The childhood epilepsy syndrome of benign familial neonatal convulsions (BFNC) exhibits the remarkable feature of clinical remission within a few weeks of onset and a favourable prognosis, sparing cognitive abilities despite persistent expression of the mutant KCNQ2 or KCNQ3 potassium channels throughout adulthood. To better understand such dynamic neuroprotective plasticity within the developing brain, we introduced missense mutations that underlie human BFNC into the orthologous murine Kcnq2 (Kv7.2) and Kcnq3 (Kv7.3) genes. Mutant mice were examined for altered thresholds to induced seizures, spontaneous seizure characteristics, hippocampal histology, and M-current properties of CA1 hippocampal pyramidal neurons. Adult Kcnq2A306T/+ and Kcnq3 G311V/+ heterozygous knock-in mice exhibited reduced thresholds to electrically induced seizures compared to wild-type littermate mice. Both Kcnq2 A306T/A306Tand Kcnq3 G311V/G311V homozygous mutant mice exhibited early onset spontaneous generalized tonic-clonic seizures concurrent with a significant reduction in amplitude and increased deactivation kinetics of the neuronal M-current. Mice had recurrent seizures into adulthood that triggered molecular plasticity including ectopic neuropeptide Y (NPY) expression in granule cells, but without hippocampal mossy fibre sprouting or neuronal loss. These novel knockin mice recapitulate proconvulsant features of the human disorder yet show that inherited M-current defects spare granule cells from reactive changes in adult hippocampal networks. The absence of seizure-induced pathology found in these epileptic mouse models parallels the benign neurodevelopmental cognitive profile exhibited by the majority of BFNC patients. Benign familial neonatal convulsions (BFNC [MIM 121200,121201]) is an autosomal dominantly inherited epileptic disorder in which newborns experience several daily partial or generalized seizures during wakefulness and sleep (Rett & Teubel, 1964;Ryan et al. 1991;Ronen et al. 1993). In the majority of cases, seizures spontaneously remit by 3-4 months yet 16% of BFNC individuals continue to experience one or more seizures during adulthood (Ronen et al. 1993;Singh et al. 2003). Mutations in either of two homologous potassium channel genes, This paper has online supplemental material.KCNQ2 and KCNQ3, have been found in patients with BFNC (Biervert et al. 1998;Charlier et al. 1998;Singh et al. 1998;Lucarini et al. 2007). The protein products of these genes colocalize and generate the M-current, a critical regulator of action potential firing and neuronal excitability (Wang et al. 1998).An intriguing phenotypic trait of BFNC is that the frequent spontaneous seizures exhibited by the neonates are not generally associated with any developmental sequelae (Ryan et al. 1991;Ronen et al. 1993). This is in stark contrast to malignant early childhood onset convulsive seizure disorders that can produce neuronal death and synaptic reorganization with subsequent (Ben-Ari & Holmes, 2006;Blume, 2006). A biological mechanism that exp...

The whole-cell patch-clamp technique was used to examine the effects of retigabine, a novel anticonvulsant drug, on the electroresponsive properties of individual neurons as well as on neurotransmission between monosynaptically connected pairs of cultured mouse cortical neurons. Consistent with its known action on potassium channels, retigabine significantly hyperpolarized the resting membrane potentials of the neurons, decreased input resistance, and decreased the number of action potentials generated by direct current injection. In addition, retigabine potentiated inhibitory postsynaptic currents (IPSCs) mediated by activation of ␥-aminobutyric acid A (GABA A ) receptors. IPSC peak amplitude, 90-to-10% decay time, weighted decay time constant, slow decay time constant, and, consequently, the total charge transfer were all significantly enhanced by retigabine in a dose-dependent manner. This effect was limited to IPSCs; retigabine had no significant effect on excitatory postsynaptic currents (EPSCs) mediated by activation of non-N-methyl-D-aspartate ionotropic glutamate receptors. A form of short-term presynaptic plasticity, paired-pulse depression, was not altered by retigabine, suggesting that its effect on IPSCs is primarily postsynaptic. Consistent with the hypothesis that retigabine increases inhibitory neurotransmission via a direct action on the GABA A receptor, the peak amplitudes, 90-to-10% decay times, and total charge transfer of spontaneous miniature IPSCs were also significantly increased. Therefore, retigabine potently reduces excitability in neural circuits via a synergistic combination of mechanisms.The novel anticonvulsant drug retigabine [D-23129; N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl ester] has been found to effectively reduce or block seizure activity in a wide variety of animal models of epilepsy (Dailey et al., 1995;Rostock et al., 1996;Tober et al., 1996). Retigabine is structurally different and has a higher protective index than many of the commonly prescribed anticonvulsants . Of particular interest is the recent finding that a primary mechanism of action of retigabine is the enhanced activation of heteromeric potassium channels composed of the KCNQ2 and KCNQ3 subunits (Main et al., 2000;Rundfeldt and Netzer, 2000b;Wickenden et al., 2000). It has been demonstrated that the channels formed by the KCNQ2/Q3 subunits underlie a neuronal potassium current commonly referred to as the M current (Wang et al., 1998;Shapiro et al., 2000). The M current is critical in determining resting membrane potential and neuronal excitability in many brain regions because of its sustained activation at potentials below the threshold for action potential generation (Marrion, 1997). Consistent with its ability to augment M channel currents, retigabine has been found to effectively hyperpolarize and reduce action potential generation in projection neurons located in layers II and III of the entorhinal cortex (Hetka et al., 1999).Previous work suggests that the mechanism of action...

