al., 1992). Mice of all three genotypes underwent
stereotaxic implantation of a bipolar electrode in the right amygdala
under pentobarbital anesthesia using the following coordinates with
bregma as reference: 0.5 mm posterior; 2.7 mm lateral; 4.5 mm below
dura. A ground wire was attached to a screw overlying the left frontal
cortex in all implanted animals (n = 37). In a subset of
these animals (n = 12), electrographic seizure activity was
recorded from an additional bipolar recording electrode placed in the
right hippocampus using the following stereotaxic coordinates relative
to bregma: 2.4 mm posterior; 2.0 mm lateral; 1.7 mm below dura. Animals
used for Timm staining and morphometric studies of hippocampus were
selected from the subset without hippocampal electrodes because of the
potential confounding factors arising from injury. After a
postoperative recovery period of at least 1 week, the afterdischarge
threshold (AD) was determined by application of a series of 1 sec
trains of 60 Hz 1 msec biphasic rectangular pulses beginning at a
current intensity of 40 µA followed by trains of increasing current
intensity administered at 1 min intervals until an AD of at least 3 sec
was observed. Seizures were classified according to a modification of
Racine (1972): 1, chewing; 2, head-nodding; 3, unilateral forelimb
clonus; 4, bilateral forelimb clonus; 5, bilateral forelimb clonus plus
falling and/or hindlimb clonus; 6, running or bouncing seizure; and 7, tonic hindlimb extension. Animals were stimulated twice daily with
interstimulus intervals of at least 4 hr until at least 10 class 4 or 5 seizures were evoked, each of which had clonic or tonic activity of at
least 12 sec. At least 10 class 4 or 5 seizures were evoked before the
animals were killed to enhance mossy fiber sprouting and facilitate
comparison of sprouting among +/+, +/, and / mice. To assure
objectivity in interpretation of electrophysiological data, the
electroencephalographs (EEGs) were coded and the duration of AD was
ascertained by an observer who was unaware of the genotype of the
subject; this was performed for the subset of the animals (Figs. 2, 4)
used for Timm staining and morphometric analyses. No differences
between the blinded and unblinded analyses were found.
Fig. 2.
Timm histochemistry. Horizontal Timm-stained
section of the dorsal dentate gyrus representative of a kindled +/+
(A) and / (C) mouse selected from animals
presented in E to show the differences measured. The area
near the asterisk is shown at higher magnification in panels
B (+/+) and D (/), respectively; the Timm
granules in the supragranular region are prominent in the +/+ but not
in the / mouse (arrows). Scale bars: A, C,
500 µm; B, D, 40 µm. E, Results of image
analysis of Timm granules in naive and kindled animals of all three
genotypes (n = 7, +/+; n = 5, +/; n = 4, /). Timm indexes from ipsilateral and contralateral hippocampi
were pooled and analyzed by two-way ANOVA with Bonferroni's
t test (post-hoc). §, p < 0.05 (vs kindled
+/+);  , p < 0.05 (vs kindled +/); *, p < 0.05 (vs kindled /); §§, p < 0.05 (vs kindled /);
, p < 0.05 (vs kindled /). When ipsilateral and
contralateral Timm scores were analyzed separately by genotype and
treatment, similar differences were found. A subset of the animals
undergoing kindling was used for these morphological studies; kindling
development in these animals was similar to the composite of animals
presented in Figure 1.
[View Larger Version of this Image (134K GIF file)]
Fig. 4.
Morphometric analyses of cell density
(A), cell number (B), and hilus volume
(C). Values represent mean ± SEM of +/+ (n = 7),
+/ (n = 5), and / (n = 4) mice. Naive
refers to unimplanted and unstimulated mice of corresponding genotypes.
Kindled refers to treatment as described in Materials and Methods.
I and C refer to the hippocampus ipsilateral and
contralateral to the stimulating electrode, respectively. Statistical
analyses were performed with two-way ANOVA with post-hoc Bonferroni's
t test for pairwise comparisons. *, p < 0.05 in
comparison to naive of corresponding genotype and side.
[View Larger Version of this Image (28K GIF file)]
Histologic analyses. To prepare brains for histologic
analysis, a subset of the animals examined for kindling development
were perfused transcardially under deep pentobarbital anesthesia at
least 3 d after the last stimulation-evoked clonic motor seizure. The
perfusion was performed at room temperature at a flow rate of 6 ml/min.
