that could increase excitability of the dentate gyrus. In pilocarpine-treated animals, the normal diffuse labeling of the ? subunit in the
chronic period. Interestingly, ? subunit labeling of many interneurons progressively increased after pilocarpine treatment. Consistent
with the observed changes in ? subunit labeling, physiological studies revealed increased excitability in the dentate gyrus of slices
obtained from the pilocarpine-treated mice and demonstrated that physiological concentrations of the neurosteroid tetrahydrodeoxy-
corticosterone were less effective in reducing excitability in the pilocarpine-treated animals than in controls. The findings support the
Studies of GABAergic inhibition in epilepsy have generally been
focused on synaptic events and synaptically localized GABAAre-
ceptor subunits. Yet, nonsynaptic GABAAreceptors could be
et al., 1996; Mody, 2001; Semyanov et al., 2004).
? Subunit-containing GABAAreceptors are critical mediators
of nonsynaptic inhibition in the dentate gyrus and are ideally
suited for this role because they have a particularly high affinity
for GABA and a slow rate of desensitization (Saxena and Mac-
appropriately positioned for such responses. The ? subunit is
localized exclusively at extrasynaptic sites in the cerebellum
aptic sites on granule cell dendrites in the dentate gyrus (Wei et
al., 2003). In both locations, ? subunit-containing receptors
could be activated by ambient levels of GABA in the neuropil or
by spillover of GABA after its synaptic release (Otis et al., 1991;
Rossi and Hamann, 1998; Wei et al., 2003).
to modulation by neurosteroids. Recent studies of cells express-
ing the ? subunit have demonstrated that the presence of the ?
subunit substantially increases the GABA receptors’ responses to
neurosteroids (Adkins et al., 2001; Belelli et al., 2002; Wohlfarth
et al., 2002). In vitro studies of the dentate gyrus have also dem-
onstrated a strong enhancement of tonic inhibition, mediated
by ? subunit-containing receptors, by tetrahydrodeoxy-
corticosterone (THDOC), a neuroactive metabolite of corti-
sone (Stell et al., 2003a).
Two other subunits, ?4 and ?2, are of particular interest be-
cause of their relationship to the ? subunit in the normal assem-
bly of GABAAreceptors. The ?4 subunit is considered a major
partner of the ? subunit in the forebrain (Sur et al., 1999),
the three subunits have been suggested by studies of ? subunit-
deficient mice, in which expression of the ?2 and ?4 subunits is
altered in precisely the same regions that normally express high
levels of the ? subunit (Tretter et al., 2001; Peng et al., 2002a).
bition and their potential involvement in epilepsy have led to the
This work was supported by National Institutes of Health Grant NS35985 to R. W. Olsen, I.M., and C.R.H. and
Lynn Fairbanks for assistance with the statistical analyses, and Mahsan Rafizadeh and John Feng for technical
Correspondence should be addressed to Carolyn R. Houser, Department of Neurobiology, CHS 73-235, David
TheJournalofNeuroscience,September29,2004 • 24(39):8629–8639 • 8629
present studies of ? subunit expression in a pilocarpine model of
acterize the changes in ? subunit expression after pilocarpine-
induced status epilepticus (SE), to determine whether ? subunit
changes were accompanied by altered expression of the ?4 and ?2
Animals and pilocarpine treatment. Young adult (6–8 weeks of age)
C57BL/6 male mice (20–27 gm; Harlan, Indianapolis, IN) were used in
this study. Sustained seizures were induced in experimental animals by
the administration of pilocarpine, a muscarinic cholinergic agonist, and
the injection protocols were similar to those used previously by our
group in rats (Obenaus et al., 1993; Esclapez and Houser, 1999). Thirty
minutes before pilocarpine administration, animals were injected with a
low dose of the cholinergic antagonist methyl scopolamine nitrate (1
mg/kg, i.p.) to reduce peripheral cholinergic effects. Animals in the ex-
perimental group then received an injection of pilocarpine hydrochlo-
ticus. Diazepam (5 mg/kg, i.p.; Abbott Laboratories, Chicago, IL) was
administered to the animals 3 hr after the onset of status epilepticus to
reduce behavioral seizures. Control animals received an identical series
of injections, except that the pilocarpine was replaced with a similar
volume of sterile saline. After the pilocarpine injection, experimental
length of the behavioral seizures.
