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The Journal of Neuroscience, July 15, 2000, 20(14):5401-5419
Selective Alterations in GABAA Receptor Subtypes in
Human Temporal Lobe Epilepsy
Fabienne
Loup1,
Heinz-Gregor
Wieser2,
Yasuhiro
Yonekawa3,
Adriano
Aguzzi4, and
Jean-Marc
Fritschy1
1 Institute of Pharmacology, University of Zurich, and
Departments of 2 Neurology, 3 Neurosurgery, and
4 Neuropathology, University Hospital Zurich, 8057 Zurich,
Switzerland
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ABSTRACT |
Temporal lobe epilepsy (TLE) is associated with impaired inhibitory
neurotransmission. Studies in animal models suggest that GABAA receptor dysfunction contributes to epileptogenesis.
To understand the mechanisms underlying TLE in humans, it is
fundamental to determine whether and how GABAA receptor
subtypes are altered. Furthermore, identifying novel receptor targets
is a prerequisite for developing selective antiepileptic drugs. We have
therefore analyzed subunit composition and distribution of the three
major GABAA receptor subtypes immunohistochemically with
subunit-specific antibodies ( 1, 2, 3, 2,3, and 2) in
surgical specimens from TLE patients with hippocampal sclerosis
(n = 16). Profound alterations in GABAA
receptor subtype expression were observed when compared with control
hippocampi (n = 10). Although decreased
GABAA receptor subunit staining, reflecting cell loss, was
observed in CA1, CA3, and hilus, the distinct neuron-specific
expression pattern of the -subunit variants observed in controls was
markedly changed in surviving neurons. In granule cells, prominent
upregulation mainly of the 2-subunit was seen on somata and apical
dendrites with reduced labeling on basal dendrites. In CA2,
differential rearrangement of all three -subunits occurred.
Moreover, there was layer-specific loss of 1-subunit-immunoreactive
interneurons in hippocampus proper, whereas surviving interneurons
exhibited extensive changes in dendritic morphology. Throughout,
expression patterns of 2,3- and 2-subunits largely followed those
of -subunit variants. These results demonstrate unique
subtype-specific expression of GABAA receptors in human
hippocampus. The significant reorganization of distinct receptor
subtypes in surviving hippocampal neurons of TLE patients with
hippocampal sclerosis underlines the potential for synaptic
plasticity in the human GABA system.
Key words:
human epilepsy; GABAA receptor; dentate
gyrus; hilus; CA2; pyramidal cells; granule cells; interneurons
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INTRODUCTION |
Fast synaptic inhibition in the
vertebrate CNS is mediated primarily by the neurotransmitter
GABA interacting with the GABAA class of
receptors. Impaired GABA transmission can lead to neuronal hyperexcitability, a condition associated with epileptogenesis. Furthermore, antiepileptic agents that enhance
GABAA receptor function are effective in treating
seizures (Olsen et al., 1999 ).
Temporal lobe epilepsy (TLE) is the most common adult seizure disorder
and, when associated with hippocampal sclerosis (HS), is the most
refractory to pharmacotherapy (Engel, 1998 ; Semah et al., 1998 ).
Evidence from human studies suggests that postsynaptic GABAA receptors contribute to the pathophysiology
of TLE with HS. Thus, alterations in GABAA
receptor function and in their allosteric modulation by ligands of the
benzodiazepine-binding site were documented in hippocampal neurons from
HS patients (Franck et al., 1995 ; Williamson et al., 1995 , 1999 ;
Isokawa, 1996 ; Shumate et al., 1998 ; Brooks-Kayal et al., 1999 ).
Furthermore, in vivo imaging and autoradiographic studies
demonstrated a decrease in central benzodiazepine receptor binding
primarily attributed to extensive cell death in the sclerotic
hippocampus of TLE patients (Olsen et al., 1992 ; Burdette et al., 1995 ;
Koepp et al., 1996 ) and additional reduction of receptor density per
remaining neuron in CA1 (Johnson et al., 1992 ; Hand et al., 1997 ; Koepp
et al., 1998 ).
The recognition of multiple GABAA receptor
subtypes and their differential expression in distinct neuronal
populations (Fritschy and Möhler, 1995 ; McKernan and Whiting,
1996 ; Sperk et al., 1997 ) permits a target-oriented analysis of
GABAA receptor dysfunction in human TLE.
GABAA receptor heterogeneity results from the
combinatorial assembly of a multitude of subunit variants encoded by at
least 20 genes ( 1-6, 1-4, 1-3, , , , , and
1-3) (Möhler et al., 1996 ; Barnard et al., 1998 ; Whiting et
al., 1999 ). If expression of specific GABAA
receptor subtypes is altered in TLE, their identification would
facilitate the development of more selective antiepileptic drugs and
ligands for in vivo imaging. Current approaches, however, have not provided the degree of spatial resolution and specificity required to characterize individual receptor subtypes and their subunit-specific alterations in human brain. Although recent studies in
animal models of TLE reported changes in the number and subunit composition of GABAA receptors in hippocampal
neurons (Schwarzer et al., 1997 ; Brooks-Kayal et al., 1998 ; Nusser et
al., 1998 ; Bouilleret et al., 2000 ), the validity of these data as
pertains to human TLE remains to be established.
In the present study, we investigated alterations in subunit
architecture and localization of GABAA receptor
subtypes in hippocampi resected from TLE patients with HS and compared
these with control tissue obtained at autopsy and with specimens from
TLE patients without HS. A protocol based on microwave irradiation and
tyramide signal amplification was used to visualize the major
GABAA receptor subunits 1, 2, 3, 2,3,
and 2 in human brain tissue using subunit-specific antisera (Loup et
al., 1998 ).
Parts of this paper have been published previously as abstracts (Loup
et al., 1997 , 1999 ).
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MATERIALS AND METHODS |
Patient selection. Twenty-one patients (mean age,
34.9 ± 2.5 years; range, 15-56 years) undergoing surgery for
medically intractable TLE were included in this study. All procedures
were performed with consent of the patients and were approved by the
Ethics Committee of the University Hospital Zurich in accordance with
the Helsinki Declaration of 1975. Presurgical assessment consisted of
detailed history and neurological examination, semi-invasive EEG
monitoring with foramen ovale electrodes, and neuropsychological
testing. Neuroradiological studies included for all cases
high-resolution magnetic resonance imaging (MRI) with special protocols
to visualize the hippocampal formation and positron emission tomography
(PET) with 18fluoro-2-deoxyglucose.