The perfusion was initiated with a 0.16% solution of 0.1 M sodium phosphate-buffered sodium sulfate, pH
7.4, for 4 min, followed by 0.1 M sodium
phosphate-buffered fixative solution of 3% glutaraldehyde, pH 7.4, for
10 min. Animals underwent perfusion at least 3 d after the last
stimulation-evoked clonic motor seizure. Only satisfactorily perfused
animals were used for image analysis. Criteria for a satisfactory
perfusion were that both forelimbs and hindlimbs were symmetrically
stiffened by perfusion and the brain was rendered symmetrically firm by
perfusion. Satisfactory perfusions were obtained in seven +/+, five
+/, and four / mice after kindling and in seven +/+, five +/,
and four / unstimulated mice; these brains were used to assess
mossy fiber sprouting and morphometric study of the dentate hilus.
After perfusion, the brain was immersed for 4 hr in the fixative buffer
at 4°C. After fixation, the brain was immersed in 15% sucrose in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C until
it sank. Horizontal sections (20 µm) were cut in a cryostat at
20°C and collected in 0.05 M Tris-HCl buffer,
pH 7.6.
Timm staining refers to a histochemical technique that labels synaptic
terminals of the mossy fiber axons of the dentate granule cells; this
was used to measure the sprouting of these axons into the supragranular
region that accompanies amygdaloid kindling (Cavazos et al., 1991).
Serial sections were collected between 1900 and 2800 µm ventral to
the dorsal surface of the brain, and the sections were mounted on
gelatin-coated slides and developed in the dark for 90-120 min in a
5:1 mixture of gum arabic (20% w/v) and hydroquinone solution (2%
w/v) containing citric acid (3% w/v) and silver nitrate (0.9% w/v).
To minimize the influence of variation of tissue thickness and of
precise horizontal level, analyses were performed on four serial
sections through the septal region of the dentate gyrus from each
animal collected between 2000 and 2080 µm ventral from the surface of
the neocortex. To facilitate meaningful comparison of sections of
different animals, slides from +/+, +/, and / animals were
included in the same slide rack and exposed to the same solutions for
the same duration of time. To assure objectivity in data analysis,
slides subsequently were coded and subjected to image analysis by an
observer who was unaware of the genotype or treatment of the
animal.
The area within the supragranular region occupied by the Timm granules
was determined by using a computer-assisted image analyzer (Image-1,
Universal Imaging) attached to a light microscope (Zeiss ICM 405) with
a high-resolution CCD camera. The assessment of sprouting was obtained
from the absolute value of the total area of Timm granules divided by
the total length of the dentate gyrus (Timm index). For each animal,
the absolute value of the Timm index is the mean of the four
sections.
Measures of hilar neuron number, density, and volume were performed as
follows. Between 2800 and 4600 µm ventral from the dorsal surface of
neocortex, every third section was stained with cresyl violet, coded,
and studied with a computer-assisted image analyzer (Neurolucida,
MicroBrightField) attached to a light microscope (Nikon FXA) with a
high-resolution CCD camera. The area of the hilus (A in
µm2) was defined in each section as the area
contained within boundaries of lines drawn along the hilar margin of
the dentate granule cell layer and from the tips of the granule cell
layer to the termination of the CA3c pyramidal cell layer within the
hilus. The total volume (VT) of the hilus
in µm3 = (A1 + A2 + A3
... . + A30) × 20 µm. Cell number
(N) was determined for each section on the video monitor
image by counting each cell with a nucleus containing a nucleolus
evident in the hilus field. The total cell number
(NT) = (N1 + N2 + N3
... . + N30). Thus hilus cell density = NT  VT for
each hilus (analyzed separately for ipsilateral and contralateral
hilus). Electrode placements were determined by a blinded observer in
sections stained with methyl green-pyronine Y or cresyl violet. Animals
in which the electrodes were not clearly located in the amygdala (and
also in the hippocampus in the subset of animals in which such
electrodes were directed) were excluded from further analysis.
Riboprobe preparation. Plasmids containing the full-length
rat c-fos cDNA insert (generously supplied by J. Morgan and
T. Curran, Roche Institute of Molecular Biology, Nutley, NJ) in
antisense or sense orientation were used to generate antisense and
sense c-fos riboprobes. Each plasmid was linearized with
XhoI, and riboprobes were generated using SP6 polymerase via
in vitro transcription in the presence of
35S-UTP. A plasmid containing nucleotides
414-184 of the rat NGFI-A cDNA (generously supplied by J. Milbrandt, Department of Pathology, Washington University School of
Medicine, St. Louis, MO) was used to generate NGFI-A
riboprobes. The 230-base pair NGFI-A sequence was cloned
into Bluescript KS containing the T7 and T3 promoters. The plasmid was
linearized with EcoRI or BglII and transcribed in
the presence of 35S-UTP by T3 or T7 to generate
sense and antisense riboprobe, respectively. A plasmid containing the
full-length mouse c-jun cDNA (American Type Culture
Collection, Rockville, MD) was used to generate c-jun
riboprobes. The plasmid was linearized with HindIII or
BglI and transcribed with SP6 or T7 to generate sense and
antisense riboprobe, respectively. The c-fos and
c-jun riboprobes were hydrolyzed to a length of ~200
nucleotides via limited alkaline hydrolysis in carbonate buffer at
60° before use in the in situ hybridization protocol.
