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Published in final edited form as:
Biochem Biophys Res Commun. 2012 March 30; 420(1): 156–160. doi:10.1016/j.bbrc.2012.02.133.
Hyperpolarization-activated cation current Ih of dentate gyrus
granule cells is upregulated in human and rat temporal lobe
epilepsy
Rainer Surgesa,b,*, Maria Kukleyc, Amy Brewsterd, Christiane Rüschenschmidta,b,
Johannes Schrammc, Tallie Z. Baramd, Heinz Becka,b,e, and Dirk Dietrichc
aDepartment of Epileptology, University of Bonn Medical Center, Sigmund-Freud-Str. 25, 53105
Bonn, Germany
bLaboratory
of Experimental Epileptology and Cognition Research, University of Bonn Medical
Center, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
cDepartment
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of Neurosurgery, University of Bonn Medical Center, Sigmund-Freud-Str. 25, 53105
Bonn, Germany
dDepartments
of Anatomy/Neurobiology and Pediatrics, University of California at Irvine, Irvine,
CA 92697-4475, USA
eDeutsches
Zentrum für Neurodegenerative Erkrankungen e.V., Ludwig Erhard Allee 2, 53175
Bonn, Germany
Abstract
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The hyperpolarization-activated cation current Ih is an important regulator of neuronal excitability
and may contribute to the properties of the dentate gyrus granule (DGG) cells, which constitute
the input site of the canonical hippocampal circuit. Here, we investigated changes in Ih in DGG
cells in human temporal lobe epilepsy (TLE) and the rat pilocarpine model of TLE using the
patch-clamp technique. Messenger-RNA (mRNA) expression of Ih-conducting HCN1, 2 and 4
isoforms was determined using semi-quantitative in-situ hybridization. Ih density was ~1.8-fold
greater in DGG cells of TLE patients with Ammon’s horn sclerosis (AHS) as compared to patients
without AHS. The magnitude of somatodendritic Ih was enhanced also in DGG cells in epileptic
rats, most robustly during the latent phase after status epilepticus and prior to the occurrence of
spontaneous epileptic seizures. During the chronic phase, Ih was increased ~1.7-fold. This increase
of Ih was paralleled by an increase in HCN1 and HCN4 mRNA expression, whereas HCN2
expression was unchanged. Our data demonstrate an epilepsy-associated upregulation of Ih likely
due to increased HCN1 and HCN4 expression, which indicate plasticity of Ih during
epileptogenesis and which may contribute to a compensatory decrease in neuronal excitability of
DGG cells.
© 2012 Elsevier Inc. All rights reserved.
*
Corresponding author at: Department of Epileptology, University of Bonn Medical Center, Sigmund-Freud-Str. 25, 53105 Bonn,
Germany. Fax: +49 228/ 28719351. rainer.surges@googlemail.com (R. Surges)..
Author contributions: R.S. has contributed to the design of the experiments, data analysis and has written the manuscript. M.K. has
performed the recordings in human hippocampus and contributed to data analysis. A.B. has done the in-situ immunhistochemistry,
T.B.Z. has contributed to the design of the experiments, data analysis and writing of the manuscript. C.R. has performed the
recordings in the rat pilocarpine model, respective data analysis and written a prior version of the manuscript. J.S. has provided
neurosurgical specimen including informed consent. H.B. and D.D. have contributed to the design of the experiments, data analysis
and writing of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2012.02.133.
Surges et al.
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Keywords
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Hippocampus; Rat pilocarpine model; Patch-clamp; h-Current; Human; Temporal lobe epilepsy
1. Introduction
The hyperpolarization-activated current Ih is a slowly activating, non-inactivating
depolarizing cationic current which is activated by hyperpolarization beyond −50 to −70
mV. These unique biophysical properties provide the basis for the diverse roles ascribed to
this current, i.e. control of resting membrane potential, passive membrane properties,
pacemaker activity, rebound burst firing in heart and brain, reduction of dendritic
summation and the presence of certain types of resonance behavior in neurons [1-8].
