1Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California 94305-5410; and 2Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523
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ABSTRACT |
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Buckmaster, Paul S. and
F. Edward Dudek.
In vivo intracellular analysis of granule cell axon reorganization in
epileptic rats. In vivo intracellular recording and labeling in
kainate-induced epileptic rats was used to address questions about
granule cell axon reorganization in temporal lobe epilepsy.
Individually labeled granule cells were reconstructed three
dimensionally and in their entirety. Compared with controls, granule
cells in epileptic rats had longer average axon length per cell; the
difference was significant in all strata of the dentate gyrus including
the hilus. In epileptic rats, at least one-third of the granule cells
extended an aberrant axon collateral into the molecular layer. Axon
projections into the molecular layer had an average summed length of 1 mm per cell and spanned 600 µm of the septotemporal axis of the
hippocampusa distance within the normal span of granule cell axon
collaterals. These findings in vivo confirm results from previous in
vitro studies. Surprisingly, 12% of the granule cells in epileptic
rats, and none in controls, extended a basal dendrite into the hilus,
providing another route for recurrent excitation. Consistent with
recurrent excitation, many granule cells (56%) in epileptic rats
displayed a long-latency depolarization superimposed on a normal
inhibitory postsynaptic potential. These findings demonstrate changes,
occurring at the single-cell level after an epileptogenic hippocampal
injury, that could result in novel, local, recurrent circuits.
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INTRODUCTION |
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Temporal lobe epilepsy is one of the most common
types of epilepsy and one of the most difficult to treat effectively
with medication (Engel et al. 1997).
Electroencephalography and response to surgical therapy suggest that
structures of the mesial temporal lobe generate the complex partial
seizures of temporal lobe epilepsy (Engel et al. 1997
;
Falconner et al. 1964
). The hippocampal formation, a
prominent component of the mesial temporal lobe, has been hypothesized to be involved in epileptogenesis by several different mechanisms (Babb et al. 1989
; Buhl et al. 1996
;
de Lanerolle et al. 1989
; During et al.
1995
; Mody and Heinemann 1987
; Peterson
and Ribak 1989
; Sloviter 1987
). Considerable
attention has focused on the dentate gyrus, in part because previous
studies have provided evidence that it normally has a high seizure
threshold and might act like a "gate," preventing seizure
propagation into the hippocampus (e.g., Fricke and Prince
1984
; Lothman et al. 1991
, 1992
;
Schweitzer et al. 1992
). The dentate gyrus is a good
candidate for involvement in chronic epileptogenesis because of the
neuropathological changes it displays in patients with temporal lobe
epilepsy (de Lanerolle et al. 1989
;
Margerison and Corsellis 1966
) and because of its remarkable potential for plasticity in response to injury
(Nadler et al. 1980
; Parent et al. 1997
).
Tauck and Nadler (1985) proposed that seizures result
from positive feedback through aberrant excitatory recurrent axon
collaterals between granule cells. Granule cell axon reorganization has
been demonstrated in tissue from patients (Babb et al.
1991
; de Lanerolle et al. 1989
; Houser et
al. 1990
; Sutula et al. 1989
) and in
experimental models of temporal lobe epilepsy (Buckmaster and
Dudek 1997b
; Cronin and Dudek 1988
; Kotti
et al. 1997
; Mello et al. 1993
; Nadler et
al. 1980
; Okazaki et al. 1995
; Represa et
al. 1993
; Sutula et al. 1988
). Most previous
studies used staining techniques that label many or all granule cell
axons; however, several have used intracellular labeling of individual
granule cells (Franck et al. 1995
; Isokawa et al.
1993
; Sutula et al. 1998
). These intracellular labeling studies employed the hippocampal slice preparation, limiting observations to that part of the axon arbor within a 400- to
500-µm-thick slice.
Important questions persist about granule cell axon reorganization.
What proportion of granule cells extends axon collaterals into the
molecular layer of the dentate gyrus? How long are the axon collaterals
that invade the molecular layer, and how selective is that invasion? Do
granule cell axon collaterals project to distant septotemporal regions
of the dentate gyrus or do they remain close to the parent cell body?
The kainate-treated rat is a widely accepted experimental model that
replicates clinical and neuropathological features of temporal lobe
epilepsy (Ben-Ari 1985; Hellier et al.
