Epilepsici, 37(9):892-901, 1996
Lippincott-Raven Publishers, Philadelphia
0 International League Against Epilepsy
Properties of a Delayed Rectifier Potassium Current in
Dentate Granule Cells Isolated from the Hippocampus of
Patients with Chronic Temporal Lobe Epilepsy
“H. Beck, TI. Blumcke, ST. Kral, $H. Clusmann, SJ. Schramm,
9U. Heinemann, and *C. E. Elger
to. D. Wiestler,
Departments of *Epileptology, fNeuropathology, and $Neurosurgery, University of Bonn Medical Center, Bonn; und
§Department of Physiology, Charit4 Berlin, Berlin, Germany
Summary: Purpose: Properties of potassium outward
currents were investigated in human hippocampal dentate
gyrus granule cells from 1 1 hippocampal specimens obtained from patients with temporal lobe epilepsy (TLE)
during resective surgery.
Methods: Dentate granule cells were isolated enzymatically and outward currents analyzed by using the wholecell configuration of the patch-clamp method. Hippocampal specimens were classified neuropathologically with
respect to severe segmental cell loss, gliosis, and axonal
sprouting (Ammon’s horn sclerosis, AHS), or the presence of a focal lesion in the adjacent temporal lobe.
Results: A delayed rectifier outward currtnt (I,), but
not an A-type potassium current (I,) or inwardly rectifying potassium currents, was observed in all cells. The
average current density of I,, the time-dependent decay
of I,, and the resting membrane characteristics were not
significantlydifferent between patients with and without
AHS. The voltage of half-maximal activation V,/z(act)was
5.4 * 1.8 mV in AHS compared with - 2.9 2 1.8 mV in
lesion-associated epilepsy (NS). In contrast, VI/2(inact)
was shifted in a hyperpolarizing direction in AHS ( - 67.7
? 0.6 mV) compared with that in hippocampi not showing
AHS (-47.7 * 2.6 mV; p = 0.0017).
C o nc lu s ions : T h e a I t e red s t e ad y - s tat e v o It age dependence of I, may result in abnormal excitability of
dentate granule cells in AHS and exert a marked influence on input-output properties of the dentate gyrus.
Key Words: Human-Acutely isolated granule cellHippocampus-Temporal lobe epilepsy-Ammon’s horn
sclerosis.
The hippocampus is a critical brain region that
has been associated with the pathogenesis of human
temporal lobe epilepsy (TLE). Considerable research has focused on morphologic and functional
alterations in the hippocampal formation in patients
with TLE. The dentate gyrus is particularly interesting for the study of excitability in the mesial temporal lobe, as it is situated critically to regulate information transfer from the entorhinal cortex to the
hippocampus proper (1). Various lines of evidence
indicate that the dentate gyrus serves as an adjustable relay station limiting excitatory neurotransmission into the hippocampus proper ( 2 4 ) . Electrophysiologic and morphologic data obtained from
surgically resected hippocampal specimens show
that the dentate gyrus is a site of major synaptic
reorganization and plasticity. Such changes include
mossy fiber sprouting into the inner molecular layer
(5-8), changes in neuronal structure (9), and regulation of neurotransmitter receptor expression
(10,ll). These changes are present to a high degree
in patients with Ammon’s horn sclerosis (AHS),
which is characterized by marked loss of principal
neurons in CA1, CA3, and CA4 with relative sparing of CA2 and the granule cell layer (12). Although
morphologic alterations and the synaptic connectivity of dentate granule cells in drug-resistant TLE
(13-15) have been\described in detail, there is little
information about the properties and density of
voltage-dependent ionic currents.
At the cellular level, potassium currents are an
important intrinsic inhibitory determinant. Indeed,
treatment of hippocampal slices or experimental animals with various potassium channel blockers results in severe and prologed epileptiform discharges
Received January 4, 1996; revision accepted May 15, 1996.
Address correspondence and reprint requests to Dr. H . Beck
at Department of Epileptology, University of Bonn Medical Center, Sigmund-Freud Str. 25, D-53105 Bonn, Germany.
892
I K I N H U M A N HIPPOCAMPAL G R A N U L E CELLS
(16-21). Potassium currents in the dentate gyrus
have been characterized in the rat. Interestingly,
granule cells show both developmental and metabolic regulation of a delayed rectifier potassium current (IK; 22,23). Similar changes in pathophysiologic conditions such as T L E would be of interest as
they could have a marked influence on input-output
characteristics of the dentate gyrus. Because of the
lack of control material, the study of epilepsyrelated alterations is not straightforward. However,
patients with AHS and those with morphologic lesions in the temporal lobe differ considerably with
respect to neuropathologic criteria (24,25). Hippocampi showing AHS display significantly greater
amounts of mossy fiber sprouting, synaptic reorganization, and neuronal cell loss. Therefore comparison of hippocampal specimens from these two
groups of patients may allow the analysis of intrinsic cellular correlates of neuropathologic changes
observed in AHS. In our study, we analyzed the
characteristics of potassium currents in acutely isolated human dentate granule cells. Acutely isolated
cells are an ideal substrate for the study of voltagedependent ion currents because they are electrotonically compact, and space-clamp artifacts are small
(26,27).
METHODS
Patient data
The mean duration of epilepsy in our patients was
19.7 k 7.7 (mean k SD) years. The mean age at
onset of seizures was 10.8 k 6.0 years. All patients
893
except one (L3, Table 1) had complex partial seizures (CPSs); the exception had only simple partial
seizures (SPSs). Seven patients also had additional
secondarily generalized seizures (sGSs). There
were no differences in the clinical characteristics
with AHS (A1-AS) and patients with lesions of the
temporal lobe ( L L L 4 ) . None of the patients had
had episodes of status epilepticus.