Intraoperative electrocorticography was performed in all subjects to
further characterize the epileptogenic zone to be resected. Based on
clinical, EEG, neuroimaging, and neuropathological data, patients were
classified into those with HS (n = 16; mean age,
36.9 ± 2.7 years; range, 17-56 years) and those without HS
(non-HS; n = 5; mean age, 28.2 ± 5.6 years;
range, 15-44 years) (Wieser et al., 1993 ). Patients from the HS group underwent selective amygdalohippocampectomy consisting in resection of
amygdala, hippocampal formation, and parahippocampal gyrus (Wieser and
Yasargil, 1982 ). The HS group included patients with severe damage to
the hippocampus as assessed by MRI and PET studies, with the
epileptogenic focus localized to the mesial temporal region, and, in
most cases, with a history of seizures from childhood. The origin of
TLE in the non-HS patients was attributed to a tumor (n = 3) or vascular malformation (n = 1) identified by
neuroimaging in an extrahippocampal location and confirmed
histopathologically, and in one case no lesion was apparent. In these
patients, the surgical procedure consisted of amygdalohippocampectomy
alone or with resection of the epileptogenic lesion or part of temporal lobe. For comparison, hippocampi from five subjects (mean age, 58 ± 6.2 years; range, 40-73 years) with no history of neurological or
psychiatric disorder were collected at autopsy between 8 and 16 hr
after death (mean postmortem interval, 11.2 ± 1.3 hr).
Hippocampal tissue was also obtained from a 56-yr-old patient with
known TLE and HS with a postmortem interval of 15 hr, who had died from multiorgan failure.
Tissue preparation. Hippocampal specimens obtained at
surgery or autopsy were cut into 7- to 12-mm-thick blocks transversely to the long axis. In surgical TLE cases, the rostrocaudal extent of the
hippocampus available for study ranged from 7.2 to 17.6 mm for HS
specimens (mean length, 12.6 ± 0.75 mm) and from 8 to 12 mm for
non-HS specimens (mean length, 9.6 ± 0.72 mm). For all specimens,
the sampled part of hippocampus comprised rostral and middle
levels. When tissue was obtained at autopsy, the whole hippocampus was
collected. After rinsing in PBS at pH 7.4 immediately on
resection in the operating room or after dissection at autopsy, tissue
blocks were immersion-fixed for 6-8 hr at 4°C under constant agitation in a mixture of 4% freshly dissolved paraformaldehyde and
15% saturated picric acid in 0.15 M phosphate buffer at pH 7.4 (Somogyi and Takagi, 1982 ). After fixation, tissue blocks were
pretreated using a modified antigen-retrieval method based on microwave
irradiation as described previously (Fritschy et al., 1998 ) and adapted
to human tissue (for details, see Loup et al., 1998 ). Tissue blocks
were cryoprotected in 10, 20, and 30% sucrose in PBS over a period of
3-4 d, frozen at 28°C in isopentane, and stored at 80°C.
Subsequently, series of 40-µm-thick sections were cut in a cryostat
and collected in ice-cold PBS. They were then either processed for
immunohistochemistry (see below) or transferred to antifreeze solution
[50 mM phosphate buffer, 15% sucrose, 30% (v/v) ethylene
glycol, and sodium azide, pH 7.4] and stored at 20°C until use.
This procedure allowed 10-12 different specimens, including tissue
from autopsies, to be processed in parallel during the same session.
Staining for the GABAA receptor subunits was
performed on consecutive sections (five series), and the space between
sections from one series was between 720 and 800 µm. An additional
adjacent series of sections was Nissl-stained for histopathological examination.
Immunohistochemistry. The following subunit-specific
antibodies were used: mouse monoclonal antibodies bd-24 and bd-17
recognizing the human GABAA receptor 1-subunit
and both the 2- and 3-subunits, respectively (Schoch et al.,
1985 ; Ewert et al., 1990 ), and polyclonal guinea pig antisera
recognizing the 2-, 3- and 2-subunits (Loup et al., 1998 ). The
preparation and characterization of the polyclonal antibodies, raised
against synthetic peptides derived from rat cDNA sequences, have been
described (for details, see Fritschy and Möhler, 1995 ). For each
subunit, the amino acid sequences used as antigens were found to be
identical in rat and human cDNAs, and the high specificity of the
GABAA receptor subunit antibodies in human brain
tissue was verified with Western blot analysis (Waldvogel et al.,
1999 ). Free-floating sections were preincubated in 1.5%
H2O2 in PBS for 10 min at
room temperature to block endogenous peroxidase activity. They were
then washed three times for 10 min in PBS and processed for
immunoperoxidase staining (Hsu et al., 1981 ) as described previously
(Loup et al., 1998 ). The dilutions of the antibodies were: 1-subunit
(monoclonal antibody bd-24), 0.2 µg/ml; 2-subunit
(affinity-purified), 1.3 µg/ml; 3-subunit (affinity-purified), 1.8 µg/ml; 2,3-subunit (monoclonal antibody bd-17), 3.8 µg/ml; and
2-subunit (crude serum), 1:1500. Control experiments for staining
specificity included the replacement of primary antibodies with
nonimmune serum and preadsorption of the antibodies with 3-5 µg/ml
of their respective peptide antigen (Fritschy and Möhler, 1995 ;
Loup et al., 1998 ). No specific staining was seen in either case. To
assess the subcellular localization of GABAA
receptor subunits in individual neurons with confocal laser-scanning
microscopy, immunofluorescence staining with tyramide signal
amplification (TSA kit; NEN Life Science Products, Brüssels, Belgium) was performed (Loup et al., 1998 ).
Data analysis. Sections were analyzed with a Zeiss (Jena,
Germany) Axioplan microscope equipped for bright-field and
epifluorescence microscopy. Photomicrographs were taken with Eastman
Kodak (Rochester, NY) T-max 100 film. Sections processed for
immunofluorescence staining were also analyzed by confocal
laser-scanning microscopy (TCS 4D; Leica, Heidelberg, Germany) using
Imaris software (Bitplane, Zurich, Switzerland) for image processing.
Nomenclature. The nomenclature of Lorente de Nó (1934)
was used except for the hilar region where the classification proposed by Amaral (1978) and later described for the human hippocampus was
adopted (Amaral and Insausti, 1990 ). Accordingly, the portion of the
pyramidal layer that inserts into the dentate hilus (CA3c and/or CA4 in
the terminology of Lorente de Nó, 1934 ) is referred to as CA3.
Neuron counts. Nissl-stained sections from all specimens
were examined to assess the presence of pathological alterations in
general and the degree of hippocampal sclerosis in epileptic samples in
particular. In addition, neurons were counted in the regions where
densitometric measurements of GABAA receptor
subunit staining intensity were obtained, using immediately adjacent
Nissl-stained sections from autopsy (n = 5), non-HS
(n = 5), and HS cases (n = 16).