In situ hybridization. In situ hybridization
was performed with 12-µm-thick frozen sections that were thaw-mounted
onto autoclaved gelatin-coated slides. The slide-mounted sections were
fixed for 10 min at room temperature in PBS (10 mM NaCl, 1.6 mM
NaH2PO4, 8.4 mM
Na2HPO4, pH 7.0) containing
4% paraformaldehyde, dehydrated in graded ethanols, and stored at
80°C. Before use, the sections were pretreated with proteinase K
(10 µg/ml) in Tris-EDTA buffer (TE: 100 mM
Tris-HCl, 50 mM EDTA, pH 8.0) at 37°C for 10 min, washed in 1× TEA, pH 8.0, for 3 min, acetylated with acetic
anhydride (0.25% v/v) in 1× TEA for 10 min, washed in 2× SSC for 2 min, and dehydrated through graded ethanols (50, 70, and 100%). The
sections were then hybridized in a solution containing 50% deionized
formamide, 10% dextran sulfate, 3× SSC, 5× Denhardt's, 10 mM DTT, 0.5 mg/ml yeast tRNA, 0.5 mg/ml
heat-inactivated salmon sperm DNA, and 60 ng/ml
35S-labeled riboprobe. After overnight
hybridization at 55°C, sections were rinsed in 2× SSC (10 min),
treated with 20 µg/ml RNase A (37°C, 30 min), rinsed in 1× SSC (10 min), washed in 0.1× SSC (55°C, 30 min × 4), and dehydrated in
graded ethanols (50, 70, and 100%). After air-drying, slides were
mounted in x-ray cassettes and apposed to  -max hyperfilm (Amersham,
Arlington Heights, IL) for at least 3 d, developed in D-19 (Kodak,
Rochester, NY) for 5 min, washed for 1 min in 1% acetic acid, and
fixed with Kodak Rapid Fixer for 5 min.
RESULTS
Development of kindling
Kindling development was assessed by monitoring both behavioral
and electrophysiological responses to brief, low intensity electrical
stimulations applied to the amygdala twice daily. Behavioral responses
consisted of limbic seizures (classes 1 and 2) and limbic and clonic
motor seizures (classes 3 through 7). Electrophysiological responses
consisted of electrographic seizure activity detected in EEG recordings
from the amygdala electrode. Both the intensity of the current required
to evoke an electrographic seizure (electrographic seizure threshold)
and the duration of the electrographic seizure were measured. These
experiments were performed in two sets of animals, and similar results
were obtained in each set; the results of both experiments were pooled
for presentation.
The development of kindling did occur in / mice (Fig.
1), but both electrographic and behavioral features of
the early stages were delayed in onset in comparison to +/+ mice. In
+/+ mice, the duration of the initial electrographic seizure was ~10
sec, and the duration of subsequent electrographic seizures invariably
exceeded this value until reaching a steady state of at least 30 sec by
the seventeenth stimulation (Fig. 1A). This steady
increase in electrographic seizure duration (ESD) was paralleled by an
increase in behavioral seizure intensity such that seizures of class 4 or 5 were evident after the eleventh stimulation (Fig. 1C).
No significant difference between +/+ and / in the duration of
electrographic seizure evoked by the first stimulation was noted;
however, in contrast to +/+ mice, in / mice the duration of
electrographic seizures failed to increase during the first nine
stimulations before steadily lengthening thereafter (Fig.
1A). Statistically significant differences in ESD
between +/+ and / mice were evident with seizures induced by
stimulations 2 through 12 (p values ranged from
<0.05 to 0.001); the differences in response to stimulation 13 and
thereafter were not statistically significant. The observed delay in
lengthening of stimulus-evoked ESD was also paralleled by less severe
behavioral seizures (Fig. 1C). In contrast to the attenuated
responses during the earlier stages of kindling development in /
mice, no differences in electrographic or behavioral seizures were
detected in the later stages of kindling development (stimulations
after 16); the numbers of stimulations required to evoke three seizures
with a duration of clonic motor activity of at least 12 sec were 15 ± 2 and 18 ± 1 for the +/+ and / mice, respectively (p > 0.05). Likewise, no significant differences between +/+ and / mice
were found in the current required to evoke the initial electrographic
seizure (not shown). Finally, no significant differences between +/+
and +/ mice were detected in either electrographic or behavioral
seizure duration (Fig. 1B,D) or any other index of kindling
development.