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The specific function of h-channels in each neuronal population depends strongly on the
voltage- and time-dependent properties of Ih. An important molecular mechanism for
generating functionally diverse Ih is the differential expression of the four underlying
HCN1-4 subunits. Homomeric channels formed by these HCN subunits display very
different kinetics, steady-state voltage dependence and sensitivity to modulation by cAMP
in heterologous expression systems [2]. Thus, regulation of the relative abundance of HCN
subunit protein is likely to represent a key mechanism for plasticity of Ih. Importantly, the
functional properties of Ih and the expression of the corresponding subunits are differentially
modulated during postnatal development as well as in both acquired and genetic forms of
epilepsy in a region- and time-dependent manner in different in vitro and in vivo models
[6,9-18]. An emerging “leitmotiv”, at least in CA1 hippocampal or neocortical neurons,
seems to be the downregulation of the HCN1-subunit along with a decrease in
somatodendritic Ih, followed, via an enhanced input resistance, by neuronal hyperexcitability
in the chronic state.
Thus, a significant body of work has implicated alteration of HCN channels expression and
altered magnitude and properties of Ih in epilepsy [reviewed in 8,19]. In temporal lobe
epilepsy (TLE), the large majority of existing studies has focused on animal models and on
the hippocampal pyramidal cell layer [20]. Remarkably, an early study suggested that
changes in HCN expression might arise also in the dentate gyrus [21]. Therefore, here, we
focus on the DGG cells, and examine somatodendritic Ih currents and the HCN expression
levels in DGG cells of people with TLE and underlying Ammon’s horn sclerosis (AHS) and
in the rat pilocarpine-model of TLE.
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2. Material and methods
2.1. Pre- and postsurgical assessment of epilepsy patients
All patients had conventional scalp EEG recordings or intracranial recordings and brain
MRI prior to neurosurgery. The histological grading of human hippocampus was performed
according to Wyler and colleagues [22]. Investigation of human cerebral tissue was
approved by the local ethics committee and written informed consent was obtained from
each patient.
2.2. Pilocarpine-induced status epilepticus (SE)
Procedures on animals were performed in accordance with local guidelines and experiments
were approved by the local animal care and use committee. Pilocarpine treatment of rats was
carried out and animals were monitored as described in Supplementary material.
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2.3. Semi-quantitative in situ hybridization (ISH) in rat hippocampus
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ISH was carried out as described previously [10,21,23,24] on specimens obtained 30-60
days after SE. For details see Supplementary material.
2.4. Electrophysiology
Electrophysiological recordings of DGG cells were performed in both human and rat
hippocampal slices using conventional patch clamp technique and established experimental
procedures. See Supplementary material for details.
2.5. Data analysis
Ih amplitudes in response to voltage steps were determined as the difference of the end of
the instantaneous current component and the current amplitude in the steady-state. The
reversal potential Vrev, was determined by analysis of tail currents and subsequent linear
regression analysis (see Fig. 1C and Supplementary material). The conductance-voltage
curves were fitted with a Boltzmann function of the form.
(1)
with V50 being the voltage of half-maximal activation.
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The activation time course of Ih was fit with exponential functions of the form.
(2)
Unpaired student’s t-test was used for statistical analyses. P values <0.05 were considered as
significant. All data are given as means ± SEM.
3. Results
3.1. Ih recordings in human TLE
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Properties of Ih recorded in DGG cells from seven TLE patients with AHS (AHS-group)
were compared to those from three patients without AHS (non-AHS). The clinical data of
the patients are summarized in Supplementary Table 1. The intrinsic membrane properties of
DGG cells were not significantly different between both groups (Supplementary Table 2).
Gross morphology was not different between groups, as described previously [25].
However, upon a hyperpolarizing voltage step from −50 to −90 mV for 5 s, we observed
slowly developing inward current which was more pronounced in the AHS-group (Fig. 1A1
and B1). These currents were blocked by the Ih-channel blocker ZD7288 (40 μM, n = 8) in
neurons from both tissues (Fig. 1A2 and B2) and had similar reversal potentials (AHS:
−38.1 mV, n = 7; non-AHS group: −39.4 mV, n = 6; Fig. 1C), consistent with reported
reversal potentials of Ih [5,13]. Application of 2 mM Cs+ in two recordings also blocked this
current component (data not shown). Taken together, these findings indicate that the
recorded time-dependent inward currents were a result of activation of Ih channels.
