Neuron-specific Kv1.1 deficiency is sufficient to cause epilepsy, premature death, and cardiorespiratory dysregulation.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality, but the precise cellular substrates involved remain elusive. Epilepsy-associated ion channel genes with co-expression in brain and heart have been proposed as SUDEP candidate genes since they provide a singular unifying link between seizures and lethal cardiac arrhythmias. Here, we generated a conditional knockout (cKO) mouse with neuron-specific deletion of Kcna1, a SUDEP-associated gene with brain-heart co-expression, to test whether seizure-evoked cardiac arrhythmias and SUDEP require the absence of Kv1.1 in both brain and heart or whether ablation in neurons is sufficient. To obtain cKO mice, we developed a floxed Kcna1 mouse which we crossed to mice with the Synapsin1-Cre transgene, which selectively deletes Kcna1 in most neurons. Molecular analyses confirmed neuron-specific Kcna1 deletion in cKO mice and corresponding loss of Kv1.1 except in cerebellum where Synapsin1-Cre is not highly expressed. Survival studies and electroencephalography, electrocardiography, and plethysmography recordings showed that cKO mice exhibit premature death, epilepsy, and cardiorespiratory dysregulation but to a lesser degree than global knockouts. Heart rate variability (HRV) was increased in cKO mice with peaks during daytime suggesting disturbed diurnal HRV patterns as a SUDEP biomarker. Residual Kv1.1 expression in cKO cerebellum suggests it may play an unexpected role in regulating ictal cardiorespiratory dysfunction and SUDEP risk. This work demonstrates the principle that channelopathies with brain-heart expression patterns can increase death risk by brain-driven mechanisms alone without a functionally compromised heart, reinforcing seizure control as a primary clinical strategy for SUDEP prevention.

Cardiorespiratory profiling reveals primary breathing dysfunction in Kcna1-null mice: Implications for sudden unexpected death in epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality, but the relative importance of underlying cardiac and respiratory mechanisms remains unclear. To illuminate the interactions between seizures, respiration, cardiac function, and sleep that contribute to SUDEP risk, here we developed a mouse epilepsy monitoring unit (EMU) to simultaneously record video, electroencephalography (EEG), electromyography (EMG), plethysmography, and electrocardiography (ECG) in a commonly used genetic model of SUDEP, the Kcna1 knockout (Kcna1-/-) mouse. During interictal periods, Kcna1-/- mice exhibited an abnormal absence of post-sigh apneas and a 3-fold increase in respiratory variability. During spontaneous convulsive seizures, Kcna1-/- mice displayed an array of aberrant breathing patterns that always preceded cardiac abnormalities. These findings support respiratory dysfunction as a primary risk factor for susceptibility to deleterious cardiorespiratory sequelae in epilepsy and reveal a new role for Kcna1-encoded Kv1.1 channels in the regulation of basal respiratory physiology.

Scn2a deletion improves survival and brain-heart dynamics in the Kcna1-null mouse model of sudden unexpected death in epilepsy (SUDEP).

People with epilepsy have greatly increased probability of premature mortality due to sudden unexpected death in epilepsy (SUDEP). Identifying which patients are most at risk of SUDEP is hindered by a complex genetic etiology, incomplete understanding of the underlying pathophysiology and lack of prognostic biomarkers. Here we evaluated heterozygous Scn2a gene deletion (Scn2a+/-) as a protective genetic modifier in the Kcna1 knockout mouse (Kcna1-/-) model of SUDEP, while searching for biomarkers of SUDEP risk embedded in electroencephalography (EEG) and electrocardiography (ECG) recordings. The human epilepsy gene Kcna1 encodes voltage-gated Kv1.1 potassium channels that act to dampen neuronal excitability whereas Scn2a encodes voltage-gated Nav1.2 sodium channels important for action potential initiation and conduction. SUDEP-prone Kcna1-/- mice with partial genetic ablation of Nav1.2 channels (i.e. Scn2a+/-; Kcna1-/-) exhibited a two-fold increase in survival. Classical analysis of EEG and ECG recordings separately showed significantly decreased seizure durations in Scn2a+/-; Kcna1-/- mice compared with Kcna1-/- mice, without substantial modification of cardiac abnormalities. Novel analysis of the EEG and ECG together revealed a significant reduction in EEG-ECG association in Kcna1-/- mice compared with wild types, which was partially restored in Scn2a+/-; Kcna1-/- mice. The degree of EEG-ECG association was also proportional to the survival rate of mice across genotypes. These results show that Scn2a gene deletion acts as protective genetic modifier of SUDEP and suggest measures of brain-heart association as potential indices of SUDEP susceptibility.