Eleven surgical specimens from patients with
drug-resistant T L E were obtained for electrophysiologic analysis. The epileptogenic focus was localized to the temporal lobe in all patients by noninvasive and invasive diagnostic procedures, as described elsewhere (28-30). In all patients, surgical
removal of the hippocampus was indicated clinically. The following surgical procedures were used:
standard temporal lobe resection (two cases), lesionectomy with amygdalohippocampectomy (three
cases), and selective amygdalohippocampectomy
(six cases): Informed consent was obtained from all
patients to perform additional histopathologic and
electrophysiologic studies. All patients except one
(L4) were seen for follow-up at 3 months. All patients except L4 were either seizure free (patients
A1-3, A5, L1-3, and D1) o r had had a markedly
reduced frequency of CPSs (>75%, patients A4 and
D2). All procedures were approved by the ethics
committee of the University of Bonn Medical Center.
Neuropathologic classification
For routine neuropathologic evaluation, a coronal block of the hippocampal specimens was immer-
TABLE 1. Clinical data of patients from which viable neurons could he ohtuined for patch-clamp analysis
Code
Pathologic
condition
Age (yr)
AHS
Al
48
A2
39
A3
29
A4
34
A5
33
Lesion
LI
14
L2
35
L3
27
L4
25
Dual pathologic condition
DI
19
D2
35
OP, side
AHS
AHS
AHS
AHS
AHS
sAHx,
sAHx,
sAHx,
sAHx,
sAHx,
Cortical dysplasia
Migrational anomaly
Porencephalic cyst
Porencephalic cyst
Lesx. sAHx
Lesx. sAHx. 1
213 TLx, I
ext. Lesx. sAHx., r
Angioma, AHS
GNH, end folium sclerosis
Lesx, sAHx. I
2/3 TLx, I
r
I
r
I
r
Onset age
(yr)
No. CPS
per mo
Seizure type
42
25
16
16
10
1-2
4-8
6 7
2-3
2wo
I
3
12
26
26
22
3-5
CPS. SGS
SPS, CPS. SGS
SPS
SPS, CPS
12
20
7
15
-4
4-1
CPS, SGS
CPS
6
14
13
18
21
L
9
I4
4 4
90-120*
CPS, SGS
SPS. CPS. SGS
SPS, CPS
SPS, CPS, SGS
SPS, CPS, SGS
Age: Age of the patients at surgery (average 31 ? 7); Onset: age at which a first epileptic seizure could be observed (average 12 ? 6);
Duration: duration of the epileptic disorder; Frequency CPS: frequency of complex partial seizures per month. One patient (L4) had only
simple partial seizures with a frequency of 9G120/month (asterisk). Patients were classified according to the presence of AHS without
evidence for a focal lesion (Al-A5) and the presence of only a focal lesion in the temporal lobe not involving the hippocampus proper
(LLL4). Two patients showed a dual pathology, i.e., an angioma in the temporal lobe and AHS (DI), or neuron loss in CA4 with
dispersion of granule cells and glioneuronal hamartia (D2).
AHS, Ammon’s horn sclerosis; GNH, glioneuronal hamartoma; sAHx, selective amygdalohippocampectomy; Lesx, lesionectomy ;
TLx, temporal lobe resection with amygdalohippocampectomy; r/l, right and left hemisphere; CPS, complex partial seizure; SPS, simple
partial seizure; SGS, secondarily generalized seizure.
Epilepsiu, Vol. 37, N o . 9, 1996
894
H . BECK ET AL.
sion-fixed in 4% buffered formalin at room temperature for 8 h to 3 days and embedded in paraffin.
Specimens were classified with respect to the presence of either a focal lesion in the temporal lobe or
a histopathologic diagnosis of AHS with severe
neuronal loss in the CAI, CA3, and CA4 subfield
and relative sparing of CA2. Two patients had dual
pathologic conditions (D1 and 2). One patient had
had an angioma in the temporal lobe with additional
AHS (DI), and another showed evidence of moderate cell loss in CA4 with dispersion of granule cells
(D2, end folium sclerosis). None of the focal lesions
directly involved the hippocampal formation. Hematoxylin and eosin, Nissl, and combined hematoxylin-eosin and luxol fast blue stains were available
for all specimens.
Immunohistochemistry for GAP-43 was performed according to standard procedures by using
the capillary gap method. In brief, a monoclonal
mouse antibody GAP-43/B-50 (Clone 91El 2) was
purchased from Boehringer Mannheim Biochemica
(Mannheim, F.R.G.). For immunohistochemistry,
it was diluted 1500. Control reactions were performed by omitting the primary antibodies. Negative controls for GAP-43 included equal concentrations of nonimmunized immunoglobulin G (IgG)
fractions or normal mouse serum (DAKO, Glastrup, Denmark). Paraffin sections were cut at 4 pm,
mounted on slides coated with 3-aminopropyltriethoxysilane (Fisher Scientific, Pittsburgh,
PA, U.S.A.), air-dried overnight in an incubator at
42"C, and stored until further use. All slides were
stained under identical conditions by using the capillary gap method as described previously (31). The
reactions were carried out in a moist chamber. The
sections were deparaffinated and incubated in 2%
hydrogen peroxide diluted in methanol for 15 min.
Phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA; Serva, Heidelberg, F.R.G.) and
0.25% BRIJ was used for the subsequent washes
and dilutions, unless otherwise specified. To improve the binding of the monoclonal antibodies, the
sections were transferred into citric acid (0.01 M,
pH = 6.0) and boiled 2 x 5 min in a microwave
oven at 600 W. Thereafter these slides were incubated for 2 x 20 min with an avidin-biotin blocking
kit according to the manufacturer's protocol (Vector Labs, Burlingame, CA, U.S.A.). In addition,
nonspecific binding of the antibodies was inhibited
with a solution containing 5% normal rabbit serum
(DAKO, Glastrup, Denmark), 3% nonfat dry milk
(Bio-Rad, Hempstead, U.K.), and 10% fetal calf serum (FCS; Seromed, Berlin, F.R.G.) for 2 h at
42°C. The primary antibodies were incubated overnight at room temperature. Bound antibodies were
Epilepsiu, Vol. 37, N o . 9 , 1996
visualized with t h e avidin-biotin peroxidase
method and an ABC Elite Kit (Vector Labs). The
reaction was developed in a substrate solution containing 0.05% 3',3-diaminobenzidine (ICN, Cleveland, OH, U.S.A.) and 0.01% hydrogen peroxide
(Merck, Darmstadt, F.R.G.) in 0.05 M TRIS-HCI,
pH = 7.4, and the sections were lightly counterstained with hematoxylin, dehydrated in ethanol,
and mounted in Corbit (1. Hecht, Kiel-Hassee,
F.R.G.).