Fig. 1.
ESD and behavioral seizure class evoked by
periodic stimulation of the right amygdala. ESD values represent mean ± SEM of wild type (+/+) (n = 16), heterozygous (+/)
(n = 11), and homozygous (/) (n = 10)
animals. Behavioral seizure class represents mean ± SEM. A,
C, Compare +/+ with / animals; B, D, compare +/+
with +/ animals. Data were subjected to analysis of variance with
Bonferroni's post-hoc t test. Single, double,
and triple asterisks refer to differences of
p < 0.05, p < 0.01, and p
<0.001, respectively, found with Bonferroni's test.
[View Larger Version of this Image (18K GIF file)]
Mossy fiber reorganization
To determine whether these defects of functional plasticity were
paralleled by defects of structural plasticity, we measured
kindling-induced sprouting of dentate granule cell mossy fiber axons
with Timm histochemistry. Significant reductions of kindling-induced
increases in Timm staining in the supragranular region of the dentate
gyrus were evident in / compared with that in +/+ mice (Fig.
2A-E). A fourfold increase in Timm
index was evident after kindling in the +/+ mice compared with that in
naive +/+ mice (p < 0.05) (Fig.
2E). By contrast, a smaller (2.4-fold) increase in
Timm index was evident after kindling of / mice compared with naive
/ mice (p < 0.05). The lower level of Timm
staining after kindling in / compared with +/+ mice (Fig.
2E; p < 0.05) was selective to the
supragranular region in that no differences in intensity were found in
the infragranular region or in stratum lucidum (Fig.
2A,C) by densitometric measures (not shown).
Moreover, in contrast to the overlapping values of Timm index among
individual naive +/+ and / animals, no overlap in Timm index was
present among individual kindled +/+ compared with kindled /
animals (Fig. 2E). Kindling produced an increase in
Timm index in +/ animals intermediate between +/+ and / animals;
this increase was more variable than that of +/+ or / animals (Fig.
2E) but was significant compared with naive +/
controls (p < 0.05) and with kindled / mice
(p < 0.05). Timm staining in the supragranular
region of naive mice was minimal and did not differ significantly among
+/+, +/, and / mice (Fig. 2E).
Because mossy fiber reorganization is thought to be caused by
pathological activity (i.e., electrographic seizure), meaningful
interpretation of the Timm results requires that the duration of
electrographic seizure be similar in the +/+, +/, and / mice. The
Timm studies were performed after completion of kindling in the initial
subset of animals under study (n = 16). These animals were
stimulated until 10-15 class 4 or 5 motor seizures had been evoked,
and the total duration of evoked electrographic seizures was equivalent
among the different genotypes. Animals were killed at least 3 d after
the last seizure and prepared for morphological study. Despite
differences in ESD early in kindling development similar to the
composite of all animals (Fig. 1A), the total
duration of evoked electrographic seizure recorded from the amygdala
electrode was summed and was not significantly different among the
three genotypes (+/+, n = 7; +/, n = 5; /,
n = 4; p = 0.41). The lack of significant
difference in the summed ESD despite differences in ESD between
genotypes early in kindling development results from the fact that the
ESDs in the early stages of kindling development (short individual
ESDs) contribute much less to the summed ESD relative to ESDs in the
later stages of kindling (much longer individual ESDs).
Morphometric analyses
Mossy fiber sprouting has been reported to occur in response to
loss of neurons in the dentate hilus (Laurberg and Zimmer, 1981; Sutula
et al., 1987). Furthermore, a reduction in hilar neuronal density has
been described after kindling, and this has been interpreted to be
attributable to loss of hilar neurons (Cavazos and Sutula, 1990;
Cavazos et al., 1994). Thus the possibility arose that the reduction in
mossy fiber sprouting after kindling in the / compared with the +/+
mice could simply be attributable to greater loss of hilar neurons in
the +/+ compared with the / mice during kindling. We therefore
attempted to answer the following questions. Does kindling produce a
reduction in hilar neuron density (as reported previously)? If so, was
the reduction in hilar neuron density attributable to a loss of
neurons? Did kindling produce a greater loss of hilar neurons in +/+
compared with / mice, thereby providing a potential mechanism for
the observed differences in mossy fiber sprouting?
The density of hilar neurons, total number of hilar neurons, and hilar
volume were measured in +/+, +/, and / mice both in unstimulated
animals (n = 16) and after kindling (n = 16).
Hilar neurons were identified by the presence of nuclei containing
nucleoli, as detected in cresyl violet-stained sections (Fig.