The M-type K ϩ current [I K(M) ] activates in response to membrane depolarization and regulates neuronal excitability. Mutations in two subunits (KCNQ2 and KCNQ3; Kv7.2 and Kv7.3) that underlie the M-channel cause the human seizure disorder benign familial neonatal convulsions (BFNC), presumably by reducing I K(M) function. In mice, the Szt1 mutation, which deletes the genomic DNA encoding the KCNQ2 C terminus and all of CHRNA4 (nicotinic acetylcholine receptor ␣4 subunit) and ARFGAP-1 (GTPase-activating protein that inactivates ADP-ribosylation factor 1), reduces seizure threshold, and alters M-channel pharmacosensitivity. Genomic deletions affecting the C terminus of KCNQ2 have been identified in human families with BFNC, and truncation of the C terminus prevents proper KCNQ2/KCNQ3 channel assembly in Xenopus oocytes. We showed previously that Szt1 mice have a reduced baseline seizure threshold and altered sensitivity to drugs that act at the M-channel. Specifically, the proconvulsant M-channel blocker linopirdine and anticonvulsant enhancer retigabine display increased and decreased potency, respectively, in Szt1 mice. To investigate the effects of the Szt1 mutation on I K(M) function explicitly, perforated-patch electrophysiology was performed in CA1 pyramidal neurons of the hippocampus in brain slices prepared from C57BL/6J-Szt1/؉ and control C57BL/6Jϩ/ϩ mice. Our results show that Szt1 reduces both I K(M) amplitude and current density, inhibits spike frequency adaptation, and alters many aspects of M-channel pharmacology. This is the first evidence that a naturally occurring Kcnq2 mutation diminishes the amplitude and function of the native neuronal I K(M) , resulting in significantly increased neuronal excitability. Finally, the changes in single-cell biophysical properties likely underlie the altered seizure threshold and pharmacosensitivity reported previously in Szt1 mice.

The electroconvulsive threshold (ECT) test has been used extensively to determine the protection conferred by antiepileptic drug candidates against induced seizures in rodents. Despite its clinical relevance, the potential of ECT to identify mouse epilepsy models in genetic studies has not been thoroughly assessed. We adopted the ECT test to screen the progeny of ethylnitrosourea treated male C57BL/6J mice. In a small-scale screen, several mutant lines conferring a low threshold to ECT minimal clonic seizures were mapped to the telomeric region of mouse chromosome 2 in independent founder families. This high incidence was suggestive of a single spontaneous event that pre-existed in the founders of mutagenized stock. Genetic and physical mapping led to the discovery that several lines shared a single mutation, Szt1 (seizure threshold-1), consisting of a 300 kb deletion of genomic DNA involving three known genes. Two of these genes, Kcnq2 and Chrna4, are known to be mutated in human epilepsy families. Szt1 homozygotes and heterozygotes display similar phenotypes to those found in the respective Kcnq2 knockout mutant mice, suggesting that Kcnq2 haploinsufficiency is at the root of the Szt1 seizure sensitivity. Our results provide a novel genetic model for epilepsy research and demonstrate that the approach of using ECT to study seizures in mice has the potential to lead to the identification of human epilepsy susceptibility genes.

Summary:Purpose: Mutations in the genes that encode subunits of the M-type K + channel (KCNQ2/KCNQ3) and nicotinic acetylcholine receptor (CHRNA4) cause epilepsy in humans. The purpose of this study was to examine the effects of the Szt1 mutation, which not only deletes most of the C-terminus of mouse Kcnq2, but also renders the Chnra4 and Arfgap-1 genes hemizygous, on seizure susceptibility and sensitivity to drugs that target the M-type K + channel. Methods: The proconvulsant effects of the M-channel blocker linopirdine (LPD) and anticonvulsant effects of the M-channel enhancer retigabine (RGB) were assessed by electroconvulsive threshold (ECT) testing in C57BL/6J-Szt1/+ (Szt1) and littermate control C57BL/6J+/+ (B6) mice. The effects of the Szt1 mutation on minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizures were evaluated by varying stimulation intensity and frequency.Results: Szt1 mouse seizure thresholds were significantly reduced relative to B6 littermates in the minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizure models. Mice were injected with LPD and RGB and subjected to ECT testing. In the minimal clonic seizure model, Szt1 mice were significantly more sensitive to LPD than were B6 mice [median effective dose (ED 50 ) = 3.4 ± 1.1 mg/kg and 7.6 ± 1.0 mg/kg, respectively]; in the partial psychomotor seizure model, Szt1 mice were significantly less sensitive to RGB than were B6 mice (ED 50 = 11.6 ± 1.4 mg/kg and 3.4 ± 1.3 mg/kg, respectively).Conclusions: These results suggest that the Szt1 mutation alters baseline seizure susceptibility and pharmacosensitivity in a naturally occurring mouse model.