Preparation of acutely isolated dentate granule cells
Hippocampal specimens were placed in ice-cold
artificial cerebrospinal fluid (ACSF; all concentrations millimolar) containing NaCI, 125; KCI, 3;
CaCI,, 2; MgCI,, 2; NaH,PO,, 1.25; glucose, 10;
and NaHCO,, 26; (pH 7.4; 95% CO,, 5% 0,) immediately after surgical removal. A 4- to 5-mm-thick
coronal segment of the corpus of the hippocampus
was prepared with a razor-blade, and the tissue
block was transferred to the stage of a vibratome
(Campden Instruments, Longborough, UK). The
corpus of the hippocampus was identified by gross
neuropathologic criteria by an experienced neuropathologist (I.B.). Coronal slices (400 Fm) were
prepared and transferred to a storage chamber with
warmed ACSF (95% O,, 5% CO,) for preparation of
acutely isolated neurons. After an equilibration period of 30 min, the first section was transferred to a
conical polystyrene tube with 10 ml of incubation
medium containing NaCI, 125; KCI, 5.4; CaCI,, 1.8;
MgCI,, I ; piperazine-N,N-bis-2-ethanesulfonicacid
(PIPES), 10; and glucose, 25 mM (35"C, pH 7.4,
100% 0,). Pronase (2-3 mg/ml) was added to the
oxygenated medium. After incubation for 30 min,
the slice was washed in ice-cold trituration medium
containing NaCI, 125; KCI, 3; CaCI,, I ; MgCI,, 10;
PIPES, 10; glucose, 10; and EGTA, 10 mM (pH 7.4,
100% 0,). The dentate gyrus was dissected under a
binocular microscope and triturated in 2 ml of icecold trituration solution with fire-polished glass pipettes. The cell suspension was then placed in a
petri dish for subsequent patch-clamp recordings.
Isolated cells showed a round or ovoid small soma
with a single process. This appearance is reminiscent of granule cell structure in situ and identical to
acutely isolated granule cells from the rat hippocampus (22). Another type of neuron occurring in
low numbers in the acutely isolated preparation
showed a multipolar structure with several processes emanating from the soma. Only neurons with
a granule cell-like structure were included in our
study. The isolated cells were superfused with an
extracellular solution containing NaCI, 120; KCI,
5.4; CaCI,, 1.8; MgCI,, 2; glucose, 25; and N-2-
I , IN HUMAN HIPPOCAMPAL GRANULE CELLS
hydoxyethylpiperazine-N-2-ethanesulfonicacid
(HEPES), 10 mM (pH 7.4). In some experiments, 1
pM tetrodotoxin (TTX) was added to the recording
solution. The amplitude of sodium currents was
measured in most cells to exclude a contamination
of the cell preparation by glial cells. Sodium currents with amplitudes < 1 .O nA were never encountered.
Patch pipettes were fabricated from borosilicate
glass capillaries (outer diameter, 1.5 mm; inner diameter, 1 mm). They usually had a resistance of 3-5
MOhm. The pipettes were filled with an intracellular solution containing KCI, 120; CaCI,, 1; MgCl,,
2; EGTA, 11.5; HEPES, 10; and glucose, 20 mM
(pH 7.4). Tight-seal whole-cell recordings were obtained according to Hamill et al. (32). Membrane
currents were recorded by using a patch-clamp amplifier (EPC9, H E K A Elektronik, Lambrecht/
Pfalz) and collected on-line with the TIDA for
Windows acquisition and analysis program. The
membrane capacitance was measured by using the
EPC9 capacitance cancellation according to Sigworth et al. (33). This estimate can yield results
slightly better than those obtained with sine-wave
techniques (33,34). The currents shown subsequently were not leak corrected. Leak components
were small and could be estimated from hyperpolarizing prepulses, because no hyperpolarizationactivated inward current could be demonstrated in
these cells.
Statistical analysis
Comparison of electrophysiologic data among the
two patient groups (AHS and lesional epilepsy) was
carried out with the aid of an analysis of variance
(ANOVA). Because this test assumes normally dis-
895
tributed samples, a nonparametric test was carried
out in addition (Mann-Whitney U , Wilcoxon Rank
test). All statistical tests were carried out with the
program SPSS Vers. 6.1.2. (SPSS Inc., Munchen,
F.R.G.). All results were expressed as mean values
SEM.
*
RESULTS
Neuropathologic characterization of
hippocampal specimens
One group of hippocampal specimens examined
in this study showed a marked loss of principal neurons in the CA1, CA3, and CA4 fields of the Ammon’s horn with relative sparing of CA2 and an
accompanying severe astrogliosis. These changes
were classified as AHS (specimens AI-A5). The
second group of hippocampal specimens had an extrahippocampal focal lesion and showed a less severe and more homogeneous pattern of neuronal
loss (specimens L1-L4, Table 1). One patient had
an angioma in the anterior temporal lobe and additional AHS (D1, Table 1). This patient showed a
pattern of hippocampal neuron loss that was comparable with that of patients with AHS only. A further patient (D2) also had dual pathologies with
moderate cell loss in CA4, some dispersion of granule cells, and a prominent glioneuronal hamartoma
of the temporal lobe. Immunohistochemical staining for growth-associated protein 43 (GAP-43) revealed a distinct staining pattern in specimens with
AHS, particularly in the molecular layer of the dentate gyrus (DG-ML). I n patients with lesionassociated TLE (L1-L4) and in one patient with
dual pathology (patient D2; Fig. lB), the supragranular zone of the DG-ML was virtually devoid of
FIG. 1. Growth-associated protein (GAP)-43
immunoreactivity in the molecular layer of
the dentate gyrus molecular layer (DG-ML).
