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- Materials and Methods
Purpose: Kv4.2 subunits contribute to the pore-forming region of channels that express a transient, A-type K+ current (A-current) in hippocampal CA1 pyramidal cell dendrites. Here, the A-current plays an important role in signal processing and synaptic integration. Kv4.2 knockout mice show a near elimination of the A-current in area CA1 dendrites, producing increased excitability in this region. In these studies, we evaluated young adult Kv4.2 knockout mice for spontaneous seizures and the response to convulsant stimulation in the whole animal in vivo and in hippocampal slices in vitro.
Methods: Electroencephalogram electrode-implanted Kv4.2 knockout and wild-type mice were observed for spontaneous behavioral and electrographic seizures. The latency to seizure and status epilepticus onset in Kv4.2 knockout and wild-type mice was assessed following intraperitoneal injection of kainate. Extracellular field potential recordings were performed in hippocampal slices from Kv4.2 knockout and wild-type mice following the bath application of bicuculline.
Results: No spontaneous behavioral or electrographic seizures were observed in Kv4.2 knockout mice. Following kainate, Kv4.2 knockout mice demonstrated a decreased seizure and status epilepticus latency as well as increased mortality compared to wild-type littermates. The background strain modified the seizure susceptibility phenotype in Kv4.2 knockout mice. In response to bicuculline, slices from Kv4.2 knockout mice exhibited an increase in epileptiform bursting in area CA1 as compared to wild-type littermates.
Discussion: These studies show that loss of Kv4.2 channels is associated with enhanced susceptibility to convulsant stimulation, supporting the concept that Kv4.2 deficiency may contribute to aberrant network excitability and regulate seizure threshold.
Kv4.2 subunits compose the pore-forming channel that contributes to the transient, rapidly activating and inactivating outward K+ current (A-current) in CA1 pyramidal cell dendrites (Kim et al., 2005; Chen et al., 2006). The A-current in this region regulates the back-propagating action potential and synaptic integration (Hoffman et al., 1997). Therefore, Kv4.2 channels are critical regulators of postsynaptic excitability, and aberrant function or loss of Kv4.2 channels is likely to facilitate hyperexcitability and potentially seizure initiation and propagation in the hippocampus.
Alterations in the Kv4.2 channel have been demonstrated in animal models of epilepsy. A decrease in Kv4.2 mRNA levels was found in rat hippocampus following pentylenetetrazol-induced seizures (Tsaur et al., 1992). In a model of cortical malformations that results in increased hippocampal excitability and a decrease in seizure threshold, there was a marked decrease in the expression of Kv4.2 channel subunits (Castro et al., 2001). Furthermore, in a rodent model of limbic seizures induced by pilocarpine, there was a significant decrease in Kv4.2 subunit expression in the hippocampus of chronically epileptic rats (Bernard et al., 2004). These studies revealed more prominent action potential backpropagation in CA1 dendrites from the epileptic compared to sham animals, indicative of increased dendritic excitability in the epileptic animals. The decrease in Kv4.2 expression in the epileptic animals was thought to underlie this effect.
Ion channelopathies have been implicated in several types of human epilepsy with complex partial seizures, including temporal lobe epilepsy (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998; Eunson et al., 2000; Klein et al., 2004; Andermann et al., 2005; Du et al., 2005). Recently in a patient with temporal lobe epilepsy, a mutation was identified in KCND2, the gene encoding Kv4.2, which resulted in a 44-amino acid truncation in the Kv4.2 carboxyl terminal (Singh et al., 2006). Expression of the Kv4.2 truncation mutant in HEK cells resulted in attenuated Kv4.2 currents (Singh et al., 2006), indicating that the truncation was functionally significant. Together, these studies suggest that loss-of-function of Kv4.2 may contribute to acute and chronic seizures.
In this report, we used Kv4.2 knockout mice to investigate whether genetic loss of Kv4.2 increases susceptibility to convulsant stimulation. Although loss of Kv4.2 at the genomic level increases hippocampal excitability and decreases the threshold for long-term potentiation (LTP) induction in area CA1, our results suggest that knockout of Kv4.2 is not associated with an epilepsy phenotype in young adult animals. However, the findings of Kv4.2 downregulation in an acute seizure model (Tsaur et al., 1992) and in chronic epilepsy (Bernard et al., 2004), and the loss-of-function Kv4.2 mutation in a patient with temporal lobe epilepsy (Singh et al., 2006) suggest that Kv4.2 may function as a candidate seizure susceptibility gene. The findings in the current study provide further support for this possibility.