3). Because previous studies have revealed that ~40%
of polymorphic neurons in the rat dentate hilus contain multiple
nucleoli (Cavazos and Sutula, 1990), cell counts were performed on
every third section. In accordance with previous reports (Cavazos and
Sutula, 1990; Cavazos et al., 1994), reductions in hilar neuron density
were evident after kindling of +/+ mice; significant reductions were
detected ipsilateral (27%; p < 0.05) and contralateral
(26%; p < 0.05) to the stimulating electrode (Fig.
4A). Significant reductions in hilar
neuron density were also found after kindling of / mice; the
magnitude of the reductions was equivalent to those found after
kindling of +/+ mice and were similar ipsilateral (23%; p < 0.05) and contralateral to the stimulating electrode (26%;
p < 0.05) (Fig. 4A). Reductions in hilar
neuron density were also evident after kindling of +/ mice, but the
differences were significant only contralateral to the stimulating
electrode (Fig. 4A).
Fig. 3.
Cresyl violet-stained section of dentate gyrus of
a +/+ mouse. Asterisk in A demarcates region
presented in higher magnification in B. Arrowhead
in B denotes a typical neuron identified by nucleolus within
the nucleus. Scale bars: A, 200 µm; B, 40 µm.
[View Larger Version of this Image (130K GIF file)]
In contrast to previous studies (Cavazos and Sutula, 1990; Cavazos et
al., 1994), no reductions in hilar neuron number were found after
kindling of +/+ mice (Fig. 4B). Likewise, no reductions in
hilar neuron number were detected after kindling of either +/ or
/ mice (Fig. 4B). Instead, to our surprise, hilar volume
in +/+ mice was found to be increased after kindling in comparison to
unstimulated +/+ mice (Fig. 4C); significant increases were
found ipsilateral (50%; p < 0.05) and contralateral (40%;
p < 0.05) to the stimulating electrode. Increases in hilar
volume were also found in / mice after kindling compared with
unstimulated / mice (Fig. 4C), similar in magnitude to
those found in +/+ mice (ipsilateral, 55%; p < 0.05;
contralateral, 50%; p < 0.05). Increases in hilar volume
were found in +/ mice after kindling, but these did not reach
statistical significance. In summary, these results replicated findings
by Cavazos and Sutula (1990) in demonstrating kindling-induced
reductions in hilar neuron density approximating 28%; however, we show
the reductions in neuron density reported here to be attributable to
increases in hilar volume and not to reductions in hilar neuron
number.
The morphometric analyses described above were performed in kindled
animals and naive animals of the corresponding genotypes. In addition
to kindling per se, the kindled animals had undergone surgical
implantation of an electrode in the right amygdala and had been handled
as a part of the kindling protocol. By contrast, naive animals had not
undergone electrode implantation or handling. To determine whether
electrode implantation and/or handling is sufficient to produce an
increase in hilar volume, hilar volume was measured in naive +/+ mice
and in +/+ mice after electrode implantation and handling with sham
stimulation for 28 sessions. No differences in hilar volume
(µm3 × 105, mean ± SEM,
left and right, respectively; p > 0.64) were detected
between these animals: naive: 7377 ± 396, 7233 ± 290; implanted and
handled: 7479 ± 539, 7055 ± 756.
Apart from differences induced by kindling, these detailed morphometric
analyses provided an opportunity to assess the effects of the null
mutation of c-fos on the anatomy of the dentate hilus. No
significant differences in hilar neuron density, neuron number, or
hilar volume were detected as a function of genotype in either
unstimulated or kindled animals (Fig. 4A-C).
Importantly, the lack of detectable hilar neuron death after kindling
argues against differential loss of neurons as the explanation for
differences between +/+ and / mice in the magnitude of
kindling-induced mossy fiber sprouting.
Assessment of seizure propagation into hippocampus
Another possible explanation of the reduction of mossy fiber
sprouting in / compared with +/+ mice after kindling could be
simply that the electrographic seizure initiated by amygdala
stimulation failed to propagate to the dentate granule cells of the
hippocampus in the / compared with the +/+ mice. This possibility
was not addressed in the initial experiments because the sole
electrographic recordings were from the electrode in the amygdala,
precluding direct measurement of activity in the hippocampus. This
possibility was tested by two different experimental approaches,
electrophysiological recordings during the stimulus-evoked seizures and
in situ hybridization for IEGs in the dentate granule
cells.