All experimental animals that developed status epilepticus after pilo-
carpine administration were used in either the histological or electro-
physiological studies (n ? 44), and control mice were included in all
30, and 60 d after pilocarpine or control treatment. Electrophysiological
studies were performed on pilocarpine and control animals at 14–21 d
After recovery from status epilepticus, pilocarpine-treated mice were
videotaped to monitor the development and occurrence of spontaneous
seizures. For six mice, the videotape recordings began the day after pilo-
carpine injections and continued for 1 week. For the other animals, vid-
hr per session. In addition, the mice were videotaped during the 24 hr
before perfusion to determine whether spontaneous seizures occurred
during this period. All animal use protocols conformed to National In-
stitutes of Health guidelines and were approved by the University of
California, Los Angeles, Chancellor’s Animal Research Committee.
Behavioral outcomes. Spontaneous behavioral seizures were observed
in all but one of the animals that were used at survival times of 14 d or
longer after the initial period of status epilepticus (n ? 25 of 26). The
spontaneous seizures typically consisted of periods of freezing, clonic
movements of the forelimbs, rearing, or rearing and falling (stage 3–5
limbic seizures) (Racine, 1972), either followed or preceded by a brief
(10–20 sec) generalized motor seizure.
A subgroup of the mice was used for densitometry studies of the time
onset were of particular interest in this group. No behavioral seizures
(excluding occasional body jerks) were observed in the mice included in
the 4 and 7 d groups and in one of the mice in the 14 d group. Sponta-
neous motor seizures were documented in all other mice in the 14–60 d
mice were deeply anesthetized with sodium pentobarbital (90 mg/kg,
i.p.) and perfused through the ascending aorta with 4% paraformalde-
hyde in 0.12 M phosphate buffer, pH 7.3. At least two control and three
pilocarpine-treated mice were studied at each time point. After perfu-
from the skull and postfixed in the same fixative for 1 hr. After thorough
rinsing in phosphate buffer, the brains were cryoprotected in a 30%
sucrose solution, blocked in the coronal plane, frozen on dry ice, and
sectioned at 30 ?m on a cryostat. Forebrain sections containing the ros-
tral half of the hippocampus were sectioned in the coronal plane. Near
the middle of the hippocampus (?2.18 mm posterior to bregma)
(Franklin and Paxinos, 1997), the brain blocks were reoriented, and the
caudal half of the hippocampus was sectioned horizontally. Sections at
300 ?m intervals were mounted on slides and stained with cresyl violet
cryoprotectant solution at ?20°C until processing.
Antisera. Subunit-specific antisera that recognize the ?, ?4, and ?2
(Bencsits et al., 1999); and ?2 (319–366) (Tretter et al., 1997). The spec-
ificity of the affinity-purified antisera has been demonstrated previously
in immunochemical (Jechlinger et al., 1998) and immunohistochemical
provided by W. Sieghart (University of Vienna, Vienna, Austria).
synthetic peptide sequence ?1 (1–16) and was generously provided by
J.-M. Fritschy (University of Zurich, Zurich, Switzerland). The specific-
ity of the antiserum has been demonstrated previously (Fritschy and
tions were incubated in 1% H2O2for 30 min to reduce endogenous
peroxidase-like activity. After a rinse in 0.1 M Tris-buffered saline (TBS),
pH 7.3, the sections were processed with water bath antigen-retrieval
incubated in 0.05 M sodium citrate solution, pH 8.6, for 30 min at room
90°C for 70 min. The sections were allowed to cool at RT for 30 min and
then were rinsed in TBS.
