The results presented here add to the previous studies which have shown Kv4.2 downregulation 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 wildtype 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 wildtype littermates. The magnitude of this decrease is 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 wildtype 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 wildtype 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 wildtype 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 wildtype 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, 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mice exhibit behavioral seizures in response to startle or acoustic stimuli, and show handling-induced seizures (unpublished observations).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 wildtype or knockout mice under basal conditions. Thus, if the animals were having experiencing epileptiform activity and seizures, it is likely to be quite rare.
Another 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 wildtype 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-type currents and GABAergic 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 in vivo
studies. There was no observable spontaneous bursting in hippocampal slices from knockout mice and the baseline evoked responses from littermate wildtype and knockout mice showed no epileptiform activity. However, convulsant stimulation augments seizure-like activity in slices from knockouts. In these slices, a low concentration of bicuculline (Wong et al., 1986
; Traynelis and Dingledine, 1988
) induced prolonged bursts with after-discharges, resembling interictal bursting activity. This bursting activity is similar to that described with higher concentrations of bicuculline (Karnup and Stelzer, 2001
) and is thought to be due to the reverbation of the hippocampal network between the CA1 and CA3 region (Hablitz, 1984
). As discussed above, 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 wildtype 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 intra-dendritically stimulated (Wong and 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
; Perreault and Avoli, 1991
; Traub et al., 1995
). However, 4-AP is a non-specific 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 were 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 and Trimmer, 2006
), and a 4-aminopyridine sensitive A-type current is present in these regions (Beck et al., 1992
; Mitterdorfer and Bean, 2002
). Thus Kv4.2 may underlie the A-type current in these regions of the hippocampus and thereby affect the excitability of these neurons. However, the relationship between Kv4.2 and the A-type 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 non-littermate versus littermate Kv4.2 knockout and wildtype 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 wildtype 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 wildtype littermates with status epilepticus died. A 10–15 % mortality rate in the wildtype animals previously has been reported in a kainate model of seizures and status epilepticus (McKhann et al., 2003
). While 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 physiological conditions at rest, the Kv4.2 knockout mice do not express a cardiac phenotype compared to wildtype 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.