The mean Ih amplitudes measured with hyperpolarizing voltage steps from −50 to −90 mV
(p = 0.0056, n = 8 non-AHS; n = 7 AHS; Fig. 1D) and Ih current densities normalized to the
cell capacitance were significantly higher in the AHS-group (p = 0.0077, n = 8 nonAHS; n =
7 AHS; Fig. 1D). The voltage-dependence of activation was significantly shifted to more
depolarized potentials in the AHS-group (non-AHS group: V50 = −85.9 ± 2.3 mV; AHSgroup: V50 = −78.2 ± 2.7 mV; p = 0.0001, Fig. 2A and D). The kinetics of Ih activation
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contained a fast and a slow component, and was examined by fitting with a bi-exponential
function. The time constants of activation did not differ between groups (Fig. 2B). Likewise,
the relative contribution of the fast current component to the total current amplitude varied
between 66% and 73% in a voltage range between −90 and −120 mV in both groups and
was not significantly different (Fig. 2C).
3.2. Ih recordings in rat pilocarpine model of TLE
Similar to the human, a slowly activating, non-inactivating inward conductance was seen
upon voltage steps to −103 mV which was completely blocked by ZD7288 (60 μM) both in
control (n = 22) and epileptic rats (n = 22, Fig. 3A, upper traces). Subtraction of the two
traces allowed isolating Ih (Fig. 3A, lower trace).
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We examined the magnitude of Ih in control and pilocarpine treated rats at two different time
points: 7–9 days following SE (latent phase, no spontaneous generalized seizures) and in the
chronic epileptic phase 35–63 days following SE (all spontaneous generalized seizures). Ih
density was enhanced in both groups, but most pronouncedly in the latent period (Fig. 3B, p
< 0.0001 and p = 0.0041 for the latent and chronic group, respectively). This increase could
not be explained by a change in driving force, as the reversal potential of Ih did not differ
between groups (controls: −35.9 ± 2.3 mV, n = 8; pilocarpine-treated rats: −36.8 ± 2.3 mV,
n = 8). The time course of activation was best fit with a mono-exponential equation with no
significant differences between controls and pilocarpine-treated animals neither at early nor
at late stages following SE (Fig. 3C).
To understand the neurobiological basis of the persistent increase in Ih in DGG cells of
epileptic rats, we investigated the expression of the genes coding for the h-channels. We
compared mRNA expression of HCN1, 2 and 4 channel isoforms in chronically epileptic,
pilocarpine-treated rats versus the control group. Whereas HCN2 expression was not
different in both groups (not shown), expression of HCN1 mRNA was significantly
increased by 22% in the pilocarpine-treated (67.9 ± 4.0 nCi/gm) compared to control rats
(55.4 ± 3.2 nCi/gm; p < 0.05, Fig. 4A). This effect was more pronounced for the HCN4
channel isoform. HCN4 mRNA level was enhanced by 52% in the granule cell layer of
epileptic (70.75 ± 6.4 nCi/gm) relative to controls rats (46.75 ± 1.0 nCi/gm; p < 0.05; Fig.
4B).
4. Discussion
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The principal findings of the current studies are that (1) Ih density is greater in granule cell
of the dentate gyrus in TLE patients with AHS as compared to those without AHS. (2)
Similar findings are observed in the rat pilocarpine-model of TLE, where Ih changes precede
the onset of chronic epilepsy. (3) Voltage-dependence but not kinetics of Ih is altered in the
epileptic state, which might result from augmented contribution of HCN1 and HCN4 to the
total HCN channels pool that conducts Ih.
4.1. Molecular mechanism of Ih upregulation
The observed upregulation of Ih in both human and rat DGG cells is most likely due to an
enhanced expression of HCN1 and, at least in rat hippocampus, HCN4 subunit, as shown in
Fig. 4 and as found previously [21]. This is in line with the observed shift of voltagedependence of Ih to more depolarized potentials in tissue from people with TLE due to AHS.
In general, h-channels composed of HCN1 subunits display faster Ih activation than those
composed of HCN2 and HCN4 subunits [13,26]. Strikingly, current kinetics was not faster
in the AHS-group or in epileptic rats as compared to controls. This might be a result of the
concomitant increase of HCN4 subunits. Furthermore, the properties of Ih in native
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hippocampus only partially overlap those of currents recorded in heterologous systems: in
human and rat hippocampus, in addition to the intrinsic biophysical properties of each HCN
isoform, a number of interacting molecules influence channel properties [18,27, reviewed in
28].