avidin-biotin-peroxidase methods (Vectastain Elite ABC; Vector Labo-
ratories, Burlingame, CA). Sections were incubated in 10% normal goat
serum (NGS) in TBS containing 0.3% Triton X-100 and avidin (200
?l/ml) for 3–4 hr to reduce nonspecific binding. The sections were in-
cubated with primary antiserum (anti-?, 1:4000; anti-?4, 1:1500; anti-
?2, 1:2000) diluted in TBS containing 2% NGS and biotin (200 ?l/ml)
overnight at RT. After rinsing, the sections were incubated in biotinyl-
ated goat anti-rabbit IgG (1:1000) at RT for 1 hr. After a thorough rinse,
the sections were incubated in avidin-biotin-peroxidase complex (1:200
and 0.006% H2O2diluted in 0.075 M PBS for 10–15 min, and immuno-
for 30 sec. Other sections were processed with a glucose oxidase-DAB-
nickel method (Shu et al., 1988) to intensify the labeling. After rinsing,
sections were mounted on gelatin-coated slides, dehydrated, and
of ? and ?1 subunits. After water bath antigen-retrieval treatment, the
sections were treated with 10% NGS in TBS at RT for 1 hr and then
incubated in a solution containing guinea pig anti-?1 (1:50,000) and
rinsing in TBS, sections were incubated in a mixture of goat anti-guinea
pig IgG conjugated to Alexa Fluor 488 and goat anti-rabbit IgG labeled
with Alexa Fluor 594 (both 1:500; Molecular Probes, Eugene, OR) at RT
slides, and coverslipped with Prolong antifade medium (Molecular
Densitometric analyses. Expression levels of ?, ?4, and ?2 subunits in
control and pilocarpine-treated animals were evaluated with densitom-
etry to determine the extent and patterns of change over time. Sections
used for these analyses were obtained from three pilocarpine-treated
animals and two controls for each time point (1, 4, 7, 14, 30, and 60 d)
time points were processed identically in the same immunohistochemi-
cal experiment. Such experiments were repeated at least three times for
8630 • J.Neurosci.,September29,2004 • 24(39):8629–8639Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsy
each subunit to ensure the reliability of the results. Digital images of
immunolabeling in the dentate gyrus were obtained with a Zeiss Axio-
light levels. The densities of labeling were then analyzed with morpho-
metric AxioVision software (version 3.0; Zeiss).
in the molecular layer, the sections were photographed with a 10? ob-
jective, and gray level values were obtained from three rectangular areas
at middle of upper blade, apex, and middle of lower blade). Thus, six
measurements were obtained for each animal. All values were corrected
callosum in the same section.
The intensity of ? subunit labeling in interneurons in the molecular
and pilocarpine-treated animals at 1 month after pilocarpine treatment
(n ? 2 animals per group; two dentate gyri per animal). All ? subunit-
labeled interneurons with a well-defined nucleus were photographed
using a 100? objective. Gray level values were measured in three small
areas (2 ?m2) within the cytoplasmic regions of the interneurons. The
nucleus of each interneuron was selected as the background reference
region, and the gray level value in this region was subtracted from the
mean gray level value obtained for the cytoplasmic labeling of the same
The densitometry measurements for diffuse labeling in the molecular
layer were analyzed with a repeated measures ANOVA (general linear
model, including subject as a within subject factor) using SPSS software
(version 12.0; SPSS, Chicago, IL). Interneuron measurements were ana-
lyzed with Student’s t test. For all analyses, p ? 0.05 was considered
significant. Graphs were prepared with Origin 7.5 software (OrigenLab,
Extracellular field recordings. Mice were anesthetized with halothane
according to a protocol approved by the University of California, Los
Angeles Chancellor’s Animal Research Committee. The brains were re-
moved and placed in ice-cold artificial CSF (ACSF) containing the fol-
NaHCO3, 10 D-glucose, pH 7.3–7.4 when bubbled with 95% O2and 5%
CO2. In ACSF containing 5 ?M GABA, field potentials were evoked
(paired pulses 20 msec apart; 0.05 Hz) by stimulating the medial per-
forant path. Bipolar electrodes delivered a constant current stimulus
(A365; World Precision Instruments, Sarasota, FL). At a stimulus width
collected over a 10 min stable baseline. The W was then varied (PG4000;
Neurodata Instruments, New York, NY) to create stimulus–response
curves by delivering two stimulationtrials(10stimulieach),withWrang-
ing from 20 to 240 ?sec (in 20 and 40 ?sec increments). After the control
trial, THDOC (10 nM) was perfused for 20 min before generating a second
Data were filtered between 0.10 and 3 KHz, and an in-house data
analysis package (EVAN, version 1.3.9) was used to fit a straight line to
the initial rising phase of the excitatory postsynaptic field potential
(fEPSP). The slope of the line was then used to represent the magnitude
of the fEPSP and plotted against W to obtain stimulus–response curves.