Nucleolar profiles of CA2 pyramidal cells and nuclear profiles of
granule cells were counted within a 12.5 × 12.5 mm ocular grid consisting of 10 × 10 squares at a magnification of 400 and
1000×, respectively. Four to six measurements per region and section were averaged where all nuclei visible in the section were counted except for those touching the top and right edges of the grid. Typically, in autopsy and non-HS cases, the width of the granule cell
layer was included in a single counted field (125 × 125 µm area), whereas in HS cases with granule cell dispersion, two or sometimes three fields were necessary to include all granule cells. To
account for this factor, granule cells were also counted within a
125-µm-wide column through the granule cell and molecular layers (Houser, 1990 ). Moreover, in some HS cases, granule cells displayed elongated cell bodies that appeared larger. Therefore, no
assumption-based methods were used, and the results were expressed as
mean number of neuronal profiles per square millimeter and
additionally, for granule cells, per column. The counts were not
performed in a stereological manner, because the intention was to
represent the distribution of these neurons in relative terms suited
for intergroup comparison and that such procedures are not amenable to
surgically collected tissue where random sampling is difficult and
tissue volume changes caused by hippocampal sclerosis cannot be estimated.
The numbers of interneurons immunoreactive for the 1-subunit were
assessed in the CA2 and CA3 areas using the grid procedure described
above. Counts were done at a magnification of 200× in strata
pyramidale, radiatum, and lacunosum-moleculare, and additionally for
CA3, in stratum lucidum. While strata pyramidale, lacunosum-moleculare, and lucidum did not show signs of major atrophy in HS cases as measured
by their respective widths, stratum radiatum had undergone variable and
at times severe reduction. For this reason and because the borders
between strata were readily delimited, counts were obtained within an
area consisting of 625 µm × the thickness of the stratum where
the cells were counted. Two to three counts per stratum and region were
made in two sections from each specimen. In a few surgical cases,
mostly non-HS, it was not possible to obtain 1-subunit-positive
interneuron counts in certain CA2 or CA3 strata because of damage
during the resection procedure.
Densitometric measurements. Sets of six adjacent sections
were considered for each case analyzed: one Nissl-stained and five immunostained sections. The intensity of labeling for the
GABAA receptor subunits 1, 2, 3, 2,3,
and 2 was measured by densitometry in sections from autopsy
(n = 5), non-HS (n = 3), and HS cases (n = 14) at equivalent midrostrocaudal levels of the
hippocampus. Sections were imaged using a high-resolution video camera
(1280 × 1024 pixels) interfaced with a Zeiss microscope under a
10× objective. Before conducting the measurements, the
computer-assisted imaging device MCID M5 (Imaging Research, St.
Catherines, Ontario, Canada) was calibrated with a set of neutral
density filters (Eastman Kodak) to automatically convert gray levels to
optical density values. Illumination and filter settings were
maintained at the same level for image acquisition and densitometric
analysis for all specimens. Optical density (OD) measurements were
recorded in the CA2 area and the dentate gyrus. For CA2 analysis, two
rectangles with an area of 68,600 µm2
each were used to measure the average OD per section in the pyramidal cell layer. In the dentate gyrus, the average OD was calculated from
measurements in five circles with an area of 1020 µm2 each in the granule cell layer, the
inner molecular layer, the outer molecular layer, and the subgranular
layer in both the upper and lower blades. Because the epileptic samples
contained no region where staining for one or more of the
GABAA receptor subunits was judged to be
unequivocally unaffected, the results were not normalized to a
reference value. Variability was greatly minimized, however, by the
highly standardized tissue preparation protocol and the
immunohistochemical processing in parallel of 10-12 different samples
from autopsy, non-HS, and HS cases.
Statistical analysis. Neuron counts and densitometric
measurements were analyzed for statistical significance using the
Kruskal-Wallis test (nonparametric ANOVA; InStat program). Data
were further compared between individual groups (at p < 0.05) with a multiple comparisons test.
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RESULTS |
Patient history
Medical records from all seizure patients were reviewed for
relevant clinical parameters. Whereas no significant difference was
found for the age at surgery (HS group: 36.9 ± 2.7 years, range,
17-56 years, n = 16; non-HS group: 28.2 ± 5.6 years, range, 15-44 years, n = 5), seizures had lasted
longer and usually started earlier in HS patients compared with non-HS
patients (duration of the epilepsy, defined as the interval between
onset of habitual seizures and surgical treatment: 28 ± 2.7 years, range, 12-44 years vs 10.4 ± 4.4 years, range, 2-26
years; age at seizure onset: 8.9 ± 1.6 years, range, 1-22 years
vs 17.8 ± 4.8 years, range, 9-30 years). A history of an initial
precipitating injury (Mathern et al., 1995a ), was documented in 11 patients in the HS group and none in the non-HS group. These events
included febrile convulsions (n = 6), infantile
meningitis/encephalitis (n = 3), and neonatal anoxia/ischemia (n = 2). Preoperatively HS and non-HS
patients were on similar antiepileptic medication. Seizure control was assessed in all surgical cases (mean follow-up period of 24 ± 1.1 months). Postoperatively, 12 patients in the HS group (75%) were
seizure-free (class 1, as classified after Engel, 1987 ), two had rare
seizures (class 2), and two patients had worthwhile improvement (class
3). In the non-HS group, four patients were seizure-free, and one
patient was unchanged. The high success rate in achieving seizure
control indicates that the epileptogenic focus was successfully removed
with selective amygdalohippocampectomy.
Comparison of patient categories
The distribution of the GABAA receptor
subunits 1, 2, 3, 2,3, and 2 was analyzed in hippocampal
specimens from TLE patients with HS (n = 16). For
comparison, two control groups were used and defined as hippocampal
tissue obtained at autopsy from patients with no evidence of
neurological disease (n = 5) and tissue surgically removed from patients with TLE, but without HS (n = 5).
Both were indistinguishable on the basis of morphological criteria
except for at most mild neuronal loss in TLE without HS, as reported in
previous studies (Babb et al., 1984 ; Kim et al., 1990 ). More importantly, the staining pattern for the GABAA
receptor subunits under study was largely similar in the two groups.
This is illustrated for the 1-subunit in Figure
1 where the postmortem specimen from a
40-yr-old man with no known neurological disorder (top left panel) was compared with the surgical specimen from a
45-yr-old-man with TLE secondary to a benign extrahippocampal tumor and
without HS (bottom left panel). The origin of control
specimens, whether from autopsy or surgery, is indicated in parentheses
for each subsequent figure.

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Figure 1.
Validation of controls. Hippocampal tissue
obtained at autopsy does not differ from hippocampal tissue removed
surgically with respect to GABAA receptor immunoreactivity.
These examples show 1-subunit staining: autopsy control, without
neurological disease (top left), surgical control, TLE
without HS (bottom left), autopsy, TLE with HS
(top right), and surgery, TLE with HS (bottom
right). Note that GABAA receptor 1-subunit
staining pattern and intensity in the TLE specimen without HS are
comparable to those in the autopsy control, yet markedly different from
both TLE specimens with HS. DG, Dentate gyrus;
H, dentate hilus; S, subiculum. Scale
bar, 1 mm.
