SWDreader: a wavelet-based algorithm using spectral phase to characterize spike-wave morphological variation in genetic models of absence epilepsy.

BACKGROUND: Spike-wave discharges (SWD) found in neuroelectrical recordings are pathognomonic to absence epilepsy. The characteristic spike-wave morphology of the spike-wave complex (SWC) constituents of SWDs can be mathematically described by a subset of possible spectral power and phase values. Morlet wavelet transform (MWT) generates time-frequency representations well-suited to identifying this SWC-associated subset. NEW METHOD: MWT decompositions of SWDs reveal spectral power concentrated at harmonic frequencies. The phase relationships underlying SWC morphology were identified by calculating the differences between phase values at SWD fundamental frequency from the 2nd, 3rd, and 4th harmonics, then using the three phase differences as coordinates to generate a density distribution in a {360°×360°×360°} phase difference space. Strain-specific density distributions were generated from SWDs of mice carrying the Gria4, Gabrg2, or Scn8a mutations to determine whether SWC morphological variants reliably mapped to the same regions of the distribution, and if distribution values could be used to detect SWD. COMPARISON WITH EXISTING METHODS: To the best of our knowledge, this algorithm is the first to employ spectral phase to quantify SWC morphology, making it possible to computationally distinguish SWC morphological subtypes and detect SWDs. RESULTS/CONCLUSIONS: Proof-of-concept testing of the SWDfinder algorithm shows: (1) a major pattern of variation in SWC morphology maps to one axis of the phase difference distribution, (2) variability between the strain-specific distributions reflects differences in the proportions of SWC subtypes generated during SWD, and (3) regularities in the spectral power and phase profiles of SWCs can be used to detect waveforms possessing SWC-like morphology.

A genetic interaction network model of a complex neurological disease.

Absence epilepsy (AE) is a complex, heritable disease characterized by a brief disruption of normal behavior and accompanying spike-wave discharges (SWD) on the electroencephalogram. Only a handful of genes has been definitively associated with AE in humans and rodent models. Most studies suggest that genetic interactions play a large role in the etiology and severity of AE, but mapping and understanding their architecture remains a challenge, requiring new computational approaches. Here we use combined analysis of pleiotropy and epistasis (CAPE) to detect and interpret genetic interactions in a meta-population derived from three C3H × B6J strain crosses, each of which is fixed for a different SWD-causing mutation. Although each mutation causes SWD through a different molecular mechanism, the phenotypes caused by each mutation are exacerbated on the C3H genetic background compared with B6J, suggesting common modifiers. By combining information across two phenotypic measures - SWD duration and frequency - CAPE showed a large, directed genetic network consisting of suppressive and enhancing interactions between loci on 10 chromosomes. These results illustrate the power of CAPE in identifying novel modifier loci and interactions in a complex neurological disease, toward a more comprehensive view of its underlying genetic architecture.

Hidden in plain sight: spike-wave discharges in mouse inbred strains.

Twenty-seven inbred strains of mice were tested for spike-wave discharge (SWD) activity by video-electroencephalographic recordings over a 24-h recording period. Eight strains had reproducible, frequent SWDs, including five strains (C57BLKS/J, CBA/J, DBA/1J, NOR/LtJ, SM/J) previously undiagnosed for this distinctive phenotype. Eighteen other strains exhibited no such activity. Spike-wave discharges usually occurred while the subject was motionless, and in a significant number of annotated instances coincided with an arrest of the subject's relatively unrestrained locomotor activity, which resumed immediately after the discharge ended. In all five new strains, SWDs were suppressed by ethosuximide administration. From the genealogy of inbred strains, we suggest that two ancestors, A and DBA, transmitted genotypes required for SWD in all positive strains. Together these strains with SWDs provide new opportunities to understand the genetic core susceptibility of this distinctive electroencephalographic activity and to explore its relationship to absence epilepsy, a human disorder for which few genes are known.

Physiological and genetic analysis of multiple sodium channel variants in a model of genetic absence epilepsy.

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8a(V929F)). Here, we confirmed the prior suggestion that Scn8a(V929F) exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8a(V929F) is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2(V752F) as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2(V752F) functional analysis revealed an overall gain-of-function and for NaV1.6(V929F) revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2(V752F) due to "functional silencing" of NaV1.6(V929F) suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6(V929F) loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy.