The critical issue addressed in these experiments was whether the ESD
recorded in the amygdala provided a reliable measure of the ESD in the
hippocampus in both +/+ and / mice. To address this issue, the
duration of electrographic seizure was compared between electrodes
placed in the stimulated amygdala and the ipsilateral hippocampus
during the development of kindling in both +/+ and / mice. We found
that each stimulation-evoked electrographic seizure in the amygdala was
associated with an electrographic seizure recorded from the hippocampal
electrode in every +/+ and / mouse. Moreover, no differences in the
total duration of electrographic seizures between amygdala and
hippocampus were evident in either the +/+ (n = 9) or /
(n = 3) mice (data not shown). Even the brief electrographic
seizure induced by the initial stimulation of the amygdala was
associated with electrographic seizure in the hippocampus in both +/+
and / mice (not shown). These electrophysiological recordings
demonstrate that the electrographic seizure recorded from the amygdala
faithfully reflected electrographic seizure propagation into the
hippocampus in both +/+ and / mice.
To determine whether electrographic seizure actually triggered
activation of immediate early gene expression in the dentate granule
cells of both +/+ and / mice, in situ hybridization
experiments were performed. Electrographic seizure has been
demonstrated to evoke dramatic increases of transcripts for multiple
IEGs in the dentate granule cells (Morgan et al., 1987; Morgan and
Curran, 1991; Kiessling and Gass, 1993; Labiner et al., 1993). We
therefore used in situ hybridization to test directly
whether a kindled seizure evoked increases of transcripts for the IEGs
NGFI-A and c-jun in both +/+ and / mice.
In situ hybridization for both of these IEGs was performed
in +/+ and / mice killed 30 min after a class 5 kindled seizure.
Striking increases in the expression of both of these IEGs were evident
in every +/+ (n = 5) and / (n = 5) mouse
examined (Fig. 5). As expected, c-fos
transcripts were increased in the dentate granule cells after seizure
in the +/+ but not the / mice (Fig. 5). Thus, these data provide
direct evidence for the seizure-induced activation of IEG expression in
the dentate granule cells of / as well as +/+ mice, and together
with the electrophysiological data (above) support the idea that
equivalent amounts of pathological activity invaded the dentate granule
cells during the kindling protocol in the +/+ and / mice. These
data also verify the absence of seizure-evoked expression of
c-fos in the / mice.
Fig. 5.
In situ hybridization for immediate
early gene transcripts in c-fos +/+ versus c-fos
/ animals. Representative in situ hybridization
autoradiograms exposed for the same amount of time from animals killed
30 min after an amygdala-kindled clonic motor seizure. Arrow
denotes right dentate gyrus. Insets show ipsilateral dentate
gyrus at higher magnification. Marked bilateral NGFI-A
induction was observed in all +/+ (n = 5) and /
(n = 5) animals (top), whereas as expected, only
+/+ animals showed c-fos induction (n = 5)
(bottom). Results similar to NGFI-A were obtained
with c-jun riboprobe in all 10 animals (not shown). Sense
riboprobes showed no specific hybridization signal (not shown).
[View Larger Version of this Image (32K GIF file)]
DISCUSSION
Two principal findings emerge from this work. First, the
development of kindling as measured by behavioral and
electrophysiological indices is partially impaired in mice carrying a
null mutation for c-fos. Second, kindling-induced granule
cell axon sprouting into the supragranular region of the dentate gyrus
as measured by Timm staining is attenuated in c-fos null
mutants. Our findings support the hypothesis that expression of IEGs
such as c-fos link fleeting changes of neuronal activity to
lasting modifications of structure and function in the mammalian
nervous system.
Defective kindling-induced axonal sprouting in null mutants
Our conclusion that there are fewer mossy fiber terminals in the
supragranular region of the dentate gyrus relies on the lower level of
Timm staining in this area after kindling in / compared with +/
or +/+ mice. The Timm method stains neural elements containing heavy
metals and stains mossy fibers in particular because of their high zinc
content (Danscher, 1981). Sutula et al. (1988) demonstrated the
presence of Timm granules in the supragranular region after kindling
and localized the aberrant Timm granules to synaptic terminals with
electron microscopy. Okazaki et al. (1995) used retrograde transport of
biocytin in slices isolated from rats after chemoconvulsant-induced
status epilepticus to demonstrate the presence of mossy fiber synapses
on granule cell dendrites in animals with robust Timm staining in the
supragranular region. On the basis of these results, we conclude that
the reduced Timm staining in the supragranular region of dentate gyrus
reflects fewer mossy fiber terminals present in / compared with
+/ or +/+ mice after kindling. The spatial selectivity of this
effect, i.e., that the lower level of Timm staining after kindling in
/ mice was confined to the supragranular region of the dentate
gyrus, argues against a global effect of the null mutation on the mossy
fiber axons and pinpoints the defect to an attenuation of the
seizure-induced sprouting of these axons.