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To test whether autopsy specimens represent valid controls for surgical
specimens, hippocampal tissue obtained at autopsy from a patient with
TLE and HS was compared with tissue obtained at surgery from patients
suffering from TLE with HS, as shown for GABAA
receptor 1-subunit staining (Fig. 1). Although the autopsy specimen
was processed after a 15 hr postmortem interval (top right
panel) and the surgical specimen immediately after resection (bottom right panel), staining patterns
were comparable, confirming that GABAA receptor
subunit antigens are stable for several hours after death (Loup et al.,
1998 ).
Regional distribution of GABAA receptor subunits in
controls and TLE with HS
To provide an overview, GABAA receptor
subunits 1, 2, 3, 2,3, and 2 in the human hippocampus
were visualized at low-power magnification in color-coded video images
(Fig. 2). Differences in staining
intensity for each subunit were assessed using a normalized scale. In
rodent brain, these subunits account for at least 80% of all
GABAA receptors (for review, see McKernan and
Whiting, 1996 ; Möhler et al., 1996 ). Whereas the 2,3- and
2-subunits are part of most GABAA receptor
subtypes, the -subunit variants represent largely distinct subtypes
with a specific pharmacological profile and distribution pattern. A
similar organization in subunit architecture was observed in normal
human hippocampus. In control specimens (Fig. 2, first
column), the 2,3- and 2-subunits exhibited a comparable
labeling pattern, with staining intensity being highest in the dentate
molecular layer and CA1, and moderate in CA2, CA3, and hilus.
Autoradiographic studies with benzodiazepine radioligands have
described a similar distribution of GABAA
receptors, confirming the ubiquitous nature of these two subunits
(Faull and Villiger, 1988 ; Houser et al., 1988 ; Hand et al., 1997 ). In
contrast, the -subunit variants showed distinct patterns of
immunoreactivity (Fig. 2, third column). For the
1-subunit, the density of immunolabeling was highest in the dentate
molecular layer and CA1. Staining was moderate in CA2 and hilus. At
this level of magnification, CA3 appeared nearly devoid of 1-subunit
immunoreactivity. These findings are in accordance with a previous
study in normal human tissue (Houser et al., 1988 ). Immunoreactivity
for the 2-subunit was most abundant in the dentate molecular layer.
Labeling was also prominent in CA2, CA3, and hilus, and moderate in
CA1, a pattern similar to that reported in rodents (Fritschy and
Möhler, 1995 ). Finally, specific 3-subunit immunoreactivity
was consistently found to be highest in CA1, moderate to low in CA2,
and virtually absent in CA3 and hilus, whereas in the dentate gyrus
immunolabeling varied among specimens (see Fig.
13E,F). Figure 2 illustrates a case with low
3-subunit immunoreactivity in the dentate molecular layer.

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Figure 2.
Cytoarchitecture and regional distribution of the
major GABAA receptor subunits in the hippocampus from a
control (autopsy, first and third
columns) and a TLE patient with HS (second and
fourth columns). Optical density of staining is
color-coded according to a normalized scale showing the strongest
signal in white and the background in dark
blue. In both specimens, adjacent sections were stained for the
subunits 1, 2, 3, 2,3, and 2, and for Nissl. In the
control, Nissl staining shows a normal distribution of neuronal cell
somata. Labeling for the 2,3- and 2-subunits is largely similar,
whereas each -subunit has a differential distribution pattern. In
TLE with HS, Nissl staining reveals prominent cell loss in CA1, CA3,
and dentate hilus with relative sparing of the dentate granule cell
layer and granule cell dispersion. The CA2 and partly CA3 pyramidal
cell layer is not shown in this section, but is visible in those
stained for the -subunits. The decrease in GABAA
receptor subunit immunoreactivity parallels cell loss in CA1, CA3, and
dentate hilus, whereas staining is increased or decreased in a
subunit-specific manner in CA2 and dentate gyrus. The
magenta spots in the dentate hilus represent surviving
mossy cells. Scale bar, 2 mm.
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In TLE with HS specimens (Fig. 2, second and fourth
columns), the characteristic pattern of gliosis and regional
neuronal loss was revealed with Nissl staining. Cell death was most
extensive in CA1 and more moderate in CA3 and dentate hilus, with areas of relative sparing in CA2 and dentate gyrus. Two main alterations in
GABAA receptor subunit expression were observed
at the regional level. First, areas of prominent cell loss, such as
CA1, CA3, and dentate hilus showed marked reduction in immunoreactivity for all subunits. These changes closely paralleled the pattern of
neuronal loss occurring in HS. Second, areas of relative cell loss,
such as CA2 and the dentate gyrus, exhibited subunit-specific, increased or decreased intensity of staining in comparison to controls,
suggesting corresponding changes in the number of receptors in
surviving principal cells. Upregulation was most prominent for the
2-subunit. Differences in staining intensity between HS specimens
and the two control groups were assessed by quantitative densitometry
in the CA2 pyramidal cell layer and dentate layers (Fig.
3, see below). In the following, results
will be described in subregions of the hippocampus in controls and TLE
specimens with HS. Unless otherwise mentioned, the distribution of the
ubiquitously present 2,3- and 2-subunits largely paralleled that
found for the -subunit variants and will not be described
further.

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Figure 3.
Mean neuronal cell counts and densitometric
measurements ± SEM in autopsy (white bars), non-HS
(shaded bars), and HS (black bars)
patient groups. A-G, Sets of six immediately adjacent
sections were stained for Nissl and the subunits 1, 2, 3,
2,3, and 2. A, B, Densities of CA2 pyramidal cells
(A) and granule cells (B).
C-G, Optical density measurements in CA2 pyramidal cell
layer (C), granule cell layer
(D), inner molecular layer
(E), outer molecular layer
(F), and subgranular layer
(G). H, I, Numbers of
1-subunit-positive interneurons per 625-µm-wide column through
each stratum in CA2 (H) and CA3
(I). Nonparametric ANOVA p
values are presented above data set, and significant post
hoc results are indicated by asterisks
(p < 0.05). *Difference compared to the
autopsy or non-HS groups; **difference compared to the autopsy and
non-HS groups.
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GABAA receptor subtypes in the CA1 area
Control specimens
Within CA1, a laminar pattern was observed for all subunits, as
illustrated in Figure
4A for the 2-subunit
and Figure 4D for the 1-subunit. Stratum
pyramidale showed highest intensity and strata oriens and
lacunosum-moleculare moderate intensity of staining. Because of the
fused hippocampal fissure, labeling of stratum lacunosum-moleculare
often appeared continuous with that of the dentate molecular layer
(Fig. 4A). The more lightly stained band represented
stratum radiatum. Immunoreactivity was diffusely present throughout the
pyramidal cell and dendritic layers. At high magnification, individual
pyramidal neurons could be discerned, outlined by discrete staining
along the surface of the somata and proximal dendrites (data not shown;
Loup et al., 1998 ). Among all subunits, 3-subunit immunoreactivity
was highest, a finding that contrasts with data from rodents, in which 3-subunit staining is absent in CA1 (Fritschy and Möhler,
1995 ; Sperk et al., 1997 ).