A: Twenty-nine-year-old patient (A3, Table 1)
with a histopathologic finding of severe Amman's horn sclerosis. GAP-43 immunoreactivity is observed throughout the entire DGML. Note the densely labeled inner molecular layer (IML) adjacent to the granule cell
layer (GC). B: Patient with a glioneuronal
hamartoma in the temporal lobe and neuron
loss in the CA4 segment (D2, Table 1). In addition, some dispersion of granule cells can
be observed. The IML shows significantly
less staining intensity than that observed in
A, whereas the outer portion of the molecular layer (OML) showed a similar staining.
This significant difference with respect to
patterns of synaptic reorganization could be
consistently observed between the two patient groups. Scale bar in B, 50 pm; scaling
in A and B is identical.
Epilepsiu, Vol. 37, No. 9 , 1996
896
H . BECK ET A L .
GAP-43 immunoreactivity with an adjacent band of
GAP-43-stained neuropil in the inner molecular
layer (DG-IML). In contrast, patients with AHS
and one patient with dual pathology (DI, Table 1)
displayed a dense and homogeneous immunolabeling throughout the entire DG-ML (Fig. IA). As patients with and without AHS were remarkably distinct with respect to neuropathologic findings, electrophysiologic characteristics were compared
between these neuropathologically defined groups.
Patient DI, who had both AHS and a focal lesion,
was very similar to patients with only AHS with
respect to neuron loss and GAP-43 immunoreactivity. This patient was therefore included in the AHS
group for electrophysiologic analyses. Patient D2
did not show neuronal cell loss in any hippocampal
subfield except in CA4. In addition, there was dispersion of granule cells, and the GAP-43 immunoreactivity pattern was markedly similar to those observed in patients with lesions only. Therefore this
patient was tentatively included in the lesionassociated epilepsy group.
Passive membrane properties of human dentate
granule cells
Because inwardly rectifying currents could not be
elicited in dentate granule cells, hyperpolarizing
voltage commands from a -50 mV holding potential can be used to calculate the input resistance of
these cells. The average input resistance was 557
142 MOhrn. The resting membrane potential was on
average -50.7 k 10.6 mV. The mean membrane
capacitance was 14.2 k 4.3 p F (n = 17, 1 I patients).
When these characteristics were determined separately for the patient groups with and without AHS,
no significant intergroup differences could be found
(see Table 2).
*
Outward current pattern in human dentate
granule cells
Recordings from acutely isolated dentate granule
cells were obtained between 5 and 30 min after isolation. The cells displayed a slowly activating and
ldq-T:----.
-.
600 ms
~
Ab
4OmV
;
-
I*
11 nA
40
0
20
40
m
80
inactivating outward current in response to depolarizing voltage commands (Fig. 2A and B). When a
conditioning prepulse of 1 s was applied before depolarization of the cell, current amplitudes were
markedly enhanced (Fig. 2A and B). As this current
shows characteristics of a delayed outward rectifier, it will be termed I,. Neither an inwardly rectifying current nor a transient A-type current could
be observed in any of the analyzed neurons. The
maximal current amplitude of 1, measured with a
voltage protocol (as shown in Fig. 2A) and command pulses to +30 mV were related to the membrane capacitance of each individual neuron, thus
yielding a measure of current density. The average
current density was 169 k 117 pA/pF (186 & 118
Lesionh
,
12.2 f 3.1
157.6 f 111.8
-50.8 2 12.0
516.5 f 108.1
All
14.2
169.3
-50.7
557.3
2
2
2
k
4.3
116.8
10.6
141.7
Data reported as mean f SEM.
AHS, Ammon's horn sclerosis; V,,,, membrane potential; Rinp,input resistance;
Cap, membrane capacitance.
" A patient with dual pathology (DI, Table I ) was included in the AHS group.
Patient D2 was included in the lesional group.
Epilcpsiu, Vol. 37, N o . 9, 1996
-20
w m m m d pulse potenti1 (mv)
260 mr
FIG. 2. Outward current families in granule cells isolated
from the hippocampus of a 35-year-old woman with lesionassociated epilepsy (patient L2, Table 1). This patient was
seizure free after the operation (follow-up, 3 months). Wholecell currents were elicited with the voltage clamp protocols,
as shown in the inserts. Recordings were performed directly
after breaking into the cell. Aa: 2,000 ms depolarizing voltage
commands preceded by a conditioning prepulse of 1,000 ms
to -120 mV, holding potential (HP) -50 rnV. Ab: Depolarizing voltage steps of 1,000-ms duration applied directly from a
holding potential of -50 mV. Note the substantial reduction
in current amplitude without a conditioning prepulse. B:
Chord condu,ctance/voltage relation. Conductance was calc.ulated by transforming peak currents (symbols in Aa and
Ab) by using the equation G, = V(V - V), where I is the peak
amplitude at a given potential (V) and V, equals the potassium-reversal potential. A calculated potassium-equilibrium
potential of -81.5 mV was used for calculation. Chord conductance (G,) of each cell was normalized to 1.0 by its maximum conductance, averaged, and plotted versus command
pulse voltage (abscissa).
AHS
17.0 f 6.8
186.8 2 118.3
-50.3 & 7.9
614.5 f 187.4
t.
-
TABLE 2. Passive membrane characteristics of human dentate
granule cells
Capacitance (pF)
I,,,,/cap (PAIPF)
V,,, (mv)
Rinp( M O W
0.4
C
tmmv
897
I , IN HUMAN HIPPOCAMPAL GRANULE CELLS
pA/pF for patients with AHS and 158 k 112 pA/pF
for patients with lesion-associated epilepsy). These
intergroup differences were not significant.