Pharmacogenetics of KCNQ channel activation in 2 potassium channelopathy mouse models of epilepsy.

OBJECTIVES: Antiseizure drugs are the leading therapeutic choice for treatment of epilepsy, but their efficacy is limited by pharmacoresistance and the occurrence of unwanted side effects. Here, we examined the therapeutic efficacy of KCNQ channel activation by retigabine in preventing seizures and neurocardiac dysfunction in 2 potassium channelopathy mouse models of epilepsy with differing severity that have been associated with increased risk of sudden unexpected death in epilepsy (SUDEP): the Kcna1-/- model of severe epilepsy and the Kcnq1A340E/A340E model of mild epilepsy. METHODS: A combination of behavioral, seizure threshold, electrophysiologic, and gene expression analyses was used to determine the effects of KCNQ activation in mice. RESULTS: Behaviorally, Kcna1-/- mice exhibited unexpected hyperexcitability instead of the expected sedative-like response. In flurothyl-induced seizure tests, KCNQ activation decreased seizure latency by ≥50% in Kcnq1 strain mice but had no effect in the Kcna1 strain, suggesting the influence of genetic background. However, in simultaneous electroencephalography and electrocardiography recordings, KCNQ activation significantly reduced spontaneous seizure frequency in Kcna1-/- mice by ~60%. In Kcnq1A340E/A340E mice, KCNQ activation produced adverse cardiac effects including profound bradycardia and abnormal increases in heart rate variability and atrioventricular conduction blocks. Analyses of Kcnq2 and Kcnq3 mRNA levels revealed significantly elevated Kcnq2 expression in Kcna1-/- brains, suggesting that drug target alterations may contribute to the altered drug responses. SIGNIFICANCE: This study shows that treatment strategies in channelopathy may have unexpected outcomes and that effective rebalancing of channel defects requires improved understanding of channel interactions at the circuit and tissue levels. The efficacy of KCNQ channel activation and manifestation of adverse effects were greatly affected by genetic background, potentially limiting KCNQ modulation as a way to prevent neurocardiac dysfunction in epilepsy and thereby SUDEP risk. Our data also uncover a potential role for KCNQ2-5 channels in autonomic control of chronotropy.

Spontaneous seizures in Kcna1-null mice lacking voltage-gated Kv1.1 channels activate Fos expression in select limbic circuits.

Mice lacking voltage-gated Kv1.1 channels as a result of deletion of the Kcna1 gene are an extensively utilized genetic model of human epilepsy and sudden unexpected death in epilepsy because of their frequent seizures and genotypic-phenotypic similarity to the human condition. Ictal behaviors, electrophysiological recordings, and gene expression studies suggest limbic circuits are critical for epilepsy in Kcna1-null mice, but the exact brain networks recruited by seizures remain unknown. In this study, Fos protein expression patterns were used to map limbic brain regions with increased neuronal activity at baseline and during spontaneous seizures in Kcna1-null mice by comparing seizing and non-seizing knockouts and wild-type controls. Basal Fos levels were unchanged in non-seizing knockout mice compared to wild types for all brain regions examined except the dentate gyrus granule cell layer which exhibited a significant decrease in Fos-positive cells. Following seizures, Kcna1-null brains exhibited significantly increased Fos labeling in the basolateral amygdala and the dentate hilus region, but not in other principal cell layers of the hippocampal formation. The selective Fos activation in the amygdala following seizures suggests that extra hippocampal limbic circuits may be critically involved with seizure generation or spread in Kcna1-null mice. Fos protein expression patterns were analyzed using immunohistochemistry to provide the first map of brain regions recruited by spontaneous seizures in mice lacking Kv1.1 channels, an extensively used genetic model of epilepsy. Seizures significantly increased Fos expression in the amygdala and hilus by about fourfold, suggesting an important contribution by extrahippocampal networks to epilepsy in this model.