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Figure 4.
Subunit-specific GABAA receptor
immunoreactivity in the CA1 area of controls (A, D,
autopsy) as compared to TLE with HS (B, C, E). A,
D, Control sections exhibit a laminar staining pattern for the
2- and 1-subunits, respectively. Whereas immunoreactivity for
both subunits is diffusely distributed in neuropil, 1-subunit
immunoreactivity is also present in numerous nonpyramidal neurons
(D, arrows). B, C, Sections from two HS
specimens stained for the 2-subunit, where in B the
partly conserved CA1 pyramidal cell layer shows 2-subunit
immunoreactivity, and in C there is almost complete
pyramidal cell loss associated with severe atrophy of the CA1 area.
E, Section from an HS specimen stained for the
1-subunit, showing the loss of neuropil staining and the
layer-specific distribution of nonpyramidal neurons. Whereas small
interneurons are conserved in stratum lacunosum-moleculare and larger
interneurons in stratum oriens, few interneurons are visible in strata
pyramidale and radiatum. Note the increase in GABAA
receptor 2- and 1-subunit expression in dentate molecular and
granule cell layers in HS specimens. al, Alveus;
gcl, dentate granule cell layer; H,
dentate hilus; hf, hippocampal fissure;
ml, dentate molecular layer; slm, stratum
lacunosum-moleculare; so, stratum oriens;
sp, stratum pyramidale; sr, stratum
radiatum. Scale bar, 200 µm.
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An important additional feature of 1-, and less prominently 2,3-
and 2-subunit immunoreactivity in the hippocampus was the intense
labeling of numerous nonpyramidal cells. Based on their size, location,
and dendritic arborization, several types of interneurons could be
distinguished. Because of the strong neuropil staining in CA1, it was
not always possible, however, to identify them all. In stratum oriens,
somata of many immunoreactive neurons were fusiform with long dendrites
that ran parallel to the alveus, or round with few, short dendrites.
These cells were superimposed on a dense network of immunoreactive
dendrites. In stratum pyramidale, large and intensely stained
multipolar cells were seen with their dendrites passing through the
strata pyramidale and radiatum before entering the stratum
lacunosum-moleculare (Fig.
5A). Most conspicuous was the
presence of small round interneurons, which were especially numerous in
stratum lacunosum-moleculare (Fig. 4D). Their
radially oriented dendrites, forming a fine network superimposed on
diffuse neuropil, conferred a stellate-like aspect to these cells.

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Figure 5.
Altered morphology of large multipolar
interneurons immunoreactive for the 1-subunit in the CA1 pyramidal
cell layer in TLE with HS. A, In the autopsy control, soma
contours are smooth with few long and straight dendrites. B,
C, In HS specimens, the soma exhibits an irregular shape, and
dendrites appear tangled, variable in their diameters, and increased in
number. At higher magnification (C), dendritic
nodulations are visible (arrows). Note the absence of
1-subunit staining in neuropil reflecting severe pyramidal cell loss
in CA1. Scale bar: A, B, 50 µm; C, 25 µm.
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TLE specimens with HS
In this series, three of 16 specimens showed segmental
conservation of the CA1 pyramidal cell layer (Figs. 2,
4B), in which atrophy was partial, and the staining
pattern in the preserved area was similar to that of control specimens.
In the other 13 specimens, there was marked atrophy of the CA1 area
(Fig. 4C, 2-subunit, E, 1-subunit). Whereas
strata pyramidale and radiatum were reduced to a thin band virtually
devoid of pyramidal cells, stratum lacunosum-moleculare largely
retained its original size. The few surviving pyramidal cells were
faintly immunoreactive for GABAA receptors (data
not shown).
In the absence of immunoreactivity in the dendritic fields of the
degenerated pyramidal cells, the interneurons expressing the
1-subunit were revealed in all specimens (Figs.
4E, 5B,C). In
stratum oriens, fusiform, round, and multipolar interneurons could be
observed within a dense network of dendrites (Fig.
4E). In strata pyramidale and radiatum, several types
of nonpyramidal cells could be distinguished with somata that were
small and round, medium-sized, or large and multipolar. The latter cell
type frequently displayed irregular soma conformations and dendrites
that appeared tangled, nodulated, variable in their diameters, and
increased in number (Fig. 5B,C). Finally, stratum
lacunosum-moleculare contained numerous small interneurons whose
dendrites formed a more intricate network than that observed in
controls. Occasional round cells of moderate size were also seen (Fig.
4E).
Because of prominent neuropil labeling in control tissue, which largely
prevented counts of 1-subunit-positive interneurons in the different
strata, it was not possible to evaluate the extent, if any, of
interneuron loss that had occurred in HS specimens. As illustrated in
Figure 4E, however, interneurons present in strata
pyramidale and radiatum were consistently less numerous than in stratum
lacunosum-moleculare in the CA1 area.
GABAA receptor subtypes in the CA2 area
Control specimens
A subunit-specific pattern of staining was observed in the CA2
area. The 2-subunit exhibited a laminar distribution comparable to
that in CA1, with intense staining in stratum pyramidale and lightest
staining in stratum radiatum (Figs. 2,
6C). 3-Subunit immunoreactivity was very different, in that it mainly outlined the
cell bodies of a few pyramidal neurons in the superficial aspect of the
stratum pyramidale. In stratum radiatum, their lightly labeled apical
dendrites formed delicate processes that ran parallel to one another
and entered the stratum lacunosum-moleculare where staining was more
diffuse (Fig. 6E). A laminar pattern was also seen
for the 1-subunit (Figs. 1, 2,
7A).

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Figure 6.
Subunit-specific alterations in GABAA
receptor immunoreactivity in the CA2 area in controls
(A, C, autopsy; E,
surgery, TLE without HS) versus TLE with HS (B, D,
F). A, B, Nissl-stained sections show
moderate pyramidal cell loss in TLE with HS (B)
as compared to control hippocampus (A). C,
D, 2-subunit immunoreactivity is increased in surviving
pyramidal cells in TLE with HS (D) in comparison
to controls (C). E, F,
Redistribution of 3-subunit immunoreactivity. In control hippocampus
(E), staining is prominent, outlining the somata
of a few pyramidal cells, but weak in their apical dendrites, whereas
in TLE with HS (F), thickened apical dendrites
show intense immunoreactivity with virtually absent labeling of somata.
Note the tangentially cut dendrites reflecting disorganization in
orientation (arrows). Abbreviations, see Figure 4. Scale
bar, 100 µm.
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Figure 7.
Layer-specific changes in interneurons stained for
the GABAA receptor 1-subunit in the CA2 area in five
different TLE specimens with HS (B, C, E, F, H,
I) versus three different controls (A,
G, autopsy; D, surgery, TLE without HS).