Steady-state inactivation behavior
The marked enhancement of I, by a conditioning
prepulse (Fig. 2A vs. B) can be attributed to a
strong influence of the holding potential on the current amplitude in the range of -50 to - 120 mV.
This reflects the steady-state voltage-dependent inactivation behavior of I,. The voltage-dependent
inactivation of I, was examined with the voltage
protocol shown in Fig. 3A. Maximal current amplitudes were normalized and averaged. Peak current
values were transformed into chord conductance
G, by using the equation
GK
=
V(V - VK)
(1)
where I represents the peak amplitude at a given
potential (V), and V, equals the assumed potassium-reversal potential. These data points could
then be fitted with a Boltzmann equation
(1 - IssYIM,t,x
=
[1
+ exp(Vl/t(inact)- v ) / K ) I ~I
(2)
where I is the total current amplitude; IMAX,the
maximal current amplitude; V the prepulse potential; VI12(inact),
the prepulse voltage of half-maximal
steady-state inactivation; and K, the slope factor. A
steady-state component I,, had to be introduced
because I, could not be fully inactivated even with
an
conditioning prepulses S +30 mV. For Vlh(inact),
average value of - 55.1 5.8 mV was determined,
1.2 (n = 16. 10
the slope factor was K = -5.98
*
*
patients). The steady-state voltage-dependent inactivation showed marked differences between patients with AHS and patients with lesion-associated
was shifted by -20 mV
epilepsy (Fig. 3B). Vli2(inact)
in a hyperpolarizing direction in AHS ( - 67.7 2 0.6
mV; n = 8, five patients) when compared with neurons isolated from patients with lesion-associated
epilepsy (-48.7 k 1 . 1 mV; n = 8, five patients).
These differences were highly significant with both
the ANOVA (p < 0.0001) and the Mann-Whitney U
test (p < 0.005; z = -3.14). In lesional epilepsy,
the residual steady-state component I,, composed
compared with 16.1
on average 32.3 k 4.4% of I,,
k 5.4% in patients with AHS (ANOVA, p < 0.01;
Mann-Whitney U test, p = 0.01; z = -2.74). As
patient D2 showed end folium sclerosis, and therefore the classification as lesional may be erroneous,
the statistical analysis was repeated after omitting
this patient from the lesional group. Differences in
VI,2(inact)
(ANOVA; p < 0.0001; Mann-Whitney U
test, p < 0.01; z = -2.86) and I,, (ANOVA, p =
0.01; Mann-Whitney U Test, p = 0.028; z =
- 2.68) remained significant. Vlh(inact)
of the voltage-dependent inactivation was markedly constant
in neurons derived from individual patients (patient
AS; Fig. 3C). A shift in the steady-state inactivation
curve in a hyperpolarizing direction after prolonged
recording in the whole-cell configuration of the
patch-clamp technique could be observed in two
neurons derived from two different patients with
lesion-associated epilepsy (patients L1 and L3; see
Fig. 3D). As this run-down phenomenon could falsify data used for intergroup comparison between
patients with AHS and those with lesion-associated
FIG. 3. Steady-state voltage-dependent inactiB
A
I
i + 6 O m V
vation of delayed rectifier outward current (IK).
A: Patient L2, as in Fig. 2 (see Table 1). Current
families were elicited 5 min after establishing
m
500 m s
the whole-cell configuration by clamping the
noAHS
membrane voltage at various values ranging
A
AHS
from - 140 to +30 mV for 5,000 ms with a subsequent depolarizing command pulse to +50
mV. B: Chord conductance-voltage relation.
-150
-100
-50
0
50
Peak conductances of each cell were normalpotential (mV)
D
ized to 1.O by the maximal conductance and averaged and plotted versus the prepulse voltage.
Conductance-voltage relations were analyzed
separately for patients with AHS (squares) and
patients with lesion-associated epilepsy (circles). C: Examples of three different neurons
from patient 5 with AHS (see Table 1). Voltage of
half-maximal activation (Vlin(inact)) was - 63.8
+
mV, - 65.7 mV, and - 67.4 mV, respectively. The
50
-150
-100
-50
0
50
data points in B and C were fitted with a Boltzpotential (mV)
mann equation l/IMAx = [I + exp(V1,2(inact)V)/K)] - 1, where I is the voltage-dependent current amplitude; IMAX, the maximal current amplitude; V, the prepulse potential,
Vl,n(,nact): the prepulse voltage of half-maximal inactivation; and K, the slope factor. Best Boltzrnann fits are superimposed on the
data points in B and C. D: Shift of Vl/2(inact)in hyperpolarizing direction in a dentate granule cell derived from patient L1 with
lesion-associated epilepsy (see Table 1). Squares, data points were collected 5 min after establishing the whole-cell configuration; circles, data points were collected 25 min after establishing the whole-cell configuration.
f
id
L
-
4
Epilepsia, Vol. 37, N o . 9 , 1996
H . BECK ET A L .
898
epilepsy, all recordings used for these comparisons
were obtained at an identical time 5 min after establishing the whole-cell configuration.
Steady-state voltage-dependent activation
For analysis of the steady-state voltage-dependent activation behavior, various voltage steps of
2,000-ms duration after a 1,000-ms conditioning prepulse to - 120 mV were used (Fig. 4). Peak current
values were then transformed into chord conductance GK by using Eq. 1 . Data points could be fitted
with a Boltzmann equation, as described previously
(see Eq. 1). The voltage of half-maximal steady1.8 mV with a
state activation Vl,z(act)was -0.2
slope factor K of - 12.5 & 1.4 (n = 18, 10 patients).
V1,2(act)
assumed slightly more depolarized values in
AHS (5.4 1.8 mV as opposed to - 2.9 f 1.8 mV
in lesion-associated epilepsy). The slope factor K
was 14.3
1.4 in AHS and 12.0
1.4 in lesionassociated epilepsy. These differences were, however, not significant (Fig. 4).