Stimulus–response curves were fit to a Boltzman equation of the form
f(W) ? [?MAX/(1 ? exp[(W ? W50)/k]) ?
MAX], where W is stimulus width, MAX is the
maximum response relative to the response
elicited by the largest W (240 ?sec) under con-
trol conditions, k is a slope factor, and W50is
the stimulus width that elicits 50% of MAX
Differences were considered significant at p ?
0.05, as determined by Student’s t test.
Although C57/BL/6 mice are apparently
kainate-induced seizures (Schauwecker
and Steward, 1997), this strain is suscepti-
ble to cell loss after pilocarpine-induced
status epilepticus (Houser et al., 2002). In
the present mouse model, the patterns of
neuronal loss in the hippocampal forma-
tion were similar to those observed in
pilocarpine-treated rats (Obenaus et al.,
1993). Extensive neuronal loss was found
in the hilus and CA3 of all pilocarpine-
treated mice in this study. In contrast,
dentate granule cells were generally well
preserved. Neuronal loss in CA1 was vari-
able but did not appear to affect the pat-
terns of receptor subunit labeling in the
ing is abundant in several forebrain re-
gions that include many thalamic nuclei,
the caudate-putamen, outer layers of the
striking in the molecular layer (M) of the dentate gyrus in the pilocarpine-treated animal (B). Labeling is also moderately de-
creased in the neocortex (Cx) and slightly decreased in the caudate–putamen (CP) in this animal. No changes in ? subunit
Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsyJ.Neurosci.,September29,2004 • 24(39):8629–8639 • 8631
diffuse ? subunit labeling in the molecular layer of the dentate
labeling were also present in the cerebral cortex. No changes in ?
subunit immunoreactivity were evident in the thalamus (Fig.
1B), and this suggested that the decreased labeling in the dentate
gyrus was not a result of global decreases in ? subunit labeling in
molecular layer was evident in caudal as well as rostral regions of
the dentate gyrus (Fig. 1C,D). In some animals, the diffuse ?
subunit labeling was also decreased in CA1 and the entorhinal
cortex (Fig. 1D). However, decreases in diffuse ? subunit immu-
noreactivity were found most consistently in the dentate gyrus,
diffuse labeling was presumably located on dendrites of granule
cells in the molecular layer and around the cell bodies of these
neurons in the granule cell layer (Figs. 2A,B, 3A,B).
In control mice, some moderately to lightly labeled interneurons
mation (Fig. 2A). Such neurons were most noticeable along the
base of the granule cell layer, within or near the pyramidal cell
layer of CA1, and in stratum lacunosum-moleculare of CA1. In
pilocarpine-treated animals at 2 weeks or longer after status epi-
lepticus, the interneurons in these locations appeared more
strongly labeled than in control mice (Fig. 2B).
At higher magnification, ? subunit-labeled neurons could be
detected along the base of the granule cell layer in control ani-
mals, but most of these interneurons exhibited only moderate or
light labeling (Fig. 3A). A few moderately labeled interneurons
were also detected within the molecular layer (Fig. 3A). In
pilocarpine-treated animals at 2 weeks or longer after status epi-
cell bodies and proximal dendrites of the interneurons (Fig. 3B).
Greater numbers of strongly labeled interneurons were evident
along the base of the granule cell layer and within the molecular
layer than in these regions of control animals (Fig. 3A,B).
The interneurons in the molecular layer were of particular
induced status epilepticus. A, In a control mouse, diffuse ? subunit labeling is high in the
few ? subunit-labeled interneurons are present in the hilus (H). B, In a pilocarpine-treated
many interneurons (arrows) is increased. Strongly labeled interneurons are prominent along
ular layer (arrows) and along the base of the granule cell layer (arrowheads) where many
the proximal dendrites in many of the labeled interneurons. A decrease in diffuse labeling is
evident within both the molecular and granule cell layers in the pilocarpine-treated animal.