Potential mechanisms for defective kindling-induced axonal
sprouting in null mutants
One possible mechanism for reductions of mossy fiber sprouting in
+/+ versus / mice could be differences in seizure-evoked hilar cell
death. Reductions of hilar neuronal density have been described after
kindling; these reductions have been interpreted as resulting from
death of hilar neurons (Cavazos and Sutula, 1990; Cavazos et al.,
1994). Thus it is conceivable that death of fewer hilar neurons in
/ compared with +/+ mice might account for defective mossy fiber
sprouting in / mice. Cavazos and Sutula (1990) described reductions
of hilar neuronal density in rats approximating 15% and 40% after 3 and 30 class 5 kindled seizures, respectively. The present findings
confirm and extend these findings by demonstrating a reduction of hilar
neuronal density of ~30% after 10-15 class 5 kindled seizures in
+/+ mice. In our study, kindling did not result in any change in the
total numbers of hilar neurons in +/+, +/, or / mice (Fig.
4B). In contrast to the findings of Cavazos and Sutula
(1990), kindling did result in increases of hilar volume in all
genotypes (Fig. 4C). Thus the reductions in hilar neuronal
density described here are attributable to increases of hilar volume
and not to loss of hilar neurons. Cavazos and Sutula (1990) did report
a small increase of hilar volume after kindling, but this was not
significant and the magnitude was not sufficient to account for the
reduction of neuronal density. The explanation for the discrepancy in
values of hilar volume may reside in methodological differences.
Cavazos and Sutula (1990) measured the areas of the dentate gyrus in
four horizontal sections and the vertical distance between these four
sections by counting the number of cryostat sections of a given
thickness; these measurements were used to calculate the volume. In the
present study, the hilar area was measured in the 30 horizontal
sections in which the neurons were counted, and the areas were
integrated to determine the volume. In similar studies, Lothman and
colleagues (Bertram et al., 1990; Bertram and Lothman, 1993) reported
kindling-induced reductions of hilar neuronal density that were
attributable to increases of hilar volume and not to loss of neurons.
Although no cell loss was detectable, we cannot exclude the possibility
that loss of a small population of hilar neurons escaped detection in
these experiments.
The kindling-induced increases in hilar volume are reminiscent of the
preferential increases in volume identified in those regions of rat
neocortex undergoing greater use during development (Purves, 1994). In
contrast to the effects of physiological activity on the developing
normal cortex, the effects described here are the consequence of
pathological activity in mature cortex. According to this developmental
analogy, we favor the idea that the increases of hilar volume are
attributable to kindling-induced neuropil elaboration. Other
possibilities, which include glial proliferation and accumulation of
fluid in the extracellular or intracellular space, warrant careful
study.
Another potential explanation for impaired mossy fiber sprouting after
kindling of / mice could be that less electrographic seizure
activity propagated to the dentate granule cells after amygdala
stimulation in / versus +/+ mice. Direct measures with recording
electrodes demonstrated that total electrographic seizure activity is
similar whether measured in the amygdala or the hippocampus.
Furthermore, in situ hybridization experiments after
amygdala-evoked kindled seizures disclosed dramatic increases in the
expression of the IEGs, NGFI-A, and c-jun in the
dentate gyrus of both +/+ and / mice (Fig. 5), implying that the
granule cells were indeed activated by electrographic seizure
regardless of genotype.
Another explanation for the attenuation of kindling-induced mossy fiber
sprouting could be that developmental absence of c-fos
modifies the later ability of the granule cells to exhibit axonal
sprouting in response to pathological activity in the mature brain.
Ontogenetic analyses of c-fos expression argue against this
possibility. First, the vast majority of dentate granule cells are born
postnatally ( 85% in the rat) (Angevine, 1965; Bayer and Altman,
1974). Second, Alcantara and Greenough (1993) examined the expression
of Fos immunoreactivity at multiple time points during embryonic and
postnatal development and found no evidence for constitutive Fos
expression in the granule cells. Rather, the dentate granule cells seem
to express c-fos in response to various stimuli (such as
electrographic seizure) only in the mature brain. Seizures in the
immature brain induce limited or no c-fos expression
(Schreiber et al., 1992) and also fail to induce mossy fiber sprouting
(Sperber et al., 1991), demonstrating that activation of this genetic
program is unique to the mature brain and suggesting that it is not
involved in the formation of mossy fiber synapses during development.
The lack of detectable differences in dentate hilar neuron density,
number, or volume and the lack of difference in Timm staining among
naive +/+, +/, and / animals also argue against a confounding
effect of development.
