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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Epilepsia. 2010 Aug;51(8):1624–1627. doi: 10.1111/j.1528-1167.2010.02554.x

Increased seizure severity and seizure-related death in mice lacking HCN1 channels

Bina Santoro 1, Janet Y Lee 5, Dario J Englot 5, Sandra Gildersleeve 3, Rebecca A Piskorowski 1, Steven A Siegelbaum 1,2,4, Melodie R Winawer 3, Hal Blumenfeld 5
PMCID: PMC2952649  NIHMSID: NIHMS199406  PMID: 20384728

Summary

Persistent down-regulation in the expression of the hyperpolarization-activated HCN1 cation channel, a key determinant of intrinsic neuronal excitability, has been observed in febrile seizure, temporal lobe epilepsy and generalized epilepsy animal models, as well as patients with epilepsy. However, the role and importance of HCN1 downregulation for seizure activity is unclear. To address this question we determined the susceptibility of mice with either a general or forebrain-restricted deletion of HCN1 to limbic seizure induction by amygdala kindling or pilocarpine administration. Loss of HCN1 expression in both mouse lines is associated with higher seizure severity and higher seizure-related mortality, independent of the seizure induction method used. Thus, downregulation of HCN1 associated with human epilepsy and rodent models may be a contributing factor to seizure behavior.

Keywords: intrinsic excitability, Ih conductance, HCN channels, limbic seizures, kindling, pilocarpine

Introduction

The intrinsic excitability of a neuron depends on the ion channels present on its plasma membrane. Alterations in channel biophysical properties, spatial distribution, or expression allow for a high degree of neuronal plasticity that can serve an adaptive role during learning or homeostatic regulation. However, plasticity in intrinsic excitability can also be maladaptive, destabilizing neuronal activity and potentially contributing to epileptogenesis.

The HCN1 isoform of the hyperpolarization-activated cation channel (Robinson and Siegelbaum, 2003) shows altered expression in patients with epilepsy (Bender et al., 2003; Wierschke et al., 2009) and several animal models of epilepsy (Brewster et al., 2002; Shah et al 2004; Jung et al, 2007; Shin et al., 2008). Both increased or ectopic HCN1 expression in brain structures that normally show little or no HCN1 expression (Bender et al., 2003; Budde et al., 2005) and decreased HCN1 expression in brain structures that normally exhibit high levels of HCN1 (Brewster et al., 2002; Shah et al 2004; Jung et al, 2007; Shin et al., 2008; Wierschke et al., 2009) have been reported. As HCN1 channels dampen excitability in cortical pyramidal neurons (Robinson and Siegelbaum, 2003), it has been suggested in particular that HCN1 down-regulation at this site may contribute to limbic epileptogenesis (but see Santoro and Baram, 2003; Brewster et al., 2006).

As a first step to address the role of HCN1 in regulating seizure activity, we examined mice in which HCN1 was deleted globally (Nolan et al., 2003) or selectively in the forebrain (Nolan et al., 2004). Since neither of these lines displays spontaneous seizures (Nolan et al., 2003, 2004; Huang et al., 2009), we examined their susceptibility to limbic seizure induction using both a chronic electrical stimulation model (amygdala kindling), and an acute chemical induction model (pilocarpine injection).

Our findings indicate that HCN1 deletion is associated with increased seizure severity and increased seizure-related death, independent of the seizure induction protocol used. Furthermore, selective loss of HCN1 channels in the forebrain is sufficient to produce the susceptibility phenotype, suggesting that HCN1 channels might play a role in limiting the spread of seizures from limbic/forebrain to hindbrain regions.

Methods

Animals

General HCN1 knockout (HCN1−/−) mice were obtained from The Jackson Laboratory (stock 005034), and crossed to wild-type B6129SF1/J mice (stock 101043) yielding heterozygous HCN1+/− mice in a mixed C57BL/6J, 129SvEv, 129SvImJ background. All experimental animals were generated from intercrossing these HCN1+/− mice to obtain homozygous HCN1−/− and wild-type HCN1+/+ littermate controls. Forebrain-restricted HCN1 knockout (HCN1f/f,cre) mice were bred as described (Nolan et al., 2004), with HCN1f/f × HCN1f/f,cre crosses yielding knockout and wild-type littermates in a mixed 129SvEv:C57BL/6J background. Only male mice were used for experiments. Mice were housed in a temperature and humidity controlled environment, with a 12 h light/dark schedule, and food and water available ad libitum. All procedures were in full compliance with approved institutional animal care and use protocols.