1998
; Nadler 1981
; Pisa et al.
1980
). The present study used an in vivo intracellular approach
to label granule cells in kainate-induced epileptic rats, examine their complete axon arbors, and record electrophysiological responses.
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METHODS |
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Animals
Male Sprague-Dawley rats (200-250 g; Harlan, Houston, TX) were
treated with kainic acid (Sigma Chemical, St. Louis, MO) dissolved in
0.9% NaCl. Kainate was administered in doses of 5 mg/kg ip at 1-h
intervals until the rat experienced 3.5 h of recurrent seizures. The
average cumulative dose was 30 mg/kg. Age-matched control rats received
a similar volume of vehicle. After recovering from acute seizures,
animals were observed
6 h/wk for spontaneous motor seizures of grade
3 or greater on the Racine scale (forelimb clonus ± rearing ± falling) (Racine 1972
). The average period between kainate treatment
and electrophysiological recording and perfusion was 143 days (range
99-207).
Electrophysiology
Rats were anesthetized with urethan (1.2 g/kg ip) and placed in
a stereotaxic frame with the nose bar set at 3.0 mm. Urethan was used
because, unlike other drugs (e.g., barbiturates) (Nicoll et al. 1975
),
it appears to have little or no direct effect on GABAergic inhibition
in the rat dentate gyrus (Shirasaka and Wasterlain 1995
). Body temperature was maintained with a heating pad, and cerebrospinal fluid was drained from the cisterna magna to improve stability. Holes were drilled through the skull, and electrodes were
directed toward the dentate gyrus and angular bundle at the following
coordinates (relative to bregma):
4.6 mm posterior and 2.2 mm lateral
for the recording electrode and
8.3 posterior and 3.6 mm lateral for
the stimulating electrode. Electrode depths were determined by
optimizing field potential responses to stimulation. A bipolar
concentric stimulating electrode (SNEX-100, Rhodes Medical Instruments,
Tujunga, CA) activated perforant pathway fibers. A glass micropipette
filled with 0.9% NaCl and broken to an inner diameter of ~15 µm
was used to measure the depth of the granule cell layer and to
determine the stimulus threshold for a population spike (T).
Then a sharp intracellular electrode, measuring 80-150 M
in vivo
and filled with 1 M potassium acetate and 2% biocytin, was
lowered toward the septal end of the dentate gyrus where granule cells
were impaled.
Membrane and field potentials were amplified (Axoprobe 1A, Axon Instruments, Foster City, CA), observed on-line, and stored on video tape (Neuro-corder, Neuro Data Instruments, Delaware Water Gap, PA) and on computer (pCLAMP, Axon Instruments) for off-line analysis. Resting membrane potential was determined by subtracting the extracellular voltage measured when the electrode was withdrawn from the cell from the resting intracellular voltage. Membrane time constant was measured from responses to 0.1- to 0.5-nA hyperpolarizing current pulses. Action potential duration and amplitude were measured from responses to depolarizing current injections just large enough to evoke one action potential. Action potential duration was defined as the time between the onset of the action potential and the return of the membrane potential to the onset value. Action potential amplitude was measured from the onset value to the peak.
Perforant pathway fibers were activated at 0.1 Hz by 150-µs constant-current pulses. Stimulus intensity was standardized to the threshold (T) of the population spike; the average value of T was 1.09 mA (range 0.08-4.80). Stimulus intensity for inhibitory postsynaptic potential (IPSP) analysis was 1.5 × T. Measurements of fast IPSPs were made at a 20-ms latency after the stimulus; slow IPSPs were measured at a 150-ms latency. IPSP reversal potentials were determined by altering the membrane potential with DC current to change the amplitude and polarity of postsynaptic potentials. IPSP amplitude was plotted against the prestimulus membrane potential, and the data were fit with a least-squares regression line to determine the IPSP reversal potential. IPSP conductance was calculated by subtracting the cell's input conductance before stimulation from its conductance during the IPSP. Conductance was the slope of I-V curves, where I was DC holding current and V was membrane potential. Input resistance was the reciprocal of input conductance. Field potential responses, recorded after the electrode was withdrawn from the cell, were subtracted from the intracellularly recorded stimulus-evoked responses.