Instead, we favor the explanation that the mechanism by which the null
mutation of c-fos attenuates kindling-induced mossy fiber
sprouting involves alterations of seizure-induced transcriptional
regulation of gene expression in the granule cells. Seizure activity in
the mature brain triggers the transcriptional activation of
c-fos and other IEGs and of genes encoding neurotrophic
factors (e.g., NGF, BDNF, bFGF), neurotrophic factor receptors (e.g.,
Trk B, FGFR-1), and axonal growth-associated proteins (e.g., GAP-43) in
the dentate granule cells (Gall and Isackson, 1989; Ernfors et al.,
1991; Morgan and Curran, 1991; Bendotti et al., 1993; Bengzon et al.,
1993; Gall, 1993; Kiessling and Gass, 1993; Labiner et al., 1993;
Meberg et al., 1993; Bugra et al., 1994; Gall et al., 1994). Such genes
are excellent candidates to contribute to seizure induction of mossy
fiber sprouting in the mature brain. The ability of BDNF or bFGF to
selectively enhance the branching of axons but not dendrites of dentate
gyrus neurons in vitro suggests that neurotrophic factors
may contribute to mossy fiber sprouting in vivo (Patel and
McNamara, 1995; Lowenstein and Arsenault, 1996). Furthermore,
transgenic mice overexpressing GAP-43 display spontaneous mossy fiber
sprouting (Aigner et al., 1995). The presence of AP-1 sites in the
regulatory elements of the bFGF (Shibata et al., 1991) and GAP-43
(Nedivi et al., 1992) promoter regions strengthens the candidacy of
these particular genes in the fos-less phenotype. Although
other mechanisms cannot be excluded, we believe that the absence of
c-fos may limit the transcriptional activation of such
growth-related genes. Alternatively, the induction of c-fos
by neurotrophic factors (Bartel et al., 1989; Collazo et al., 1992)
raises the possibility that the absence of c-fos may
diminish responsiveness to seizure-induced neurotrophic factor
expression. The potential for compensatory or adaptive effects of other
members of the FOS family such as fra-1, fra-2,
or fos B may explain why inhibition of this structural and
functional plasticity is partial instead of complete (Pennypacker et
al., 1995).
Relationship of defective mossy fiber sprouting to defective
kindling development
The defective sprouting of granule cell axons may contribute to
the defective kindling development of the fos-less mice.
Structural or functional modifications of the granule cells may
influence kindling development evoked by amygdala stimulation, because
granule cell destruction is known to retard amygdaloid kindling
(Dasheiff and McNamara, 1982). Synaptic reorganization of granule cell
axons identified in kindled animals provides a plausible mechanism for
part of the hyperexcitability of kindling (Sutula et al., 1988).
Morphological and electrophysiological evidence supports the notion
that at least part of the aberrant projection of the granule cell axons
innervates the granule cell dendrites, thereby forming a recurrent
excitatory circuit (Frotscher and Zimmer, 1983; Tauck and Nadler, 1985;
Sutula et al., 1988; Cronin et al., 1992; Okazaki et al., 1995).
Although a subpopulation of these aberrant axons may activate
inhibitory neurons (Cronin et al., 1992; Sloviter, 1992), the enhanced
synaptic excitation of the granule cells resulting from the remaining
axons (Tauck and Nadler, 1985; Cronin et al., 1992) could promote the
progressive intensification of evoked seizures in the kindling model.
It therefore seems plausible that the attenuation of mossy fiber
sprouting may contribute to the functional defects observed with
kindling development; however, detailed analyses of the spatial pattern
of c-fos expression during kindling development in the rat
have demonstrated that c-fos expression is induced in a
highly specific anatomic pattern restricted to the medial and cortical
amygdaloid nuclei and pyriform cortex ipsilateral to the stimulating
electrode during the first several stimulation-induced electrographic
seizures (Hosford et al., 1995). If similar patterns occur in mouse and
rat, perhaps the failure of the ESD to lengthen after several
stimulations is attributable in part to the absence of c-fos
expression in these amygdaloid nuclei and pyriform cortex. The absence
of c-fos expression in the granule cells and reduced mossy
fiber sprouting may then contribute to the shorter electrographic
seizures and less intense behavioral seizures evoked by later
stimulations. Although this explanation is plausible, our experiments
do not prove that the attenuation of mossy fiber sprouting itself
caused or contributed to the defect of kindling development.
FOOTNOTES
Received Jan. 23, 1996; revised March 25, 1996; accepted April 2, 1996.
We thank Dr. L. Lerea for expert technical assistance. This work was
supported by National Institutes of Health Grants HD24926 (R.S.J.,
B.M.S.), HD27295 (R.S.J., B.M.S., V.E.P.), NS17771 (J.O.M.), and
NS32334 (J.O.M.).
Correspondence should be addressed to Dr. James O. McNamara, 401 Bryan
Research Building, Duke University Medical Center, Durham, NC
27710.
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