Amygdala kindling

Kindling was performed as described (Blumenfeld et al. 2009). Briefly, animals were stereotaxically implanted with bipolar electrodes in the right amygdala, with electrode positioning confirmed by cresyl violet staining at the conclusion of the experiment. One week after surgery, afterdischarge thresholds were determined for each animal, by titrating the current stimulus starting at 40 µA and increasing by 20 µA until an afterdischarge lasting ≥ 3 s was observed on the EEG. The stimulus train consisted of square biphasic (1 ms each phase) pulses at 60 Hz with train duration of 1 s. The threshold stimulus for each animal was then administered twice daily until full kindling was achieved. For behavioral seizure rating, a modified Racine scale was employed, as described in Butler et al. (1995): 1. Immobility, facial clonus; 2. Head nodding; 3. Unilateral forelimb clonus; 4. Bilateral forelimb clonus; 5. Rearing and limb clonus with loss of postural control; 6. Running or bouncing seizure; 7. Tonic hindlimb extension; 8. Tonic hindlimb extension culminating in death. Animals were considered fully kindled when they had three consecutive class 5 (generalized) seizures.

Pilocarpine injection

Seizure induction was performed as described in Winawer et al. (2007). Briefly, mice were administered atropine methyl nitrate (5 mg/kg, intraperitoneally) 30 min before pilocarpine (300 mg/kg, intraperitoneally). Animals were observed continuously for 2 h after pilocarpine administration, and seizure behaviors recorded with their time of onset in minutes from injection. Pilocarpine induced seizures were rated using a modification of the Racine scale, as described (Winawer et al., 2007): 1. Immobility; 2. Tremors, not continuous; 3. Continuous body tremor; 4. Rearing, limb clonus with loss of postural control, and/or bouncing seizures; 5. Tonic hindlimb extension.

Results

We first used amygdala kindling to examine the effect of the general deletion of HCN1 channels on the initiation and/or development of kindling-induced epileptogenesis. We compared the average afterdischarge threshold, number of stimuli required to obtain full kindling, and maximum seizure severity between HCN1−/− (KO) mice and their HCN1+/+ wild-type littermate controls. Animals were age matched, to avoid confounding effects of age on seizure susceptibility (Age, mean ± s.e.m.: HCN1+/+, 127 ± 3 days, range 105–135, n=10; HCN1−/−, 129 ± 5 days, range 105–146, n=8).

We did not detect significant changes in the afterdischarge threshold or number of stimuli needed to obtain full kindling, defined as three consecutive seizures with behavioral ratings of at least class 5 (Afterdischarge threshold, mean ± s.e.m.: HCN1+/+, 304 ± 52 µA, n=10; HCN1−/−, 277 ± 41 µA, n=8. Number of stimuli, mean ± s.e.m.: HCN1+/+, 13.8 ± 2.3, n=10; HCN1−/−, 11.8 ± 1.7, n=8) (Fig. 1). However, maximum seizure severity was significantly increased in the HCN1−/− group (mean ± s.e.m.: HCN1+/+, 5.9 ± 0.1, n=10; HCN1−/−, 7.1 ± 0.4, n=8; P < 0.02, t-test). This increase is due to the high incidence of tonic hindlimb extensions followed by death (class 8 seizures) in the knock-out animals, a behavior not observed in wild-type littermates (Mortality: HCN1+/+, 0/10 animals; HCN1−/−, 5/8 animals; P < 0.01, chi-sq; Figure 1D).

Figure 1.

Figure 1

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Amygdala kindling results in higher seizure severity and death in HCN1−/− mice. No differences were observed in mean afterdischarge threshold (A) or mean number of stimuli needed to obtain full kindling (B) between HCN−/− (knock-out) and HCN1+/+ (control) animals. However mean maximum seizure class reached (C) was higher in the HCN−/− group, due to the high incidence of tonic hindlimb extension followed by death (62.5%, shown in D). This behavior was absent in the control HCN+/+ group. Error bars indicate s.e.m. See text for numeric values.

The relevance of the general HCN1 KO to epilepsy is potentially complicated by the fact that HCN1 is normally expressed at high levels in parts of the midbrain, cerebellum, inferior olive and brainstem (Notomi and Shigemoto, 2004), areas in which HCN1 downregulation has not been implicated in epilepsy. To strengthen the potential relevance of our findings to epilepsy, we performed a second set of experiments using a forebrain-restricted HCN1 KO mouse (Nolan et al., 2004), where the pattern of HCN1 deletion more closely matches the pattern of HCN1 down-regulation observed in temporal lobe epilepsy. In addition, to assess the relevance of our results to other acute epilepsy models, we examined seizures induced by intraperitoneal injections of pilocarpine.

We compared seizure progression, including latency to first generalized seizure, and maximum seizure severity between HCN1f/f,cre (KO) mice and their HCN1f/f wild-type littermate controls. As above, animals were age matched to avoid confounding effects of age on seizure susceptibility (Age, mean ± s.e.m.: HCN1f/f, 86.5 ± 2.4 days, range 74–106, n=15; HCN1f/f,cre, 86.1 ± 2.4 days, range 72–104, n=15).