A-C, Overview; D-F, stratum pyramidale;
G-I, stratum lacunosum-moleculare. A, D,
G, In controls, several types of nonpyramidal cells are found
in strata pyramidale and radiatum, whereas predominantly small
interneurons are present in stratum lacunosum-moleculare. B,
C, In TLE with HS, the loss of interneurons in strata
pyramidale and radiatum is either extensive (B)
or virtually complete (C), whereas the small
interneurons are conserved in stratum lacunosum-moleculare. E,
F, In stratum pyramidale, surviving interneurons are large and
display prominent somatic and dendritic alterations, whereas other
types of interneurons are no longer apparent. H, I, In
stratum lacunosum-moleculare, the small interneurons are preserved,
exhibiting an intricate dendritic network. Abbreviations, see Figure 4.
Scale bars: A-C,100 µm; D,
E, 50 µm; F-I, 25 µm.
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In addition, as in CA1, 1-subunit immunoreactivity revealed a
population of interneurons that were readily visible because of the low
to moderate 1-subunit-positive neuropil in CA2. In stratum oriens,
strong immunolabeling of fusiform and large multipolar somata was
detected. Their dendrites mostly ran parallel to the alveus and
frequently formed a dense fiber network. In stratum pyramidale, three
types of nonpyramidal neurons were identified (Fig. 7A,D):
(1) large, intensely stained multipolar interneurons with long apical
and basal dendrites, (2) medium-sized, oviform cells with apical
dendrites that could be followed into stratum radiatum, and (3) small
round interneurons. In stratum radiatum, a few round and medium-sized
1-subunit-positive interneurons were seen. In addition, strongly
stained individual, or fascicles of, dendritic processes passed through
stratum radiatum and entered stratum lacunosum-moleculare (Fig.
7A). Stratum lacunosum-moleculare contained a number of
small stellate-shaped interneurons with four to eight dendrites forming
a delicate network against a lightly stained diffuse neuropil (Fig.
7A,G). At the border between stratum lacunosum-moleculare
and the dentate molecular layer, occasional medium-sized cells were
observed with smooth dendrites oriented in a horizontal plane (data not shown).
TLE specimens with HS
Considerable reorganization in GABAA
receptor subtype distribution was observed in the CA2 area. Despite an
average 40% loss in pyramidal cells (Figs. 3A,
6B), stronger 2-subunit immunoreactivity than in
control tissue was present in the surviving pyramidal cells and
dendritic fields (Fig. 6D). At high-power
magnification, intense staining was seen to outline their somata, which
were frequently deformed in shape and arranged in a disorganized
manner. Furthermore, a striking rearrangement of 3-subunit
immunoreactivity occurred. Intense immunolabeling adorned individual
thickened apical dendrites passing through stratum radiatum, whereas
pyramidal cell bodies were virtually unstained (Fig.
6F). Tangentially cut processes could be observed in
strata radiatum and lacunosum-moleculare, indicating a change in
direction of some apical dendrites along the rostrocaudal axis.
Alterations in 1-subunit immunoreactivity markedly differed in
pyramidal versus nonpyramidal cells. Pyramidal cells and dendritic layers displayed decreased and more diffuse staining in comparison to
control tissue (Figs. 1, 7B,C). In contrast, extensive
layer-specific changes in 1-subunit-positive interneurons were seen
for all HS specimens. In strata pyramidale and radiatum, profound loss of the small, round and medium-sized interneurons immunopositive for
the 1-subunit was observed (Fig. 7B,C,E), whereas the
large multipolar nonpyramidal cells remained for the most part.
However, their somata were deformed, and the dendritic arborization was altered (Fig. 7E,F). In contrast, in stratum
lacunosum-moleculare the numerous small, round interneurons were
preserved and surrounded by a dense dendritic network (Fig.
7B,C,H,I). Furthermore, the fusiform somata of
interneurons present at the border between stratum lacunosum-moleculare
and the dentate outer molecular layer frequently appeared larger and
more irregular than in control tissue, and their dendrites were more prominent.
Quantitative analysis
In addition to neuron counts performed in adjacent Nissl-stained
sections (Fig. 3A), differences in staining intensity
between the autopsy, non-HS, and HS groups were assessed quantitatively by densitometry for each of the five subunits in the CA2 pyramidal cell
layer (Fig. 3C). The following observations were made: (1) HS specimens showed decreased neuron densities when compared to autopsy
(p < 0.01), and a similar trend was observed
when compared with non-HS cases, whereas the difference between the
autopsy and non-HS groups was not significant. (2) There were
subunit-specific differences in OD for the -subunit variants 1,
2, and 3. Compared with the autopsy group, 2-subunit OD in HS
was significantly increased (p < 0.01), whereas
OD for the 1- and 3-subunits was significantly decreased
(p < 0.05, p < 0.01, respectively). Furthermore, OD for the 1- and 3-subunits was
significantly decreased compared to the non-HS group
(p < 0.01, p < 0.05, respectively). (3) No significant difference in OD was found for all
three -subunit variants between the autopsy and non-HS groups. (4)
As for the ubiquitously present 2,3- and 2-subunits, no
significant difference in OD was found between the three patient
categories, which may reflect a change in receptors containing these
subunits, whereby a decrease in one subtype of receptor ( 1 or 3)
is compensated for by an increase in another ( 2). These findings
suggest that while the total number of receptors may not change
significantly in the CA2 area in HS specimens, the relative proportion
of 2-subunit-containing GABAA receptors is
markedly increased.
To verify the observed loss or preservation of 1-subunit-positive
interneurons in specific strata, counts were performed in 625-µm-wide
columns through each stratum in the three patient categories (Fig.
3H). In strata pyramidale and radiatum, the HS group
showed decreased numbers of 1-subunit-positive interneurons when
compared with the autopsy or non-HS specimens (stratum pyramidale: p < 0.01, p < 0.05, respectively;
stratum radiatum: p < 0.05 for both). In contrast, in
stratum lacunosum-moleculare, the number of 1-subunit-positive
interneurons was not significantly different in the HS group when
compared with the autopsy and non-HS specimens. For all strata, no
significant difference in interneuron counts was found between the
autopsy group and non-HS cases. These statistical data confirm the
observation that, in the HS group, a significant number of
1-subunit-positive interneurons are lost in strata pyramidale and
radiatum while they are preserved in stratum lacunosum-moleculare.
GABAA receptor subtypes in the CA3 area
Control specimens
In the CA3 area, abundant 2-subunit immunoreactivity was
distributed in a laminar pattern largely similar to that in CA1 and CA2
(Fig. 8A). Neuropil
staining was, however, more prominent and less diffuse, except in
strata radiatum and lucidum where it was fainter. Immunolabeling for
the 3-subunit was virtually absent (Fig. 2). Likewise, faint or no
1-subunit staining was observed on the pyramidal cell bodies and
dendritic fields (Figs. 2,
9A). In contrast, numerous
interneurons expressing this subunit were detected in CA3 (Figs.
