Kv4.2 knockout mice demonstrate increased susceptibility to convulsant stimulation

Abstract

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.

Figure 1

Electrographic seizures and status epilepticus in Kv4.2 wildtype and knockout littermates. Representative simultaneous cortical (Ctx) and hippocampal (Hip) EEG recordings are shown for (A) Kv4.2 wildtype (+/+) and (B) Kv4.2 knockout (−/−) littermates before and after intraperitoneal injection of kainate (40 mg/kg). (A and B) The top two traces shown for each genotype are samples of cortex and hippocampal baseline EEG recordings done thirty minutes prior to kainate injection in the animals. The 2nd, 3rd and 4th pairs of traces below the top pair for the Kv4.2 knockout and wildtype mice are continuous EEG recordings of electrographic seizures that began after kainate injection. The electrographic elements of the seizures are similar in both genotypes except for differences in the location and latency of seizure onset. In both wildtype and knockout mice, seizure onset is marked by a 30 Hz recruiting rhythm which is immediately followed by high voltage synchronous 4–6 Hz generalized spike and wave activity and a period of post-ictal depression. However, in the trace for the wildtype mouse there is seizure onset focally in the cortex, while in the trace for the knockout mouse there is seizure onset diffusely in the cortex and hippocampus. This latter finding was not seen in any of the wildtype mice, while 50% of the knockout mice showed this diffuse seizure onset (see results section). Electrographic status epilepticus is marked by continuous high voltage 2.5–3 Hz generalized spike and wave activity. The latency to first electrographic seizure for the wildtype mouse shown is 108 seconds and to status epilepticus is 30 minutes after kainate injection (A). The latency to electrographic seizure onset for the knockout mouse is 42 seconds and to status epilepticus is 15 minutes after kainate injection (B).

Figure 2

Kv4.2 knockout littermates show increased susceptibility to kainate stimulation. Following kainate (40 mg/kg) injection intraperitoneally in Kv4.2 wildtype (+/+) (n = 8) and Kv4.2 knockout (−/−) (n = 6) littermates, the animals were monitored with video-EEG for electrographic and behavioral seizures according to the Racine scale. (A) Knockout animals developed electrographic seizures significantly faster than wildtype mice (n=6–8). One hundred percent of the knockout and 80% of the wildtype mice developed seizures in response to kainate (data not shown). (B) Electrographic status epilepticus developed significantly faster in the knockout mice compared to wildtype littermates (n=6–8). (C) The latency to onset of forelimb clonus was significantly shorter in the knockout compared to wildtype mice. (D) Behavioral status epilepticus also developed significantly faster in the knockout versus wildtype littermates (n=6–8). Thus, both electrographic and behavioral seizures occurred significantly earlier in the knockout compared to the littermate wildtype mice. *p < 0.05, **p < 0.01, Student’s t test.

Figure 3

Kv4.2 deficiency is associated with high mortality after convulsant stimulus. Kv4.2 knockout (−/−) and Kv4.2 wildtype (+/+) littermate mice were given kainate (40 mg/kg) intraperitoneally. Acutely following the onset of status epilepticus, 100% (6/6) of the Kv4.2 knockout mice and 25% (2/8) of the littermate wildtype mice died. *p < 0.05, chi-square test.

Figure 4

Enhanced seizure susceptibility phenotype in Kv4.2 knockout mice when compared to non-littermate wildtype mice. Following kainate (20 mg/kg) injection intraperitoneally into the 129S6/SvEvTac purchased wildtype (+/+) and inbred non-littermate Kv4.2 knockout (−/−) mice and littermate wildtype and Kv4.2 knockout mice, behavioral seizures were scored according to the Racine scale. (A) Non-littermate knockout mice developed forelimb clonus significantly faster than the wildtype 129S6/SvEvTac mice (n = 8, *p < 0.05, Student’s t test). (B) After backcrossing the Kv4.2 knockout mice with the 129S6/SvEvTac background strain to the F6 and higher generations, the seizure susceptibility studies were repeated with the same dosage of kainate using littermate knockout and wildtype mice. At this dose of kainate (20 mg/kg) there was no significant difference in seizure susceptibility between the littermate knockout (−/−) and wildtype (+/+) mice (n = 8).

Figure 5

Bicuculline-induced excitability is enhanced in hippocampal slices from Kv4.2 knockout mice. (A) Representative evoked field potential traces from transverse hippocampal slices prepared from Kv4.2 wildtype (+/+) and knockout (−/−) littermate mice before (Control) and after bicuculline (5 μM; Bicuculline) application are shown. Bicuculline application led to bursting in response to orthodromic stimulation in both genotypes; however, the response was more prolonged in the slices from the Kv4.2 knockout compared to littermate wildtype mice. A summary of the evoked response durations after bicuculline application is shown for both Kv4.2 knockout (n = 15) and wildtype (n = 12) littermates. *p < 0.05, Student’s t test. (B) Representative spontaneous field potential responses following bicuculline (5 μM) application to slices from the Kv4.2 wildtype (+/+) and knockout (−/−) littermate mice are shown. Bursting is more prolonged in the slices from the Kv4.2 knockout compared to wildtype mice. Summary data for bicuculline-induced spontaneous burst duration for slices from the Kv4.2 wildtype (n = 5) and knockout (n = 7) animals are shown. *p < 0.05, Student’s t test.