Comparison of ? subunit-labeled interneurons in the dentate gyrus of control
8632 • J.Neurosci.,September29,2004 • 24(39):8629–8639 Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsy
interest because they were difficult to detect in normal animals
but were quite evident in the pilocarpine-treated animals. To
verify that the increased visibility of these neurons was attribut-
able to increased ? subunit expression within the interneurons
rather than to decreased diffuse labeling within the neuropil, the
metric measurements demonstrated significantly stronger label-
ing in the cytoplasm of interneurons in the pilocarpine-treated
animals than in the control animals (control gray level value,
14.3 ? 1.0, n ? 13; pilocarpine gray level value, 27.1 ? 1.0, n ?
37; p ? 0.01). The number of labeled interneurons that could be
with increased ? subunit expression in many interneurons that
be visualized readily. Many of the labeled interneurons in the
molecular layer were probably molecular layer perforant path
(MOPP) cells that have extensive axonal arborizations in the
to modulate the effects of perforant path input through feedfor-
1993; Freund and Buzsaki, 1996).
(control gray level value, 18.6 ? 1.1, n ? 29; pilocarpine gray level
value, 38.2 ? 1.2, n ? 45; p ? 0.01). Many of these interneurons
cell bodies and proximal dendrites of the granule cells (Ribak and
In confocal microscopy studies of ? subunit labeling of inter-
neurons in the dentate molecular layer, the neurons were inde-
pendently identified by immunohisto-
chemical labeling of the ?1 subunit of the
in these neurons was then compared in
control and pilocarpine-treated animals.
Distinct ?1 subunit labeling of many in-
terneurons was present in control mice,
and similar labeling was observed in the
pilocarpine-treated animals (Fig. 4A,D)
(Z.P. and C.R.H., unpublished findings).
In control mice, ?1 subunit-labeled inter-
neurons in the molecular layer showed a
low level of ? subunit labeling within the
cytoplasm (Fig. 4B). In contrast, virtually
all ?1 subunit-labeled interneurons in the
molecular layer of pilocarpine-treated an-
imals exhibited strong ? subunit labeling
within the cytoplasm (Fig. 4E). Labeling
in the pilocarpine-treated animals, suggest-
ing that the ? subunit was increased along
the plasma membrane as well as within the
Interestingly, interneurons in the
dentate gyrus did not appear to be la-
beled for ?4 in sections from either ex-
perimental or control animals. This
observation and the frequent finding of
double labeling of interneurons for the
?1 and ? subunits raise the possibility
that the ? subunit may be associated predominantly with the
?4 subunit in principal cells, as is generally recognized, but
with the ?1 subunit in interneurons.
To determine the time course of the ? subunit changes, sections
from animals at several time points between 1 and 60 d were
processed in parallel in the same immunohistochemical experi-
ments. By 24 hr after status epilepticus, little change in diffuse ?
subunit labeling was evident within the molecular layer (Fig. 5,
compare A and B). However, at this early time point, labeling of
interneurons along the base of the granule cell layer appeared to
be decreased (Fig. 5B). At 4 d after status epilepticus, diffuse
labeling in the molecular layer was decreased below control lev-
els, and labeling of interneurons in the dentate gyrus also re-
mained decreased (Fig. 5C). At 1 week after status epilepticus,
diffuse labeling in the molecular layer was lower, but the ?
subunit-labeled interneurons along the base of the granule cell
layer and within the molecular layer were more evident than at
earlier time points (4 d) or in control tissue (Fig. 5D). At 14, 30,
and 60 d after pilocarpine-induced seizures, diffuse labeling in
the molecular layer remained substantially lower than that in
control animals, and strongly labeled interneurons continued to
be observed at these later time points (Fig. 5E,F).
The identification of progressive decreases in diffuse ? subunit
labeling over time led to questions about changes in potentially
interrelated GABAAreceptor subunits during the same period.
the same groups of animals and at the same time points as those
used in the ? subunit studies.
relatively low in the cytoplasm of the interneuron (B, arrow), and no ? subunit labeling can be detected on the surface of the
interneuron (C). D–F, In a pilocarpine-treated animal, distinct ?1 subunit labeling is evident on the surface of interneurons
Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsyJ.Neurosci.,September29,2004 • 24(39):8629–8639 • 8633
In control animals, diffuse ?4 subunit labeling was moder-
ately high throughout the molecular layer of the dentate gyrus
(Fig. 6A). In pilocarpine-treated animals, ?4 labeling was de-
creased by 24 hr and appeared even lower at 4 d after status
epilepticus (Fig. 6B). By 7 d after pilocarpine-induced seizures,
?4 labeling was only slightly lower than that in control animals.