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The results presented here add to the previous studies, which have shown Kv4.2 down-regulation in acute seizure (Tsaur et al., 1992) and limbic epilepsy (Bernard et al., 2004; Birnbaum et al., 2004 for review) models. Here, we have demonstrated that Kv4.2 knockout compared to wild-type littermate mice have increased sensitivity to convulsant stimulation at the whole animal level in vivo and at the network level in vitro. Systemic application of a chemoconvulsant resulted in a decreased latency to seizures and status epilepticus in Kv4.2 knockout versus wild-type littermates. The magnitude of this decrease was genetic background dependent. In concordance with the studies performed in vivo, hippocampal slice preparations from Kv4.2 knockout mice exhibited a greater increase in epileptiform activity compared to slices from wild-type mice following convulsant stimulation.
Loss of Kv4.2 channels in knockout mice was predicted to augment convulsant-induced hippocampal seizures. Indeed, convulsant stimulation with systemic kainate resulted in a decreased latency to seizures and status epilepticus in the Kv4.2 knockout compared to wild-type mice. This is consistent with the recent report showing that a decreased stimulation threshold is required to induce maximal hippocampal LTP in Kv4.2 knockout compared with wild-type mice (Chen et al., 2006). Furthermore, the simultaneous unilateral onset of electrographic seizures in the hippocampus and cortex in half of the knockout compared to none of the wild-type mice demonstrates that the loss of Kv4.2 channels leads to seizure onset with more diffuse hemispheric involvement in some animals. This finding is supported by work showing that Kv4.2 is involved in the modulation of excitability and synaptic responses in both the hippocampus and cortex (Nerbonne et al., 2008).
In this study, video-EEG monitoring of naive young adult Kv4.2 knockout mice did not reveal spontaneous seizures or epileptiform activity. However, we have observed that aged Kv4.2 knockout mice exhibit behavioral seizures in response to startle or acoustic stimuli, and show handling-induced seizures (unpublished observations). We cannot definitively exclude the possibility that the younger mice used in our studies have epileptiform activity with intermittent EEG monitoring. Continuous monitoring is currently unavailable in our animal facility; however, we used prolonged and frequent intermittent monitoring of the mice and were unable to detect any epileptiform activity in the wild-type or knockout mice under basal conditions. Therefore, if the animals were experiencing epileptiform activity and seizures, it is likely to be quite rare.
A possible explanation for the lack of spontaneous seizures in the Kv4.2 knockout mice may be related to compensatory mechanisms in these animals (Chen et al., 2006). Our findings indicate that the threshold for forebrain excitability in the Kv4.2 knockouts is lower compared to the wild-type mice, but this may not be enough to generate spontaneous epileptiform activity or seizures in the presence of compensatory mechanisms. Indeed, compensatory mechanisms have recently been reported in the Kv4.2 knockout animals (Chen et al., 2006; Andrasfalvy et al., 2008; Nerbonne et al., 2008). In the hippocampal CA1 region of Kv4.2 knockout mice, non-Kv4.2 A currents and γ-aminobutyric acid (GABA)ergic responses are elevated (Chen et al., 2006; Andrasfalvy et al., 2008). When the increase in GABAergic compensation was blocked, action potentials were more likely to be followed by burst firing in hippocampal area CA1 of the Kv4.2 knockout mice. In the cortex of Kv4.2 knockout mice, inhibitory currents IK (delayed rectifier current) and Iss (late component of the outward potassium current) are increased (Nerbonne et al., 2008). By robustly increasing excitability, such as with convulsant stimulation, the compensatory mechanisms are likely overcome, and the increase in excitability due to the loss of Kv4.2 is expressed.
The results from experiments in vitro in the hippocampal slices parallel that of the studies in vivo. There was no observable spontaneous bursting in hippocampal slices from knockout mice, and the baseline evoked responses from littermate wild-type and knockout mice showed no epileptiform activity. However, convulsant stimulation augmented seizure-like activity in slices from knockouts. In these slices, a low concentration of bicuculline (Wong et al., 1986; Traynelis & Dingledine, 1988) induced prolonged bursts with afterdischarges, resembling interictal bursting activity. This bursting activity is similar to that described with higher concentrations of bicuculline (Karnup & Stelzer, 2001) and is thought to be due to the reverbation of the hippocampal network between the CA1 and CA3 regions (Hablitz, 1984). As discussed previously, compensatory mechanisms in the knockout mice probably prevent hyperexcitability in the excitatory neuron and the local hippocampal network under basal conditions. However, when the local network is challenged with a convulsant agent, the compensatory mechanisms may not be adequate to prevent the hyperexcitability. Together, these measures indicate increased hippocampal network reverberation in the hippocampal slices from Kv4.2 knockout relative to those from wild-type animals. These findings provide further support for a role of Kv4.2 channels in regulating hippocampal excitability.