Morphology
After obtaining intracellular recordings, cells were labeled with 2% biocytin (Sigma) by passing 300-ms pulses of 0.4- to 1.0-nA hyperpolarizing current for an average of 8 min. However, cells began being labeled with biocytin even before the onset of the labeling procedure because they were impaled and current was passed for a period while they stabilized and while electrophysiological recordings were obtained. The rat then was perfused through the ascending aorta with 300 ml of 0.37% sodium sulfide and 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed, postfixed overnight, and cryoprotected in 30% sucrose in 0.1 M PB. The hippocampus was isolated, straightened, frozen, and sectioned (nominally at 60 µm) perpendicular to the septotemporal axis.
Serial sections of the septal 75% of the hippocampus containing the labeled cell were mounted on gelatin-coated slides and exposed to 0.3-0.5% hydrogen peroxide and 10% ethanol in 0.1 M PB for 1 h and then 0.3% Triton X-100 in 0.1 M PB for 1 h before incubating in ABC solution (1:150, Vector Laboratories, Burlingame, CA) in 0.1 M PB with 0.1% Triton X-100 for 2-4 h at room temperature or overnight at 4°C. After thorough washing in PB and 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (TB, pH 7.6), sections were exposed to 0.04% diaminobenzidine (Sigma) and 0.3% NiCl for 3 min. Hydrogen peroxide was added to result in a 0.0025% solution, and sections reacted for 0.5-1.0 h. The reaction was stopped in washes of 0.1 M TB, and the sections were dehydrated and coverslipped.
Axon arbors of 26 granule cells were drawn with a camera lucida, and two-dimensional axon lengths were measured from the drawings. This subgroup of our total sample of 64 labeled granule cells included the best cells used for axon reconstruction and measurement. These 26 cells used for quantitative analysis appeared to be completely labeled because their axons were darkly labeled, even at the extremes of the arbor. Cells with lightly labeled axons and cells in hippocampi with more than four labeled cells were excluded because overlapping axon collaterals might impede accurate axon reconstruction and measurement.
Granule cell axon arbors of 4 of the 26 cells (2 in kainate-induced epileptic rats and 2 in controls) were reconstructed three dimensionally with a Neurolucida system (MicroBrightField, Colchester, VT; Fig. 3). The complexity of intermingled axon arbors in preparations with more than one labeled cell precluded the use of the Neurolucida system in many cases. However, axon lengths measured from the two-dimensional camera-lucida representations were adjusted for three dimensionality by a correction factor (1.25), which was determined using values from the cells analyzed both ways. Axon lengths were adjusted for tissue shrinkage, which occurred during processing, by correction factors determined by measuring the distance between stereotaxically located fast green dye injections. Fast green dye (1%) was ejected iontophoretically from a field potential electrode, using 20 µA DC current for 10 min. In the transverse plane, tissue shrank to 94% of its original size (correction factor = 1.06); along the septotemporal axis it shrank to 51% (correction factor = 1.96). Because some hippocampi contained multiple labeled granule cells, average axon length per cell was estimated by dividing the summed length of all axon collaterals by the number of labeled cells. This would not affect the results, which were averaged and plotted by experimental group. The number of labeled somata was verified by determining the number of mossy fibers that projected through the CA3 field, there being only one mossy fiber in CA3 for each labeled granule cell. The dendritic trees of granule cells were reconstructed three dimensionally with the Neurolucida system.
Sections of the temporal 25% of the hippocampus were mounted on
gelatin-coated slides and processed by a modified Timm staining protocol (Babb et al. 1991). Sections were placed in a
solution consisting of 180 ml of 50% gum arabic, 30 ml of 2.25 M
citrate buffer, 45 ml of 0.5 M hydroquinone, and 50 ml of 0.04% silver lactate. Sections were developed for ~70 min in darkness at room temperature, then they were rinsed in water, lightly counterstained with cresyl violet, dehydrated, and coverslipped.
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RESULTS |
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Animals
Thirty rats were used in this study: 18 treated with kainic acid
and 12 age-matched vehicle-treated controls. All of the kainate-treated rats displayed spontaneous, recurrent, motor seizures, which first were
observed 70 ± 9 days after treatment (mean ± SE; range
25-168). The average seizure frequency over the period between the
first observed seizure and use in an experiment was 0.34 ± 0.04 seizures/h (range 0.11-0.62). No control rats were observed to have a
seizure. The rats used in this study came from the same pool of
identically treated animals that were used in previously published
studies (Buckmaster and Dudek 1997a,b
; Hellier et
al. 1998
; Patrylo and Dudek 1998
; Wuarin
and Dudek 1996
).