8C,E, 9A). The types of nonpyramidal neurons
labeled and the distribution pattern in CA3 were largely similar to
those in CA2. Their darkly stained dendrites were of variable calibers,
usually running toward stratum lacunosum-moleculare and stratum oriens,
but also without preferred direction. In addition, interneurons with
round to oval cell bodies and fine dendrites were observed in stratum
lucidum (Fig. 8C). Conspicuously, interneurons were most
numerous at the border between strata pyramidale and lucidum.

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Figure 8.
Subunit-specific changes in GABAA
receptor immunoreactivity in the CA3 area in controls
(A, E, autopsy; C,
surgery, TLE without HS) as compared to TLE with HS (B, D,
F). A, B, 2-subunit, overview.
C-F, 1-subunit with part of CA3 bordering CA2
(C, D) and part of CA3 inserting into the dentate hilus
(E, F). A, A control section
showing the laminar staining pattern with prominent labeling in stratum
pyramidale and lighter labeling in strata lucidum
(sl) and radiatum. B, Section from
a HS specimen with segmental, moderate to light staining reflecting the
moderate to severe pyramidal cell loss. C, In the
control, whereas different types of 1-subunit-positive interneurons
are present in strata pyramidale and radiatum, small oviform neurons
are found in stratum lucidum. D, In TLE with HS, there
is marked loss of interneurons in these strata. E, In
stratum pyramidale of the control, large multipolar interneurons with
dendrites oriented perpendicularly to the pyramidal cell layer stand
out against a fine dendritic network. F, In TLE with HS,
the few surviving interneurons located in stratum pyramidale have
undergone extensive morphological transformation. Their fine dendrites
appear tangled, increased in number, and radially oriented.
Abbreviations, see Figure 4. Scale bar: A,
B, 200 µm; C-F, 100 µm.
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Figure 9.
Differential distribution of 1- and
2-subunit immunoreactivity in dentate hilus and CA3 in hippocampal
tissue obtained at autopsy. A, Moderate 1-subunit
staining is present in the hilus, whereas the CA3 area appears faintly
labeled. The abrupt change in staining intensity allows the boundary
between the hilus and the CA3 area to be readily distinguished
(dashed line). B, In contrast, moderate
2-subunit staining is equally present in both regions where the
lighter spots represent neuronal cell bodies. Even at this low-power
magnification, a trilaminate pattern can be discerned within the hilus.
A and B are adjacent sections. Scale bar,
0.5 mm.
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TLE specimens with HS
In contrast to the increased GABAA receptor
2-subunit immunoreactivity described for the CA2 area, staining
intensity for this subunit paralleled the degree of pyramidal cell loss
in CA3, suggesting that no upregulation occurs in CA3 pyramidal cells (Fig. 8B). Considerable cellular disorganization was
apparent in stratum pyramidale, where somata were poorly aligned to
each other and exhibited various shapes. In three cases, pyramidal neurons were largely preserved, and staining was similar to control tissue (data not shown). In the other specimens, pyramidal cell loss
was moderate to severe and frequently segmental, mostly in the part of
CA3 inserting into the dentate hilus (Fig. 8B).
Layer-specific changes were observed for 1-subunit-positive
interneurons, which were comparable to those in CA2. Marked loss of
these interneurons was observed in strata pyramidale, lucidum, and
radiatum, and especially at the border between strata pyramidale and
lucidum (Fig. 8D). When large multipolar interneurons
were preserved in stratum pyramidale, their somata were frequently encrusted with intense 1-subunit staining (Fig.
8F). Instead of exhibiting a few long dendrites
oriented perpendicularly to the pyramidal cell layer as in controls
(Fig. 8E), these interneurons displayed more
numerous, shorter dendrites that ran in a radial direction. In
contrast, small, round interneurons morphologically identical to those
found in CA1 and CA2 were also preserved in CA3 stratum
lacunosum-moleculare.
Quantitave analysis
As in CA2, the numbers of 1-subunit-positive interneurons were
quantitatively assessed in distinct strata in the three patient groups
(Fig. 3I). Strata pyramidale and lucidum, however,
were considered together, because most interneurons were present at the
border between both strata and, therefore, could not be attributed unambiguously to one or the other stratum. In strata pyramidale-lucidum and radiatum, the HS group showed decreased numbers of
1-subunit-positive interneurons when compared with the autopsy or
non-HS specimens (stratum pyramidale-lucidum: p < 0.01, p < 0.05, respectively; stratum radiatum:
p < 0.01, p < 0.05, respectively). In
contrast, in stratum lacunosum-moleculare, the number of
1-subunit-positive interneurons was not significantly different in
the HS group as compared to the autopsy or non-HS specimens. For all
strata, no significant difference in interneuron counts was found
between the autopsy group and non-HS cases. Thus, statistically
significant layer-specific changes in the number of
1-subunit-positive interneurons in HS were present in CA3 as well.
GABAA receptor subtypes in the dentate hilus
Control specimens
In the dentate hilus, the GABAA receptor
distribution pattern was found to be subunit-specific, as in the
hippocampal fields (Fig. 2). The boundary between the hilus and CA3,
which often is quite difficult to delineate, was readily recognized as
an abrupt transition of 1-subunit staining. Thus, 1-subunit
immunoreactivity was moderate in dentate hilus but weak in CA3 (Figs.
2, 9A). In contrast, 2-subunit immunoreactivity was of
relatively similar intensity in these two regions (Fig. 9B).
Within the dentate hilus, a trilaminate pattern was observed for both
the 2- and 1-subunits, as well as the ubiquitously present
2,3- and 2-subunits (Figs. 4A, 9A,B,
10D,G). First, a
layer of prominent diffuse neuropil staining, the subgranular layer,
was observed immediately subjacent to the granule cell layer and
represented the basal dendrites of granule cells (see
"GABAA receptor subtypes in the dentate gyrus").
Second, a weakly labeled neuron-sparse zone of variable thickness could
be identified. Third, a region that comprised the majority of the hilar
cells, the polymorphic layer, was situated deep to this zone. In the
case of 2-subunit staining, the hilar neurons frequently appeared as
white spots at low-power magnification, surrounded by a moderately
immunoreactive neuropil (Fig. 9B). In contrast,
1-subunit-positive cell bodies and an associated network of
dendrites were intensely stained and surrounded by a moderately to
lightly immunoreactive neuropil (Figs. 9A,
10D).

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Figure 10.