Results are illustrated in Figure 2. Seizure progression, measured by maximum seizure class reached in each successive bin of 15 minutes (Figure 2A), was not significantly different between the two groups, despite a trend for faster progression in knock-out animals. However, latency to first generalized seizure was decreased for knock-outs relative to controls (Average latency to class 4, mean ± s.e.m.: HCN1f/f, 58 ± 2’, n=15; HCN1f/f,cre, 41 ± 2’, n=15; P < 0.025, t-test). In addition, maximum seizure severity was significantly higher in the KO mice, with a much greater incidence of tonic hindlimb extension followed by death in the knock-out animals compared to controls (Mortality: HCN1f/f, 2/15 animals; HCN1f/f,cre, 8/15 animals; P < 0.025, chi-sq). Specifically, whereas animals in either group are equally likely to progress to generalized class 4 seizures, once that occurs, the HCN1f/f,cre animals rapidly escalate into tonic hindlimb extension and death (class 5). In contrast, most HCN1f/f animals remain in “status epilepticus”, alternating between class 3 and 4 through the end of the experiment (up to 60 min).

Figure 2.

Figure 2

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Limbic seizure development following pilocarpine administration in mice with forebrain restricted HCN1 channel deletion. (A) Seizure progression, illustrated as mean maximum seizure class reached by 15 min, 30 min, 45 min, 60 min or 90 min after pilocarpine administration. Error bars indicate s.e.m. Note that only about half the animals in either group progress into generalized seizures at the dose of 300 mg/kg pilocarpine used in this experiment. (B) Incidence of maximum seizure class reached during the course of the experiment. Only a small percentage of wild-type (HCNf/f) animals progress to tonic hindlimb extension and death (class 5) compared to knock-out (HCNf/f,cre) animals. See text for numeric values.

Discussion

Our results showing that general or forebrain-restricted deletion of HCN1 increases seizure intensity support the view that HCN1 downregulation observed in humans with epilepsy may be a contributing factor to disease progression. The higher seizure severity phenotype in the KO mice is characterized by an increased occurrence of seizures with tonic hindlimb extension (THE), which have been shown to be associated with activation of diencephalic, mesencephalic and brainstem structures, an activation not seen during limbic seizures (Barton et al., 2001). Accordingly, a failure or disruption in the brainstem networks that regulate autonomic function might be the origin of the mortality associated with THE seizures. As local loss of HCN1 channels in the forebrain is sufficient to increase the incidence of THE seizures and death, HCN1 channels may normally limit the spread of seizures out of the limbic/forebrain region into hindbrain regions. Additionally, abnormal neuronal excitability in the insular cortex, which has normally high levels of HCN1 (Notomi and Shigemoto, 2004), might directly contribute to seizure-related mortality due to disruption of autonomic function (Britton and Benarroch, 2006).

In contrast to the striking effects of HCN1 channel loss on maximum seizure severity and death, we found no significant difference in the threshold for seizure initiation between knockout and wild-type animals. It is possible that a small but genuine difference in threshold was obscured in our experiments by variability in the genetic background due to the use of F2 hybrid experimental animals (129SvEv:C57BL/6J; see Methods). Indeed, a recent study using HCN1 general knockout animals bred on a pure129SvEv background revealed a clear influence of the HCN1 genotype on limbic epileptogenesis and seizure susceptibility following kainate induced status epilepticus (Huang et al., 2009).

The concordance of findings obtained across three different seizure induction modalities (kindling, pilocarpine, kainate) strongly supports a model in which decreased HCN1 expression in cortical forebrain neurons is a maladaptive, pro-epileptogenic alteration. Still, these results need to be evaluated with caution due to limitations inherent to the use of acute seizure models. Moreover, while no such alterations have been reported to date in the restricted HCN1 KO used here, knockout mice may undergo compensatory changes unrelated to the knockout itself. They also have a more drastic reduction in HCN1 compared to that observed in epilepsy, and do not allow us to assess the effect of HCN1 loss in the context of other plastic changes that occur during epilepsy. Finally, HCN1 was deleted in both principal neurons and interneurons in the two mouse lines examined here. Future experiments, using newly generated mouse lines with inducible deletions limited to excitatory or inhibitory neurons will help clarify these issues, further refining our understanding of the role of HCN1 channels in the initiation, propagation and development of temporal lobe seizures.

Acknowledgments

This work was supported by a Research Grant from the Epilepsy Foundation (B.S.), NIH grants NS 366058 (S.A.S), NS 049307 (H.B.), NS 050429 and NS 061991 (M.R.W.).

Footnotes

Conflict of interest: We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. None of the authors have any conflicts of interest to disclose.

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