Timm stain
All rats in this study displayed dark Timm staining in the hilus
and in stratum lucidum of CA3. Control rats showed very little Timm
staining in the molecular layer of the dentate gyrus (Fig. 1A), except at the temporal
pole of the hippocampus as reported previously (Buckmaster and
Dudek 1997b; Cavazos et al. 1992
). In contrast,
all of the kainate-induced epileptic rats displayed dark Timm staining
in the inner third of the molecular layer (Fig. 1B). This
finding confirmed that granule cell axon reorganization occurred in all
of the kainate-induced epileptic rats in this study. This finding also
suggests that there was hilar neuron loss in kainate-induced epileptic
rats because aberrant Timm staining is correlated with hilar neuron
loss (Babb et al. 1991
; Buckmaster and Dudek
1997b
; Masukawa et al. 1995
).
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Granule cells
MULTIPLE LABELING.
The total number of labeled granule cells was 31 and 33 for the control
and kainate-induced epileptic groups, respectively. The number of
labeled granule cells per hippocampus ranged from one to six, and the
average was similar in the control (2.8 cells) and kainate-induced
epileptic groups (2.5 cells). Multiple labeling occurred because
repeated penetrations with biocytin-containing recording electrodes
were made to obtain electrophysiological recordings. In addition,
multiple-labeled cell bodies were closely apposed in some cases,
suggesting that some multiple filled cells resulted from
electrode-induced injury (Claiborne et al. 1990) or from
gap junction connections between granule cells (MacVicar and
Dudek 1982
).
AXONS.
Granule cells were more likely to project axon collaterals into
the granule cell layer and molecular layer of the dentate gyrus in
kainate-induced epileptic rats versus controls. In all cases, granule
cells extended a primary axon from the cell body, through the hilus,
and into stratum lucidum of CA3. Granule cell axon arbors were examined
in 11 hippocampi of 11 control rats. In five control hippocampi (45%)
at least one axon collateral entered the granule cell layer, and in one
control hippocampus (9%), one axon collateral entered the molecular
layer. Granule cell axon arbors were examined in 12 hippocampi of 10 kainate-induced epileptic rats. In 11 of these hippocampi (92%) at
least one axon collateral entered the granule cell layer and molecular
layer (Figs. 2 and
3). Differences between the control
and kainate-induced epileptic groups, with respect to the proportion of
hippocampi with an axon collateral entering the granule cell layer or
molecular layer, were significant (P < 0.025 and
P < 0.005, respectively, 2
test).
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DENDRITES.
Granule cells in kainate-induced epileptic rats were significantly more
likely to have a basal dendrite than those in controls (Figs.
3C and 5). In the control
group, 0/31 labeled granule cells had a basal dendrite compared with
4/33 granule cells (12%) in the kainate-induced epileptic group
(P < 0.05, 2 test). One of the basal
dendrites branched, the other three did not. All of the basal dendrites
were covered with spines (Fig. 5A, inset). In cells
that had them, the average length of the basal dendrite was 0.23 mm or
6% of the total dendritic length per cell. The average total length of
dendrites per cell for those cells with a basal dendrite (3.90 ± 0.22 mm) was not significantly different from that of the cells without
basal dendrites in kainate-induced epileptic rats (3.56 ± 0.51 mm) or in controls (3.60 ± 0.10 mm) (t-test). These
dendritic length results are similar to those reported previously for
granule cells in normal rats (mean = 3.22-3.66 mm)
(Claiborne et al. 1990
; Desmond and Levy
1982
).
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ELECTROPHYSIOLOGY.
Intrinsic electrophysiological properties of granule cells were not
significantly different in control and kainate-induced epileptic rats.
Both groups displayed strongly negative resting membrane potentials,
overshooting action potentials, and similar input resistances and
membrane time constants (Table 1; Fig. 6A). These results are
similar to previously published values for rat granule cells recorded
in vivo (Buckmaster and Schwartzkroin 1995).