in the molecular layer was higher than that in control animals
Immunolabeling of the ?2 subunit was distributed through-
dendritic regions of CA1. In pilocarpine-treated animals, little
change in the level of labeling was evident at 24 hr, and only a
d after status epilepticus, increased ?2 subunit labeling was ob-
served throughout the hippocampal formation. The increases
(Fig. 6F). Increased immunolabeling of the ?2 subunit was ob-
extent of the increase varied among animals at the same poststa-
comparatively large increase in ?2 subunit expression.
Densitometry measurements were conducted to provide a semi-
subunits over time. The results were consistent with the qualitative
analysis of each subunit, and similar results were obtained among
The density of immunolabeling for the ? subunit was signifi-
cantly decreased by 4 d after status epilepticus and continued to
be lower than control values at all later time points. Differences
were statistically significant at all times after 1 d (Fig. 7A).
Density measurements of ?4 subunit labeling confirmed an
initial decrease in the intensity of ?4 labeling in pilocarpine-
treated animals compared with their paired controls at 1 and 4 d
after status epilepticus (Fig. 7B). Differences were less marked at
7 d, suggesting a return toward control values (Fig. 7B). The
intensity of labeling in the pilocarpine-treated animals was in-
creased above control levels at 2 weeks, and the increased inten-
sity of labeling continued to be present at 30 and 60 d after status
epilepticus. Differences were statistically significant at 1 d ( p ?
0.05) and 30 d ( p ? 0.01).
Density measurements of ?2 subunit labeling showed mild to
moderate increases in labeling over time in the pilocarpine-
treated animals. No significant differences were found at 1 and
4 d after status epilepticus (Fig. 7C). The density of ?2 subunit
labeling in pilocarpine-treated animals was higher than that of
paired control animals at all later times, from 7 to 60 d (Fig. 7C).
The differences were statistically significant at 60 d ( p ? 0.05).
To facilitate comparisons of the patterns of change for the
three subunits over time, densitometric values from the
pilocarpine-treated animals were converted to percentages of
by 100%. The patterns are described graphically in Figure 8.
In summary, the density of ? subunit labeling decreased in
pilocarpine-treated mice and remained significantly lower than
control values from 4 to 60 d after status epilepticus. Although
labeling for the ?4 subunit decreased initially, it then increased
above control levels and remained increased from 7 to 60 d after
pilocarpine treatment. The ?2 subunit showed a general, al-
though comparatively small, increase above control values that
epilepticus, the latest time point studied, ? subunit labeling was
decreased by 38.4%, whereas the labeling for the ?4 and ?2 sub-
units was increased by 29.7 and 19.2%, respectively (Table 1).
is necessary for modulation of dentate gyrus granule cells by
the decreased expression of ? subunit-containing receptors in
granule cells of pilocarpine-treated mice should result in de-
creased neurosteroid modulation of these cells. To test this,
evoked fEPSPs were used to generate stimulus–response curves
before and after application of 10 nM THDOC to slices from
control and pilocarpine-treated mice. After application of the
153.8 ? 12.0 ?sec; n ? 5; p ? 0.05) (Fig. 9A), indicating that
THDOC decreased the excitability of dentate gyrus granule cells
in control mice (consistent with Stell et al., 2003a). However, the
stimulus–response curves generated in slices from pilocarpine-
treated animals were unaffected by THDOC (pilocarpine W50,
122.1 ? 4.4 ?sec vs pilocarpine plus THDOC W50, 117.9 ? 5.0
?sec, n ? 6; p ? 0.05) (Fig. 9A). Furthermore, analysis of stimu-
lus–response curves from both the control and pilocarpine-
treated animals under control conditions indicated that slices
ally decreased (B, C). At 7 d (D), 30 d (E), and 60 d (F) after status epilepticus, the diffuse ?