What are the molecular mechanisms and pathways involved in the upregulation of Ih and
HCN channels in DGG cells? Although this question has not been directly addressed in the
present work, a number of recent studies provide insights into regulation of HCN expression
in association with seizures and epilepsy. In the kainic acid-induced SE model of TLE,
downregulation of HCN1 expression in CA1 pyramidal region was caused by an
upregulation of a repressing transcription factor, neuron-restrictive silencer factor (NRSF)
which suppresses transcription of a number of genes, amongst them the HCN1 gene [20]. In
addition, a reduction of HCN1 expression has been shown to involve transient Ca2+permeable AMPA receptor activation with subsequent activation of Ca2+/calmodulindependent protein kinase II during in-vitro experiments in hippocampal slices [29]. Little is
known, however, about mechanisms of upregulation of HCN expression. Reduction of
NRSF levels might take place, or the enhanced expression might result from a myriad of
cellular mechanisms that influence ion channel expression [30].
4.2. Significance for the pathophysiology of TLE and potential clinical implications
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In previous studies, Ih expression appears to be differentially modulated in acquired and
genetically caused epilepsies in a region- and time-dependent manner. For instance, in rat
models of genetically caused absence epilepsies, impaired Ih with a concomitant decrease of
HCN1 expression was found in cortical neurons [12], whereas thalamocortical neurons
showed an decrease in cAMP responsiveness, explained by an increase of HCN1 expression
[31,32]. Both phenomena favor onset of absence seizures. In models of acquired TLE, Ih
currents and expression of underlying HCN1 and HCN2 subunits were temporarily reduced
in dendrites of the entorhinal cortex following kainate-induced SE [11]. A further study
found sustained reduction in HCN1 and 2 expression following kainate-induced SE in the
CA1 region [16]. Likewise, in the rat pilocarpine model of TLE, dendritic Ih of CA1
pyramidal cells was progressively reduced during epileptogenesis, because of permanent
reduction of HCN1-subunit expression and a persistent alteration of Ih-modulating
phosphorylation pathways [6,15,18]. The significance of HCN1 downregulation is further
strengthened by the observation that a primary loss of the HCN1 subunit facilitates onset of
seizures and of epilepsy [33,34]. In a model of seizure-inducing hypoxia, somatic Ih in rat
CA1 pyramidal was decreased [14], and a complex region-specific regulation of HCN1 and
2 in an in vitro model of epilepsy has been described, with commensurate alteration of burst
activity [17].
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Taken together, downregulation of Ih seems to be a leitmotiv in the early phases of
epileptogenesis and epilepsy. However, relatively few studies in animal models have
explored potentially compensatory alterations in HCN isoform expression and Ih in the
chronic and even ‘end stage’ of TLE. Even fewer yet are studies in human tissue. In human
neocortical neurons of patients with TLE, Wierschke and co-workers reported on reduced Ih
density in patients with higher seizure frequencies; these observations support the notion
that Ih contributes importantly to the regulation of neuronal excitability [35].
Focusing on the dentate gyrus, a major gateway into the hippocampal formation, we report
here a robust, epilepsy-associated upregulation of Ih in DGG of human and rat hippocampus.
Our findings are in line with a recent study reporting enhanced Ih in DGG of people with
TLE with severe AHS as compared to those with mild or moderate AHS [36]. Whereas the
mechanisms leading to this increase of HCN1 expression and Ih density are not fully
understood, the teleological purpose is intriguing: augmented Ih in the epileptic dentate
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gyrus might serve as an attempt to compensate for abnormal hyperexcitability in the circuit
for example, by dampening dendritic summation [1]. Alternatively, in response to
augmented inhibition (hyperpolarizing), the increase in Ih might promote rebound
depolarization and contribute to hyperexcitability of epileptic tissue [5,9]. Whereas other ion
channels are also altered in epileptic DGG cells [37,38], the changes in Ih reported here
should contribute to neuronal firing behavior, and might provide a drug target to both
existing and future medications (see below).
4.3. Temporal profile of Ih upregulation
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In human tissue, it is not possible to time the onset of HCN expression and Ih changes,
because tissue is only available from chronic epileptic tissue. Therefore, the current studies
employed also tissue from animal models. In the rat pilocarpine model employed here, an Ih
upregulation was already present in the latent stage preceding the onset of spontaneous
seizures, and may thus contribute to their onset. This increased Ih was associated with, and
hence is likely a result of, an upregulation of both HCN1 and HCN4 mRNA in neurons of
the dentate gyrus. Given the importance of Ih in attenuating postsynaptic potentials at
dendrites and limiting their spread in pyramidal neurons of CA1 region and layer five of the
somatosensory cortex [39,40], our data suggest that upregulation of Ih might be a protective
mechanism resulting in maintained dentate gyrus gating. Dendrites of DGG neurons in rats
without epilepsy have no or little Ih [41]. In view of the considerable increase of Ih, it is
tempting to speculate that somatodendritic Ih in DGG neurons plays a role in signal
integration only under pathophysiological circumstances such as epileptogenesis and chronic
epilepsy, but not under physiological conditions.