Alterations in GABAA receptor 2-
and 1-subunit immunoreactivity in the dentate hilus in TLE with HS
(B, C, E, F, H, I) versus controls
(A, D, autopsy; G,
surgery, TLE without HS). 2-subunit (A-C),
1-subunit (D-I). A, In the
control, 2-subunit labeling discretely outlines the somata of
pyramidal-shaped neurons (double arrows), whereas
intense staining reveals their axon initial segments (large
arrowheads). B, C, HS specimens. In
B, oval neurons with intense 2-subunit
immunoreactivity along the somata resulting in a thickened outline are
superimposed on decreased neuropil staining. In C, a
surviving medium-sized neuron exhibits 2-subunit staining against
the white background because of severe loss of neuropil and cells.
D, G, In controls, light neuropil staining is present
with several cell types immunoreactive for the 1-subunit, including
mossy cells (arrows) and large multipolar interneurons
with long dendrites (small arrowheads). E,
F, In TLE with HS, neuropil staining is absent, and cell loss
has occurred; among the surviving cells, mossy cells (E,
arrows) and large interneurons with irregular somata and
disorganized tangled dendrites can be recognized. G-I,
The altered morphology of large multipolar interneurons located in the
subgranular layer is shown at higher magnification in two HS specimens
(H, I) as compared to a control specimen
(G). In TLE with HS, these interneurons often
display changes in soma shape and dendritic arborization including
coiling, nodulation, strong variation in calibers, and increased
ramification. Scale bar: A-C, I, 25 µm; D-F, 100 µm; G, H, 50 µm.
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The dentate hilus contains a variety of neuronal cell types with unique
morphological features, as reported in rat (Amaral, 1978 ) and human
brain (Braak, 1974 ; al-Hussain and al-Ali, 1995 ; Blümcke et al.,
1999 ). In the present study, a large number of hilar cells
immunopositive for either the 1-, 2-, or 3-subunit, or both
the 1- and 2-subunits were identified, based on the comparison of
adjacent sections stained for these subunits. Unexpectedly, previously
undescribed pyramidal-shaped neurons immunoreactive for the
2-subunit were observed well within the hilar borders. At higher
magnification, discrete staining occurred along the surface of the
somata and proximal dendrites, and intense staining revealed darkly
stained longitudinal processes 20- to 40-µm-long (Fig.
10A). Using confocal laser-scanning microscopy, these
structures were putatively identified as axon initial segments
intensely immunoreactive for the 2-subunit (Loup et al., 1998 ). The
most prominent type of hilar cell was the mossy cell, which exhibited strong staining for both the 2- and 1-subunits (Fig.
10D; Loup et al., 1998 ). The mossy cells were readily
distinguished by the presence of characteristic "clusters of
spheres" that form the mossy cell thorny excrescences, as described
by Amaral (1978) . Finally, a number of putative interneuron cell types
immunopositive for the 1-subunit were seen, including large
multipolar cells. These were localized in the polymorphic and
subgranular layers (Fig. 10D,G), and their
dendrites traveled for long distances within the hilus or ascended far
into the dentate molecular layer. Occasionally large multipolar
interneurons with long dendrites were observed that were stained
lightly for the 3-subunit. Otherwise, no specific staining was
detected in the polymorphic layer for this subunit.
TLE specimens with HS
In the polymorphic layer, the degree of neuronal loss was
variable, with cell loss being mild to moderate in five cases and severe in the other specimens. In cases of lesser cell loss, the neuropil showed decreased 2-subunit immunoreactivity, and round hilar cells could be seen with their somata frequently outlined by
intense staining (Fig. 10B). These neurons were
unstained for the 1-subunit in adjacent sections (data not shown).
In cases of marked cell loss, staining intensity of the neuropil for
both the 1- and the 2-subunits was reduced to virtually
nondetectable levels. Surviving hilar neurons and their dendritic
network thus appeared darkly stained against the pale background and
were easily recognized (Fig. 10C,E,F). Preserved cell
types included: (1) 1- and 2-subunit-positive mossy cells (Figs.
2, fourth column, 10E). Despite severe
loss, mossy cells were the most numerous among the surviving
2-subunit-positive hilar neurons and represented a sizable portion
of the surviving 1-positive hilar neurons; (2) 2-subunit-positive
oviform cells with dendrites extending over long distances (Fig.
10C); and (3) 1-subunit-positive large multipolar
interneurons, with irregular cell bodies and dendrites that appeared
tangled, nodulated, and increased in number (Fig. 10E,F,H,I). No specific soma staining was
visible for the 3-subunit against the white background.
GABAA receptor subtypes in the dentate gyrus
Control specimens
Cell type-specific GABAA receptor subunit
distribution was also observed in the dentate granule cells. Staining
for the 1-, 2-, 2,3-, and 2-subunits was prominent in the
apical dendritic field forming the molecular layer and appeared
stronger in the inner as compared to the outer part (Fig. 2,
first and third columns). The dentate molecular
layer displayed a higher intensity of staining than any other area of
the hippocampus (Fig. 2, first and third columns). In the subgranular layer, diffuse moderate labeling was
observed in the basal dendritic field (Figs. 4A,
9A,B,
11A,C). In contrast
to the marked staining of both apical and basal dendritic trees,
labeling barely outlined the somata of the tightly packed granule cells
(Figs. 11A,C,
12, 13A,D). Whereas the 1-,
2-, 2,3, and 2-subunits showed a largely similar distribution
pattern in the dentate gyrus of all specimens, 3-subunit expression
was variable. Despite similar intensity of staining in the CA1 area for
all cases (as exemplified in Fig. 2, third column), in
granule cells this subunit showed patchy labeling ranging from very
weak (Fig. 11E) to high levels of density (Fig.
11F) along the somata and basal and apical dendritic
fields. These case-specific variations were not dependent on age,
postmortem interval, or general quality of tissue, suggesting a
propensity for plasticity in 3-subunit immunoreactivity in dentate
granule cells.

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Figure 11.
Changes in subunit-specific GABAA
receptor immunoreactivity in the dentate gyrus in TLE with HS
(B, D, G, H) versus controls (A,
C, surgery, TLE without HS; E, F, autopsy).
A, B, 1-subunit; C, D, 2-subunit;
E-H, 3-subunit. A, C, In control
specimens, the somata of granule cells are outlined by faint or no
staining for the 1-subunit (A) and the
2-subunit (C), whereas apical and basal
dendritic fields are moderately labeled. B, D, In TLE
with HS specimens, strongly increased 1-subunit
(B) and 2-subunit immunoreactivity
(D) is seen surrounding the somata of granule
cells and in the molecular layer, whereas only few basal dendrites
remain. Note the granule cell dispersion into the molecular layer in HS
(B, arrows). E, F, Two control specimens
illustrate the range of 3-subunit immunoreactivity. Whereas in
E staining is absent from the granule cells and their
dendritic fields, in F staining is moderate to intense,
outlining the granule cell somata and apical dendrites. G,
H, In these TLE specimens with HS, 3-subunit
immunoreactivity is either absent in dentate gyrus
(G) or, in H shows the same
staining pattern as the  |
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