Progressive changes in immunohistochemical labeling of the ? subunit in the
8634 • J.Neurosci.,September29,2004 • 24(39):8629–8639 Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsy
from control animals were less excitable than slices from
pilocarpine-treated animals (control W50, 143.9 ? 8.3 ?sec vs
iological studies confirmed lower levels of ? subunit labeling of
the dentate molecular layer in all pilocarpine-treated animals
than in their paired controls. The physiological findings indicate
that the decrease in ? subunit-containing receptors in
pilocarpine-treated mice substantially reduced the neurosteroid
to the increase in general excitability of this region.
Our major finding is that expression of the ? subunit of the
GABAAreceptor was altered in ways that could contribute to
increased excitability of the dentate gyrus in a mouse model of
temporal lobe epilepsy. Diffuse labeling of the ? subunit on den-
drites of dentate gyrus granule cells was decreased, whereas ?
subunit expression in interneurons was increased during the
gyrus was increased in extracellular field re-
excitability in the pilocarpine-treated ani-
The findings are unique in the follow-
ing ways: (1) focusing attention on
GABAAreceptor subunit alterations in-
volved in nonsynaptic, tonic inhibition;
(2) demonstrating differential changes in
GABAAreceptor subunit expression in
principal cells and interneurons; and (3)
suggesting that the observed subunit
changes can limit the effectiveness of
neurosteroids in enhancing inhibition in
an epilepsy model.
A decrease in diffuse labeling of the ? sub-
unit in the dentate molecular layer oc-
riod. These results are in agreement with
some but not all previous reports. De-
and protein was found at several intervals
from 12 hr to 30 d after kainate-induced
seizures in rats (Schwarzer et al., 1997; Tsu-
croarray analysis identified the ? subunit
In contrast, single-cell mRNA amplifi-
cation methods in dissociated granule
subunit mRNA during the chronic period
in the rat pilocarpine model (Brooks-
Kayal et al., 1998). Currently, there is no
explanation for the differences between
study and other previous reports, but the
discrepancies do not appear to be attributable to major variables
such as the animal model or species.
Interestingly, mutations of the gene for the ? subunit of the
forms of generalized epilepsy (Dibbens et al., 2004). Recombi-
nant receptors with at least one of the identified mutations have
decreased GABAAreceptor current amplitudes, suggesting that
the mutated ? subunit could contribute to increased neuronal
excitability (Dibbens et al., 2004).
Although the ? subunit is the major subunit associated with
tonic inhibition in the dentate gyrus, the ?5 subunit may be
involved in nonsynaptic inhibition in CA1 (Crestani et al., 2002;
Caraiscos et al., 2004), and decreased labeling of the ?5 subunit
mRNA and protein in CA1 was previously observed in a rat pilo-
preserved (Houser and Esclapez, 2003). The decreased expres-
sion of these two putative nonsynaptic subunits contrasts with
the more frequent finding of increased expression of other
the molecular layer is increased and is stronger than that in the control mouse (compare F and D). Illustrated sections were
Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsy J.Neurosci.,September29,2004 • 24(39):8629–8639 • 8635
GABAAreceptor subunits in several epilepsy models (Schwarzer
et al., 1997; Tsunashima et al., 1997; Brooks-Kayal et al., 1998;
Nusser et al., 1998a; Fritschy et al., 1999) and human temporal
lobe epilepsy (Loup et al., 2000).
In this study, ? subunit expression was differentially altered in
granule cells and interneurons, and we hypothesize that these
changes could converge to increase excitability in this mouse
model of temporal lobe epilepsy.
presumably at perisynaptic and extrasynaptic locations, could
lead to reduced responsiveness to GABA spillover or a reduction
in tonic inhibition. Such alterations could directly reduce the
effectiveness of the dentate “gate” that normally limits the
amount of excitatory input that enters the hippocampus (Loth-
effective in allowing excitation through the perforant path to
invade the hippocampus (Carlson et al., 2003).