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DISCUSSION |
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This study describes morphological changes that occur in granule cells after epileptogenic hippocampal injury and examines intrinsic and synaptic electrophysiological properties of granule cells in epileptic rats.
Chronic morphological changes of granule cells after epileptogenic hippocampal injury
The morphological changes of granule cells in kainate-induced
epileptic rats are summarized in Fig. 9.
The biggest change was in mean axon length, which was twice that of
controls, corroborating a previous in vitro study (Sutula et al.
1998). Dentate granule cells are not the only cell type to
sprout axon collaterals in response to epileptogenic treatments. Axon
sprouting has been reported for inhibitory interneurons in the dentate
gyrus (Davenport et al. 1990
; Mathern et al.
1995
), CA3 pyramidal neurons (McKinney et al.
1997
), CA1 pyramidal neurons (Perez et al.
1996
), and layer V pyramidal neurons in neocortex (Salin
et al. 1995
), suggesting that this might be a general response
of neurons to injury. The present study focused on granule cells in the
septal end of the dentate gyrus; results from the temporal dentate
gyrus might be different.
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Quantitatively, the largest morphological change was in axon length,
but the most dramatic difference was in the distribution of axon
collaterals in epileptic versus control rats. At least one-third of the
granule cells in epileptic rats had axon projections into the molecular
layer (compared with 1 of 31 cells in controls), and the averaged
summed length of these projections was 1 mm per cell. Previous
ultrastructural analyses have shown that these aberrant axon
projections form synaptic contacts with other granule cells
(Babb et al. 1991; Franck et al. 1995
;
Okazaki et al. 1995
; Represa et al.
1993
), and there is evidence that they could synapse with
inhibitory interneurons, too (Kotti et al. 1997
;
Sloviter 1992
).
Despite this dramatic change in circuitry, new axon collaterals
respected most of the boundaries of normal granule cell axons. Along
the longitudinal axis of the hippocampus, the axon arbors in epileptic
rats remained confined to a relatively thin lamella of the dentate
gyrus and did not extend to distant septotemporal regions. The
septotemporal span of the granule cell axon arbor within the dentate
gyrus in control and epileptic rats was ~1 mm, whereas the length of
the rat hippocampus is 10 mm (Amaral and Witter 1989).
In the transverse plane of the hippocampus, axon collaterals that
entered the molecular layer remained in the inner third and did not
extend into the middle or outer thirds. These findings corroborate
previous reports (Franck et al. 1995
; Isokawa et
al. 1993
; Represa et al. 1993
; Sutula et
al. 1998
; but see Okazaki et al. 1995
) and
suggest that there are predictable rules of granule cell axon growth
after epileptogenic injury.
In addition to changes in axon structure, there were changes in
dendrites. Granule cells in adult rats do not normally have basal
dendrites (Claiborne et al. 1990; Seress and
Pokorny 1981
). We were surprised to find that they occurred in
12% of the granule cells in kainate-induced epileptic rats. Recently,
it was reported that basal dendrites occur in a similar proportion of
granule cells in another experimental model of temporal lobe epilepsy (Spigelman et al. 1998
). Granule cells in immature rats
have basal dendrites (Seress and Pokorny 1981
),
suggesting the recent generation of granule cells with basal dendrites
in adult epileptic rats (Parent et al. 1997
). Basal dendrites normally
occur in some granule cells in nonepileptic adult humans (Seress
and Mrzljak 1987
), but they are reported to be more common in
patients with temporal lobe epilepsy (Franck et al.
1995
; but see Von Campe et al. 1997
). It is not
known presently if aberrant basal dendrites of granule cells in
epileptic tissue receive synaptic input from neighboring granule cells.
Possible functional effects of morphological changes in granule cells
Granule cell axon projections into the molecular layer and basal
dendrites that extend into the hilus offer possible avenues for
recurrent excitation that normally are rare or nonexistent. Previous
studies provide evidence for recurrent excitation between granule cells
in kainate-treated and kindled rats (Buckmaster and Dudek
1997a; Cronin et al. 1992
; Golarai and
Sutula 1996
; Masukawa et al. 1992
;
Patrylo and Dudek 1998
; Tauck and Nadler 1985
; Wuarin and Dudek 1996
). The finding of
late depolarizations in granule cells of epileptic but not control rats
is consistent with a recurrent excitatory mechanism. It is unlikely
that these depolarizations are due to intrinsic electrophysiological
properties of granule cells because those are similar in control and
kainate-induced epileptic rats. However, we cannot exclude the possible
contribution of differences in neuronal circuits outside of the dentate
gyrus (e.g., Du et al. 1995
) or alterations in receptor
pharmacology of granule cells (e.g., Mody and Heinemann
1987
). The late depolarizations in granule cells resemble
responses of CA3 pyramidal neurons (McKinney et al.