Kv1.1 deficiency alters repetitive and social behaviors in mice and rescues autistic-like behaviors due to Scn2a haploinsufficiency.

BACKGROUND: Autism spectrum disorder (ASD) and epilepsy are highly comorbid, suggesting potential overlap in genetic etiology, pathophysiology, and neurodevelopmental abnormalities; however, the nature of this relationship remains unclear. This work investigated how two ion channel mutations, one associated with autism (Scn2a-null) and one with epilepsy (Kcna1-null), interact to modify genotype-phenotype relationships in the context of autism. Previous studies have shown that Scn2a+/- ameliorates epilepsy in Kcna1-/- mice, improving survival, seizure characteristics, and brain-heart dynamics. Here, we tested the converse, whether Kcna1 deletion modifies ASD-like repetitive and social behaviors in Scn2a+/- mice. METHODS: Mice were bred with various combinations of Kcna1 and Scn2a knockout alleles. Animals were assessed for repetitive behaviors using marble burying, grooming, and nestlet shredding tests and for social behaviors using sociability and social novelty preference tests. RESULTS: Behavioral testing revealed drastic reductions in all repetitive behaviors in epileptic Kcna1-/- mice, but relatively normal social interactions. In contrast, mice with partial Kcna1 deletion (Kcna1+/- ) exhibited increased self-grooming and decreased sociability suggestive of ASD-like features similar to those observed in Scn2a+/- mice. In double-mutant Scn2a+/- ; Kcna1+/- mice, the two mutations interacted to partially normalize ASD-like behaviors associated with each mutation independently. CONCLUSIONS: Taken together, these findings suggest that Kv1.1 subunits are important in pathways and neural networks underlying ASD and that Kcna1 may be a therapeutic target for treatment of Scn2a-associated ASD.

Kv1.1 potassium channel subunit deficiency alters ventricular arrhythmia susceptibility, contractility, and repolarization.

Epilepsy-associated Kv1.1 voltage-gated potassium channel subunits encoded by the Kcna1 gene have traditionally been considered absent in heart, but recent studies reveal they are expressed in cardiomyocytes where they could regulate intrinsic cardiac electrophysiology. Although Kv1.1 now has a demonstrated functional role in atria, its role in the ventricles has never been investigated. In this work, electrophysiological, histological, and gene expression approaches were used to explore the consequences of Kv1.1 deficiency in the ventricles of Kcna1 knockout (KO) mice at the organ, cellular, and molecular levels to determine whether the absence of Kv1.1 leads to ventricular dysfunction that increases the risk of premature or sudden death. When subjected to intracardiac pacing, KO mice showed normal baseline susceptibility to inducible ventricular arrhythmias (VA) but resistance to VA under conditions of sympathetic challenge with isoproterenol. Echocardiography revealed cardiac contractile dysfunction manifesting as decreased ejection fraction and fractional shortening. In whole-cell patch-clamp recordings, KO ventricular cardiomyocytes exhibited action potential prolongation indicative of impaired repolarization. Imaging, histological, and transcript analyses showed no evidence of structural or channel gene expression remodeling, suggesting that the observed deficits are likely electrogenic due to Kv1.1 deficiency. Immunoblots of patient heart samples detected the presence of Kv1.1 at relatively high levels, implying that Kv1.1 contributes to human cardiac electrophysiology. Taken together, this work describes an important functional role for Kv1.1 in ventricles where its absence causes repolarization and contractility deficits but reduced susceptibility to arrhythmia under conditions of sympathetic drive.

Directed Connectivity Analysis of the Neuro-Cardio- and Respiratory Systems Reveals Novel Biomarkers of Susceptibility to SUDEP.