*
*
*
*
Time-dependent inactivation
The data in Fig. 2 demonstrate that I, shows a
time-dependent inactivation during the command
pulse. To study the properties of time-dependent
inactivation of I,, we investigated decay kinetics of
outward currents evoked by 2-s voltage commands
after a 1-s hyperpolarizing prepulse to - 120 mV
(Fig. 5A). The decay of the current traces during a
2,000-ms command pulse could be fitted with the
following biexponential equation:
I(t) = A,
,
+ A , x exp( - t/T,) + A,
X
exp( - t/T2)
(3)
with A, being constant and A , + A, representing
the amplitudes of I, with the decay time constants
TI and T,. Best fits are shown superimposed on the
time-dependent decay of I, (Fig. 5B). This procedure yielded time constants of 254 2 120 ms and of
command pulse potential
FIG. 4. Steady-state voltage-dependent activation of delayed rectifier outward current (IK). Chord-conductance/
voltage relation. Peak current values were obtained from
voltage protocols as in Fig. 2. The chord conductance was
determined as stated previously. Normalized and averaged
values were fitted with a Boltzmann equation, according to
Eq. 1.
Epilepsiu, Vol. 37, N o . 9, 1996
A
B
z3.0
51.6
0
-\
oa---
1 .o
2 .o
command pulse dumtion ( t )
FIG. 5. Time-dependent inactivation of delayed rectifier outward current &). A: Time-dependent decay was studied during a 2-s voltage command after a 1-s hyperpolarizing prepulse to -120 mV. B: Decay of the current traces in A were
fitted with the biexponential equation: I(t) = A,, + A, x
exp( - tlT,) + A, x exp( - tlT,) with A0 being constant and A,
+ A, being the amplitudes of IKwith the decay time constants
TI and T., Best fits are superimposed on current traces.
*
1,372 397 ms on average (n = 18, 10 patients).
There were no significant differences between neurons isolated from hippocampi with AHS or those
associated with a lesion. The time-dependent activation properties were not investigated in these experiments because -a contamination by an inward
sodium current could not be excluded in most neurons.
DISCUSSION
In this study, we analyzed the kinetic properties
of potassium currents in acutely isolated human
dentate granule cells from patients with temporal
lobe epilepsy. A delayed rectifier current (I,) could
be observed in all granule cells, but A-currents or
inwardly rectifying currents were absent. Although
resting membrane characteristics did not show
characteristic differences beween patients with
AHS and lesion-associated epilepsy, the steadystate voltage dependence of I, was substantially
different.
Various voltage- and calcium-dependent potassium conductances are thought to play an important
role in controling neuronal excitability, and alterations of these membrane currents may be involved
in epileptogenesis. Indeed, blocking potassium currents in in vitro slice preparations (16-19) and in
intact animals (20,21) results in pronounced seizurelike activity. Only a few studies have addressed the
possibility of chronically altered voltage-dependent
potassium conductances in epileptogenic tissue.
The properties of delayed rectifier potassium currents in pyramidal cells of the CA1 region seem
to be unchanged after kindling epileptogenesis in
rats (35). On the other hand, messenger RNAs
(mRNAs) for delayed rectifier potassium channels
are downregulated in the hippocampus after seizure-like activity (36).
Comparison between human and rat dentate
granule cells
The current pattern in acutely isolated human
granule cells was comparable to that found in nor-
I , IN HUMAN HIPPOCAMPAL GRANULE CELLS
ma1 adult rat dentate granule cells. Whereas immature dentate granule cells possess a transient A-type
current in addition to I,, adult rat dentate granule
cells show a dominating delayed rectifier component (22). As in rat dentate granule cells (37), inwardly rectifying potassium current components
could not be found in human hippocampi. Theoretically, absence of I, and of inwardly rectifying currents may be attributable to rundown of these current components. However, lAremains remarkably
stable toward whole-cell perfusion in acutely isolated rat granule cells (22,37). The steady-state voltage-dependent inactivation behavior of I, in human
dentate granule cells is likewise reminiscent of data
obtained in rats (22,37), showing a marked increase
in I, current amplitudes on application of hyperpolarizing prepulses before depolarization of the cell.
As in rats, changes in membrane potential to more
hyperpolarized levels could thereby exert a strong
control over the amplitude of repolarizing outward
currents. This could result in a marked influence of
steady-state membrane potential on the integration
properties of these cells during excitatory input.
V,,2(,nact)
of inactivation was somewhat more depolarized than were juvenile rat dentate granule cells
( - 70 mV) directly after establishing the whole-cell
configuration (22).
Interestingly, time-dependent hyperpolarizing
could be observed in two granule
shifts of VI,2(,nact)
cells from two different patients with lesionassociated epilepsy. Similar shifts have been observed for I, in cells similar to stellate cells isolated
from the entorhinal cortex (38) and rat dentate granule cells and have been attributed to the washout of
a regulatory cofactor. Future analysis with perforated-patch recordings will clarify whether this is
also the case in human dentate granule cells. The
observed potential for metabolic regulation of IK
both in rat and human granule cells raises the question whether the observed differences in voltage dependence can be attributed to regulation of I, or
rather to the regulation of potassium-channel subunit expression. This issue can be decided only by
further study of the mechanisms governing potassium-current expression and regulation in the human dentate gyrus.
Differences between AHS and
lesion-associated epilepsy
A marked shift of half-maximal activation and inactivation of I, in a hyperpolarizing direction could
be observed in hippocampi showing AHS compared
with hippocampi associated with a lesion. These
differences in steady-state inactivation of I, should
result in less repolarizing potassium current being
899
available in a critical membrane potential range of
- 80 to - 30 mV in AHS. The reduced availability
of 1, may facilitate propagation of epileptiform discharges in the dentate gyrus. In contrast to the voltage-dependent inactivation behavior, other kinetic
characteristics of 1, showed no significant differences between the two neuropathologically defined
patient groups. Similarly, resting membrane characteristics did not show any significant intergroup
differences and were not significantly different from
those measured in acutely isolated rat dentate granule cells (unpublished observations). The membrane potential measured with sharp microelectrodes in the kindling model of epilepsy (39) o r in
hippocampal slices from patients with T L E (13,40)
was slightly more hyperpolarized but did not show
any significant differences from that in normal
mammalian controls.