The reason for the decreased threshold in network excitability in the hippocampal slice of knockout animals is likely to be at the cellular level. In area CA1, the pyramidal dendrites exhibit a tendency to fire in burst mode when intradendritically stimulated (Wong & Stewart, 1992; Golding et al., 1999). However, this burst firing normally does not propagate into the somatic region. The dendritic A-current is a potential mechanism underlying this observation. Blocking A-current with 4-aminopyridine (4-AP) causes seizure-like activity both in vivo and in vitro (Glover, 1982; Perreault & Avoli, 1991; Traub et al., 1995). However, 4-AP is a nonspecific K+ channel blocker, which in addition to A-current, affects other presynaptic and postsynaptic K+ channel currents (Sheng et al., 1992; Klee et al., 1995; Pedarzani et al., 1998). In the Kv4.2 knockout mice used in our studies, the A-current is selectively eliminated in the CA1 hippocampal dendrites, with the preservation of non-Kv4.2 somatic A-currents (Chen et al., 2006). Indeed, when the compensatory increase in GABAergic responses was eliminated in the CA1 region of the knockout animals, action potentials were followed by burst firing (Andrasfalvy et al., 2008). Apart from the CA1 region, Kv4.2 is well-expressed in the principal neurons in other subfields of the hippocampus (Menegola & Trimmer, 2006), and a 4-AP sensitive A-current is present in these regions (Beck et al., 1992; Mitterdorfer & Bean, 2002). Therefore, Kv4.2 may underlie the A-current in these regions of the hippocampus and thereby affect the excitability of these neurons. However, the relationship between Kv4.2 and the A-current in these regions is not yet characterized. The participation of these neurons in the seizure circuit of the knockout animals could contribute to the increase in seizure susceptibility in the hippocampal slice.
Genetic background is known to affect seizure susceptibility in both animals and humans. For instance, 129S6/SvEv mice require twice as much kainate (75 mg/kg) as other mouse strains, such as C57, to maintain high-grade seizures, including status epilepticus (McKhann et al., 2003). Human, studies have shown that the phenotype of a channelopathy can be modulated by the genetic background (Schulze-Bahr et al., 1999; Mulley et al., 2003). Furthermore, the discordant expression of temporal lobe epilepsy in the patient with the Kv4.2 loss-of-function mutation, while the patient’s father who has the identical mutation does not have epilepsy (Singh et al., 2006), suggests that other factors influence the expression of seizures in this genotype. The seizure resistance of the 129S6/SvEvTac background strain (McKhann et al., 2003) of the Kv4.2 knockout mice may contribute to the phenotype of the Kv4.2 knockout mice. Perhaps in a less seizure-resistant genetic background, knockout of Kv4.2 might result in spontaneous seizures. The findings from our studies comparing susceptibility to convulsant stimulation in nonlittermate versus littermate Kv4.2 knockout and wild-type mice indicate that the susceptibility to convulsant stimulation with kainate in this genotype is modulated by the background mouse strain. These findings underscore the importance of using littermate wild-type controls for studies with knockout and transgenic mice, and highlight the importance of the genetic background as a modifying factor in the expression of seizures.
Kv4.2 knockout was associated with 100% mortality during status epilepticus. In comparison, 25% of the wild-type littermates with status epilepticus died. A 10–15% mortality rate in the wild-type animals previously has been reported in a kainate model of seizures and status epilepticus (McKhann et al., 2003). Although the mechanism underlying the high mortality rate in the Kv4.2 knockout mice is unknown, the prominent role of Kv4.2 in the early repolarization phase of the rodent myocyte action potential (Snyders, 1999) suggests the possibility of a cardiac mechanism for sudden death during status epilepticus in these mice. Under physiologic conditions at rest, the Kv4.2 knockout mice do not express a cardiac phenotype compared to wild-type animals (Guo et al., 2005). However, during frequent seizures and status epilepticus, autonomic hyperactivity may result in a progressive cardiac dysrhythmia (Walton, 1993). Under these conditions, genetic absence of Kv4.2 in rodents may have lethal consequences.
The findings reported here strengthen the coupling of a channelopathy with seizures. Kv4.2 deficiency in the setting of an appropriate genetic background predisposes to increased forebrain excitability and a reduction in the seizure threshold. Therefore, enhancement of Kv4.2 function may represent a novel therapeutic strategy in the treatment of seizure disorders.