Goal: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality and its pathophysiological mechanisms remain unknown. We set to record and analyze for the first time concurrent electroencephalographic (EEG), electrocardiographic (ECG), and unrestrained whole-body plethysmographic (Pleth) signals from control (WT - wild type) and SUDEP-prone mice (KO- knockout Kcna1 animal model). Employing multivariate autoregressive models (MVAR) we measured all tri-organ effective directional interactions by the generalized partial directed coherence (GPDC) in the frequency domain over time (hours). When compared to the control (WT) animals, the SUDEP-prone (KO) animals exhibited (p < 0.001) reduced afferent and efferent interactions between the heart and the brain over the full frequency spectrum (0-200Hz), enhanced efferent interactions from the brain to the lungs and from the heart to the lungs at high (>90 Hz) frequencies (especially during periods with seizure activity), and decreased feedback from the lungs to the brain at low (<40 Hz) frequencies. These results show that impairment in the afferent and efferent pathways in the holistic neuro-cardio-respiratory network could lead to SUDEP, and effective connectivity measures and their dynamics could serve as novel biomarkers of susceptibility to SUDEP and seizures respectively.

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Central myelin gene expression during postnatal development in rats exposed to nicotine gestationally.

Abnormal myelin gene expression in the central nervous system (CNS) is associated with many mental illnesses, including psychiatric disorders and drug addiction. We have previously shown that prenatal exposure to nicotine, the major psychoactive component in cigarette smoke, alters myelin gene expression in the CNS of adolescent rats. To examine whether this effect is specific for adolescents, we examined myelin gene expression in the CNS of juveniles and adults. Pregnant Sprague-Dawley rats were treated with nicotine (3 mg/kg/day; GN) or saline (GS) via osmotic mini pumps from gestational days 4-18. Both male and female offspring were sacrificed at postnatal day P20-21 (juveniles), P35-36 (adolescents), or P59-60 (adults). Three limbic brain regions, the prefrontal cortex (PFC), caudate putamen (CPu), and nucleus accumbens (NAc), were dissected. The expression of genes encoding major myelin components was evaluated using quantitative RT-PCR. We found that GN altered myelin gene expression in juveniles with brain region and sex differences. The pattern of alteration was different from that observed in adolescents. Although these genes were expressed normally in male adults, we observed decreased expression in GN-treated female adults, especially in the CPu. Thus, GN altered myelin gene expression throughout postnatal development and adulthood. The effect on adolescents was quite different from that at other ages, which correlated with the unique symptoms of many psychiatric disorders during adolescence.

CD-1 mice Show Individual Differences in Nicotine Preference in a Modified Two-Bottle Oral Self-Administration Model.

Although both animal and human studies reveal significant contributions of genetics to smoking addiction, many human studies were underpowered or biased by potential confounding variables, and animal genetic studies are challenged by limited genetic variations and lack of convincing phenotypes. To address these concerns, we used non-sibling outbred CD-1 mice to evaluate individual differences in nicotine preference with a modified two-bottle oral self-administration model. Animals were first given free access to two bottles, one filled with nicotine dissolved in 2% saccharin and the other with saccharin only. Under this regular two-bottle choice condition, the majority of animals avoided the nicotine solution with limited individual differences. However, when we modified the model by introducing 4 days of exposure to 5% saccharin in the drinking water, the animals significantly increased nicotine consumption in the two-bottle choice test, with about 30% animals showing a nicotine preference. Nicotine preference after 5% saccharin treatment remained elevated throughout the 28 days of the experiment. Further, we found there existed striking individual differences in nicotine consumption after exposure to 5% saccharin, with a range of 0-100% of total liquid consumption. The enhanced individual differences and the ratio of nicotine consumption were observed at different concentrations of nicotine (10-80 µg/ml) and in both adolescents and adults. Further examination on the induction mechanism showed that the long-lasting nicotine preference was not correlated with nicotine consumption before the induction, 5% saccharin consumption, or weight gain during the induction. Although liquid consumption during the 4 days of 5% saccharin exposure was decreased by about 30%, comparable liquid restriction alone for 4 days did not induce nicotine preference. Together, this study showed a strong and stable nicotine preference in CD-1 mice, which was induced by a short-term high concentration of saccharin in the drinking water. Considering the nature and heterogeneity of CD-1 mice, the striking individual differences imply that genetics plays an important role in nicotine preference observed in these animals.