The differences in the steady-state properties of
I, are of particular interest, considering that alterations of neurotransmitter receptor density and synaptic reorganization in the DG-ML are present to a
markedly higher degree in AHS as compared with
lesion-associated epilepsy (24,25). This could be
confirmed in each individual specimen by GAP-43
immunohistochemistry, showing markedly increased immunoreactivity in the DG-IML in patients with AHS. A correlation between mossy fiber
sprouting into the DG-ML and electrophysiologic
characteristics of granule cells has been demonstrated with sharp microelectrodes both in human
subjects with TLE (13,41) and kainate-treated rats
(42,43). In both cases, excitability of granule cells
was significantly enhanced, as evidenced by an increased sensitivity to antidromic stimulation of dentate granule cells under conditions of y-aminobutyric acid (GABA,) blockade. Although the observed stimulation-induced and spontaneous (42)
burst discharges are compatible with an increased
intrinsic and synaptic excitability, the significance
of altered voltage-dependent ion channels for the
excitability of individual neurons in TLE remains
unclear. Additional studies using intracellular recordings will be necessary to assess the impact of
altered properties of potassium currents on granule
cell excitability. Caution must be exercized in discussing the causal relation of alterations in I, to
neuropathologic alterations, as both changes could
represent a consequence as well as the cause of the
epileptogenic process. In addition, the small numbers of patients included in the study and other
methodologic considerations preclude generalization of these results. For instance, all patients with
AHS underwent a selective amygdalohippocampectomy, whereas patients with lesion-associated epiEpilepsiu, V o l . 37, No. 9, 1996
H . BECK ET AL.
900
lepsy underwent different surgical procedures (see
Table l ) , possibly resulting in differing times of
ischemia. Nevertheless, we speculate that phenomena of synaptic reorganization are accompanied by
simultaneous changes in voltage-dependent membrane currents that may further modulate excitability in the dentate gyrus.
Acknowledgment: This research was supported by a
grant from the Ministry of Science and Education,
Northrhine-Westfalia, and a University of Bonn Center
grant BONFOR 11 1/2. I.B. is supported by the Helmholtz
Foundation of the German Ministerium fur Forschung
und Technologie. We thank Prof. Zentner for providing
neurosurgical specimens and B. Scheffler for excellent
technical assistance.
REFERENCES
I . Andersen P, Holmqvist B, Voorhoeve PE. Entorhinal activation of dentate granule cells. Actu Physiol Scund 1966;66:
448-60.
2. Collins WF, Davis BM, Mendell LM. Modulation of EPSP
amplitude during high frequency stimulation depends on the
correlation between potentation, depression and facilitation.
Bruin Res 1988;442:161-5.
3. Heinemann U, Clusmann H, Dreier J , Stabel J. Changes in
synaptic transmission in the kindled hippocampus. A d v Exp
M e d Biol 1990;268:445-50.
4. Dreier JP, Heinemann U. Regional and time dependent variations of low magnesium induced epileptiform activity in rat
temporal cortex. Exp Bruin Res 1991;87:581-96.
5. De Lanerolle NC, Kim JH, Robbins RJ, Spencer DD. Hippocampal interneuron loss and plasticity in human temporal
lobe epilepsy. Bruin Res 1989;495:387-95.
6. Sutula TP, Cascino G , Cavazos J, Parada I , Ramirez L.
Mossy fiber synaptic reorganization in the epileptic human
temporal lobe. Ann Neurol 1989;26:321-30.
7. Houser CR, Miyashiro JE, Swartz BE, Walsh GO, Rich JR,
Delgado-Escueta VA. Altered patterns of dynorphin immunoreactivity suggest mossy fiber reorganization in human
hippocampal epilepsy. J Neurosci 1990;10:267-82.
8. Babb TL, Kupfer WR, Pretorius JK, Crandall PH, Levesque
MF. Synaptic reorganization by mossy fibers in human epileptic fascia dentata. Neuroscience 1984;42:351-63.
9. Isokawa M, Levesque MF, Babb TL, Engel J Jr. Single
mossy fiber axonal systems of human dentate granule cells
studied in hippocampal slices from patients with temporal
lobe epilepsy. J Neurosci 1993;13(4):151 1-22.
10. Geddes JW, Cahan LD, Cooper SM, Kim RC, Choi BH,
Cotman CW. Altered distribution of excitatory amino acid
receptors in temporal lobe epilepsy. Exp Neurol 1990;108:
214-20.
11. Hosford DA, Crain BJ, Cao Z, et al. Increased AMPAsensitive quisqualate receptor binding and reduced NMDA
receptor binding in epileptic human hippocampus. J Neurosci 1991;11:428-34.
12. Margerison JH, Corsellis JAN. Epilepsy and the temporal
lobes: a clinical, electroencephalographic and neuropathological study of the brain in epilepsy, with particular reference to the temporal lobes. Bruin 1966;89:499-530.
13. Isokawa M, Avanzini G, Finch DM, Babb TL, Levesque
MF. Physiological properties of human dentate granule cells
in slices prepared from epileptic patients. Epilepsy Res 1991;
9:242-50.
14. Masukawa LM, Higashima M, Hart GJ, Spencer DD,
O’Connor MJ. NMDA receptor activation during epileptiform responses in the dentate gyrus of epileptic patients.
Bruin Res 1991;562: 176-80.
Epilepsio, V o l . 37, N o . 9 , 1996
15. Urban L, Aitken PG, Friedman A, Somjen GG. An NMDAmediated component of excitatory synaptic input to dentate
granule cells in “epileptic” human hippocampus studied in
vitro. Bruin Res 1990;515:319-22.