1997
) and layer V pyramidal neurons in neocortex (Prince and Tseng 1993
) after axon sprouting was induced in those
regions by epileptogenic treatments.
Whether there is enough recurrent excitation to lower seizure threshold
will depend in part on inhibitory control mechanisms (Miles and
Wong 1987). Previous studies suggest that recurrent inhibition
is enhanced after granule cell axon reorganization (Buckmaster
and Dudek 1997a
,b
; Milgram et al. 1991
). In the
present study, inhibition was quantified by evoking IPSPs with
standardized stimuli and measuring their conductance during the fast
and slow components. However, there are many different types of
inhibition [e.g., feedforward vs. feedback,
-aminobutyric acid-A
(GABAA)-receptor-mediated vs.
GABAB-receptor-mediated vs. non-GABA-receptor-mediated,
presynaptic vs. postsynaptic, etc.], and inhibition is a dynamic
process making it difficult to assess comprehensively. Nevertheless,
robust IPSPs in granule cells, found in all but one kainate-induced
epileptic rat, demonstrated strong inhibition. Prominent IPSPs occur in principal cells in other experimental models after axon sprouting is
induced by epileptogenic treatments (McKinney et al.
1997
; Prince and Tseng 1993
), and inhibition is
intact in tissue from many patients with temporal lobe epilepsy
(Colder et al. 1996
; Franck et al. 1995
;
Uruno et al. 1995
; Williamson et al.
1995
). Granule cell axon elongation, especially in the hilus
where inhibitory interneurons concentrate and where granule cells
normally synapse preferentially with interneurons (Acsády
et al. 1998
), could provide a mechanism for increasing
excitatory synaptic drive of interneurons, as has been shown in kindled
rats (Buhl et al. 1996
).
Epileptogenic injuries vary in severity, and the most severe injuries
not only induce granule cell axon sprouting but also kill inhibitory
interneurons. In those cases where a critical population of
interneurons is missing, inhibition of granule cells will be
compromised (Buckmaster and Dudek 1997b). The granule cell that responded to afferent stimulation with repetitive burst discharges and without an IPSP may demonstrate the effect of recurrent excitation between granule cells that is no longer masked by
inhibition, because blockade of synaptic inhibition of normal granule
cells does not result in repetitive burst discharges in response to afferent stimulation (Buckmaster and Dudek 1997a
;
Fricke and Prince 1984
). In other regions of the brain,
where epileptogenic treatments induce axon sprouting, pharmacological
blockade of inhibition results in similar burst discharges to afferent
stimulation (Hoffman et al. 1994
; McKinney et al.
1997
).
Summary
This study provides detailed data on the morphological changes of granule cells in kainate-induced epileptic rats. Measurements of the complete axon arbors of granule cells in vivo have confirmed results from previous in vitro studies. Changes in axonal and dendritic structure offer possibilities for the formation of novel, local, recurrent circuits within the dentate gyrus. Electrophysiological data are consistent with strong synaptic inhibition masking recurrent excitation between granule cells most of the time. These findings are similar to those from epileptogenic treatments in other brain regions and suggest that a general response of neurons to injury is to increase connectivity according to potentially predictable rules.
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ACKNOWLEDGMENTS |
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We are grateful to A. Kuhn for excellent technical assistance and Dr. Bret Smith for helpful comments on the manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-01778 and NS-16683. P. S. Buckmaster is a recipient of a Burroughs Wellcome Fund Career Award.
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FOOTNOTES |
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Address for reprint requests: P. S. Buckmaster, Dept. of Comparative Medicine, Stanford University School of Medicine, Stanford, CA 94305-5410.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 soley to indicate this fact.
Received 3 September 1998; accepted in final form 23 October 1998.
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REFERENCES |
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