4.4. Implications for epilepsy therapy
Ih is a target for the anticonvulsants acetazolamide, gabapentin and lamotrigine, all of which
increase Ih [42-44]. It is therefore also possible that the observed upregulation of Ih renders
its modulation by these antiepileptic drugs more effective, thereby dampening excitability.
Such a mechanism operating in DGG cells may be particularly relevant in chronic epilepsy,
when granule cells are one of the major preserved cell populations after the profound loss of
neurons in the CA1, CA3 and CA4 subfields. Therefore, defining and understanding the
changes in Ih and in its ‘building blocks’, the HCN channels, both in humans and animal
models is important. The knowledge is a prerequisite for using this important channel as
target for epilepsy therapy and prevention in the future.
Supplementary Material
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Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by the German–Israel collaborative research program of the Bundesministerium für
Bildung und Forschung (BMBF), the Ministry of Science (MOS), the Deutsche Forschungsgemeinschaft (DFG)
Projects SFB-TR3 (H.B., D.D.), DI 853/3 and DI 853/4 (D.D.), the BONFOR Program of the University of Bonn,
and NIH Grant NS35439. The sponsors had no role in study design, in the collection, analysis and interpretation of
data or in the writing of the report. The authors declare no conflict of interest.
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Biochem Biophys Res Commun. Author manuscript; available in PMC 2013 March 30.
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Fig. 1.
Ih density is greater in DGG neurons of people with TLE due to AHS. Ih is elicited by
voltage steps from −50 to −90 mV in both DGG neurons from the non-AHS (A1) and AHSgroup (B1). Scaling 10 pA/0.5 s. The inward current is blocked by ZD7288 in both groups
(A2, B2). Scaling 50 pA/0.1 s. (C) Reversal potential was around −39 mV in both conditions
(upper panel, tail currents indicated with an asterisk, Scaling 100 pA/0.5 s; bottom panel;
filled symbols: non-AHS group; open symbols: AHS-group). (D) Ih amplitudes (left panel)
and current densities at −90 mV (right panel) were significantly greater in people with AHS.
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Fig. 2.
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Activation properties of Ih in human DGG neurons. (A) A family of current traces was
elicited by stepping the voltage from −50 mV to more hyperpolarized potentials in 10 mVsteps (from −60 to −120 mV) in the non-AHS (upper panel) and AHS-group (lower panel).
Scaling 100 pA/1 s. (B) Fast and slow time constants were not different in both conditions at
different command potentials (filled symbols: non-AHS group; open symbols: AHS-group).
(C) The amplitudes from the fast (the upper data points under each condition) and the slow
time component (the lower data points under each condition) were derived from the biexponential fitting paradigms. There was no significant difference in the relative
contributions of the fast and slow current component between both conditions. Symbols as
in (B). (D) The voltage-dependence of activation was significantly shifted to more
depolarized potentials in the AHS-group. Symbols as in (B).
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Fig. 3.
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Ih density is upregulated in DGG cells of rat hippocampal slices in the latent and chronic
phase following pilocarpine-injection. (A) Inward currents elicited upon a voltage step from
−63 to −103 mV in the absence and presence of ZD7288 (upper panel, indicated by an
arrow). Lower trace obtained after subtraction of traces in the absence and presence of
ZD7288. Recordings from a control animal. Scaling 10 pA/0.5 s. (B) Ih current densities
during the latent and chronic phase (sham-control animals: black bars, SE-experienced
animals: white bars). (C) Time constants at a potential of −103 mV were not different in
controls and pilocarpine rats during latent (p = 0.49, n = 9 and 10 cells) and chronic phase (p
= 0.96, n = 13 and 12 cells). Symbols as in (B).
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Fig. 4.
Expression of both HCN1 and HCN 4 subunits is increased in epileptic animals following
pilocarpine-induced SE. As depicted in the photomicrographs (A and B lower panels),
HCN1 and HCN4 subunit mRNA signals were significantly increased in the granule cell
layer (GCL) of the pilocarpine-group (A and B upper panels), with relatively little change in
expression over the pyramidal cell layer of CA3. Brains from 4-9 animals were used per
group. Asterisk indicates p < 0.05.
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