Increased expression of ? subunits in interneurons could also
have powerful effects on excitability within the dentate gyrus if
the changes were to increase the tonic inhibition of inhibitory
tonic GABAAreceptor conductances in interneurons in CA1 of
the normal guinea pig hippocampus and have emphasized the
potential importance of such inhibition in regulating excitability
within the hippocampus (Semyanov et al., 2003, 2004). Strong
tonic inhibition has also been found in some interneurons in the
dentate gyrus of normal mice (W. Wei and I.M., unpublished
findings), and studies of tonic inhibition in interneurons of
pilocarpine-treated mice are planned.
rons that most likely included MOPP cells in the molecular layer
and basket cells along the base of the granule cell layer. Increased
tonic inhibition of these and other interneurons throughout the
of the perforant path. Both functional changes could compro-
mise inhibitory control of the principal cells.
The decreased responsiveness of the dentate gyrus to neuroste-
roid modulation in the pilocarpine-treated mice strongly sug-
gests that the altered ? subunit expression has functional conse-
quences. In the normal dentate gyrus, the enhancement of tonic
inhibition by physiological concentrations of neurosteroids is
mediated primarily by ? subunit-containing GABAAreceptors
(Stell et al., 2003a). Accordingly, physiological concentrations of
THDOC decreased the excitability of the
dentate gyrus in control animals but were
essentially ineffective in reducing excit-
ability in slices from the pilocarpine-
These findings are consistent with a
previous report of diminished allopreg-
nanolone sensitivity of GABAAreceptor
currents in acutely isolated granule cells
from chronically epileptic rats (Mtch-
edlishvili et al., 2001). At the time of the
study, the relationship between the ? sub-
but it now appears quite possible that a
decrease in ? subunit expression could
have been responsible for the reduced re-
sponse to the neurosteroid.
In the current study, decreased labeling of
the ? subunit was accompanied by in-
creased expression of the ?4 and ?2 sub-
pvalue Control Pilo
ticus. Control values are represented as 100% (dotted line) for all subunits. Intensity of ?
subunit labeling is below control values at 4–60 d after status epilepticus. In contrast, after
8636 • J.Neurosci.,September29,2004 • 24(39):8629–8639Pengetal.•GABAAReceptor?SubunitAlterationsinEpilepsy
units. Similar increases in ?4 and ?2 subunit expression have
been observed in other temporal lobe epilepsy models. In
kainate-treated rats, changes in these subunits as well as the ?
or ?4 subunit (Brooks-Kayal et al., 1998; Fritschy et al., 1999).
Similar, potentially interrelated changes in the ?, ?4, and ?2
subunits have also been observed in a chronic, intermittent, eth-
anol model of alcohol withdrawal (Cagetti et al., 2003) that ex-
hibits decreased inhibition and a decreased threshold for penty-
lenetetrazol seizures (Kokka et al., 1993; Kang et al., 1998; Liang
?2 subunit expression may predominate in several animal mod-
els with increased seizure susceptibility.
with each other for partnership with the ?4 subunit in the fore-
brain and the ?6 subunit in the cerebellum (Tretter et al., 2001;
Peng et al., 2002a). In our epilepsy model, ? subunit expression
decreased progressively in the days after status epilepticus,
whereas, after initial mild decreases, the ?4 and ?2 subunits in-
creased over the same time course. Such patterns are consistent
with the previously proposed model of subunit assembly and
with altered subunit composition of some GABAAreceptors in
epilepsy (Coulter, 2001; Fritschy and Bru ¨nig, 2003).
suggestion. Significant changes in ? subunit expression were not
observed at 24 hr but developed during the first 2 weeks after
status epilepticus. Importantly, the ? subunit changes developed
before the occurrence of spontaneous behavioral seizures, con-
sistent with the ? subunit changes contributing to (rather than
resulting from) the development of spontaneous seizures.
targets for alterations that could contribute to epilepsy, because
2002; Stell et al., 2003a), ethanol (Wallner et al., 2003), and pH
principal cells could lead to less effective enhancement of inhibi-
tion by such modulators. Furthermore, alterations in the normal
dynamic regulation of ? subunit-containing receptors by these
modulators could contribute to the fluctuations in seizure sus-
ceptibility that characterize many forms of epilepsy.
Additional studies are necessary to demonstrate directly that
tonic GABAergic inhibition is decreased in this epilepsy model
and that such deficits are not compensated for by other GABAA
receptors or non-GABAergic mechanisms, such as occurs in the
2001). However, the present findings support the enticing possi-
bility that alterations in subunits associated with nonsynaptic
inhibition may contribute to temporal lobe epilepsy, despite
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