16. Rutecki PA, Lebeda FJ, Johnston D. 4-Aminopyridine produces epileptiform activity in hippocampus and enhances
synaptic excitation and inhibition. J Neurophysiol 1987;57:
191 1-24.
17. Rutecki PA, Lebeda FJ, Johnston D. Epileptiform activity in
the hippocampus produced by tetraethylammonium. J Neurophysiol 1990;64: 1077438.
18. Louvel J , Heinemann U. Changes in [Ca”],,, [K+],, and
neuronal activity during oenanthotoxin-induced epilepsy in
cat sensorimotor cortex. Electroencephulogr CIin Neurophysiol 1983;56:457-66.
19. Baranyi A, Feher 0. Convulsive effects of 3-aminopyridine
on cortical neurones. Electroencephulogr CIin Neurophysiol
1979;47:745-5 1.
20. Velluti JC, Caputi A, Macadar 0. Limbic epilepsy induced
in the rat by dendrotoxin, a polypeptide isolated from the
green mamba venom. Toxicon 1987;25:649-57.
21. Silveira R, Siciliano J , Abo V, Veira L , Dajas F. lntrastriatal
dendrotoxin injection: behavioural and neurochemical effects. Toxicon 1988;26:1009-15.
22. Beck H, Ficker E, Heinemann U. Properties of two voltagedependent potassium currents in acutely isolated juvenile rat
dentate gyrus granule cells. J Neurophysiol 1992;68:208699.
23. Heinemann U, Albrecht D, Beck H, Ficker E, von Haebler
D, Stabel J . Delayed potassium regulation and potassium
current maturation as factors of enhanced epileptogenicity
during ontogenesis of the hippocampus of rats. In: Engel J
Jr, Wasterlain C, Cavalheiro EA, Heinemann U , Avanzini
G, eds. Molecular neurobiology of epilepsy. Epilepsy Re.?
Suppl 1992;9:107-14.
24. Kim JH, Guimareas PO, Shen MY, Masukawa LM, Spencer
DD. Hippocampal neuronal density in temporal lobe epilepsy with and without gliomas. A c t u Neuroputhol 1990;80:
41-5.
25. Fried I , Kim J H , Spencer DD. Limbic and neocortical gliomas associated with intractable seizures: a distinct clinicopathological group. Neurosurgery 1994;34:8 15-23.
26. Kay AR, Wong RKS. isolation of neurons suitable for patchclamping from adult mammalian central nervous systems. J
Neurosci Methods 1986;16:227-38.
27. Mody I , Salter MW, MacDonald JF. Whole-cell voltageclamp recordings in granule cells acutely isolated from hippocampal slices of adult or aged rats. Neurosci Lett 1989;
96:7&5.
28. Presurgical evaluation: University of Bonn. In: Engel J , ed.
Surgicul freutrnent of the epilepsies. 2nd ed. New York:
Raven Press, 1993:Appendix II, 740-2.
29. Wyllie E, Awad I. lntracranial EEG and localization studies.
In: Wyllie E, ed. Treutment of epilepsy: principles und pructice. Philadelphia: Lea & Febiger, 1993:1023-38.
30. Behrens E , Zentner J , Van Roost D, Hufnagel A, Elger CE,
Schramm J. Subdural and depth electrodes in the presurgical
evaluation of epilepsy. A c t u Neurochir 1994;128:84-7.
31. Blumcke I , Wolf HK, Hof PR, Morrison JH, Wiestler OD.
Regional distribution of the AMPA glutamate receptor subunits GluR2(4) in human hippocampus. Bruin Res 1995;682:
23944.
32. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pj7uger.s Arch 1981;391:85-100.
33. Sigworth FJ, Affolter H , Neher E . Design of the EPC-9, a
computer-controlled patch-clamp amplifier. 2. Software. J
Neurosci Methods 1995;56:203-15.
34. Gillis KD. Techniques for membrane capacitance measurements. In: Neher E, Sakmann B, eds. Single chunnelrecording. New York: Plenum Press, 1994:xx-xx.
35. Vreugdenhil M, Wadman WJ. Ionic currents in CAI pyra-
I , IN HUMAN HIPPOCAMPAL GRANULE CELLS
midal neurons in the rat after kindling epileptogenesis. Epilepsy Res Suppl (in press).
36. Tsaur ML, Sheng M, Lowenstein DH, Jan YN, Jan LY.
Differential expression of potassium channel mRNAs in rat
brain and downregulation in the hippocampus following seizures. Neuron 1992;8:1055-67.
37. Stabel J, Ficker E, Heinemann U . Young CAI pyramidal
cells of rats, but not dentate gyrus granule cells, express a
delayed inward rectifying current with properties of I,. Neurosci Lett 1992;135(2):2314.
38. Eder C, Ficker E , Gundel J, Heinemann U . Outward currents in rat entorhinal cortex stellate cells studied with conventional and perforated patch recordings. Eur J Ncurosci
1991;3: 1271-80.
39. Mody I, Stanton PK, Heinemann U . Activation of NMDA
receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling. J Neurophysiol 1988;50:1033-54.
901
40. Williamson A, Spencer DD, Shepherd GM. Comparison between the membrane and synaptic properties of human and
rodent dentate granule cells. Bruin Res 1993;622(1,2):194202.
41. Masukawa LM, Uruno K, Sperling M, O’Connor MJ, Burdette LJ. The functional relationship between antidromically
evoked field responses of the dentate gyrus and mossy fiber
reorganization in temporal lobe epileptic patients. Bruin Res
1992;579:119-27.
42. Cronin J, Obenaus A, Houser CR, Dudek FE. Electrophysiology of dentate granule cells after kainate-induced synaptic
reorganization of the mossy fibers. Bruin Res 1992;474:
1814.
43. Dudek FE, Obenaus A, Schweitzer JS, Wuarin JP. Functional significance of hippocampal plasticity in epileptic
brain: electrophysiological changes of the dentate granule
cells associated with mossy fiber sprouting. Hippocumpus
I994;4(3):259-65.
Epilepsiu, Vol. 37, No. 9, 19%