Etiology of a genetically complex seizure disorder in Celf4 mutant mice.

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adults. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at the age of 7 weeks. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice.

Szt2, a novel gene for seizure threshold in mice.

In a chemical mutagenesis screen we identified Szt2 (seizure threshold 2) as a gene that confers low seizure threshold to mice and may also enhance epileptogenesis. The semidominant phenotype was mapped to Chromosome 4 and narrowed further to a critical interval of approximately 650 kb. A novel large (> 10 kb) transcript in the critical interval was found to have fourfold increased steady-state expression at the RNA level in Szt2 homozygous mutant brain. The corresponding 72 exon gene encodes a 378-kD protein with no significant or suggestive sequence similarities to any other protein. The mutant allele of Szt2 contains a splice donor mutation after exon 32, predicting transcriptional read-through, translational frameshift and premature stop. A second Szt2 allele, containing a gene-trap mutation in exon 21, also conferred a low seizure threshold and increased RNA expression, but unlike the original allele, some gene-trap homozygotes died embryonically. Szt2 is transcribed in many tissues, with the highest expression in brain, and it is also expressed during embryonic development. Szt2 is highly conserved in evolution, with a clear, single orthologue found in all land vertebrates and in many invertebrates. Interestingly, in mammals the Szt2 gene resides in a highly conserved head-to-head configuration with Med8 (which encodes a Mediator complex subunit), separated by only 91 nt. While the biological function of Szt2 remains unknown, its high conservation, unique structure and effect on seizure threshold suggest that it serves an important role in the central nervous system.

Genetic complexity of absence seizures in substrains of C3H mice.

Absence epilepsy is a common form of idiopathic generalized epilepsy whose etiology is poorly understood because of genetic and phenotypic heterogeneity. The inbred mouse strain C3H/He exhibits spontaneous absence seizures characterized by spike and wave discharges (SWD) on the electroencephalogram concomitant with behavioral arrest. Previous studies using the C3H/HeJ (HeJ) substrain identified a mutation in the Gria4 gene as a major susceptibility locus. In the present study, we found that two closely related substrains C3H/HeOuJ (OuJ) and C3H/HeSnJ, which have a similar SWD incidence as HeJ, do not contain the Gria4 mutation. Further analysis of backcross mice segregating OuJ and C57BL/6J alleles shows that, unlike the HeJ substrain, OuJ does not have a major locus for SWD but has suggestive loci at best that would explain only a fraction of the phenotypic variance. These results illustrate how the genetic etiology of a common neurological disorder can differ between substrains with similar phenotypes. We infer that all C3H strains are sensitized to SWD and that additional mutations affecting SWD arose or were fixed independently in the years since the substrains diverged.

Two novel alleles of tottering with distinct Ca(v)2.1 calcium channel neuropathologies.

The calcium channel CACNA1A gene encodes the pore-forming, voltage-sensitive subunit of the voltage-dependent calcium Ca(v)2.1 type channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine, episodic ataxia 2 and spinocerebellar ataxia type 6. The mouse homologue, Cacna1a, is associated with the tottering, Cacna1a(tg), mutant series. Here we describe two new missense mutant alleles, Cacna1a(tg-4J) and Cacna1a(Tg-5J). The Cacna1a(tg-4J) mutation is a valine to alanine mutation at amino acid 581, in segment S5 of domain II. The recessive Cacna1a(tg-4J) mutant exhibited the ataxia, paroxysmal dyskinesia and absence seizures reminiscent of the original tottering mouse. The Cacna1a(tg-4J) mutant also showed altered activation and inactivation kinetics of the Ca(v)2.1 channel, not previously reported for other tottering alleles. The semi-dominant Cacna1a(Tg-5J) mutation changed a conserved arginine residue to glutamine at amino acid 1252 within segment S4 of domain III. The heterozygous mouse was ataxic and homozygotes rarely survived. The Cacna1a(Tg-5J) mutation caused a shift in both voltage activation and inactivation to lower voltages, showing that this arginine residue is critical for sensing Ca(v)2.1 voltage changes. These two tottering mouse models illustrate how novel allelic variants can contribute to functional studies of the Ca(v)2.1 calcium channel.

A null mutation in inositol polyphosphate 4-phosphatase type I causes selective neuronal loss in weeble mutant mice.

Weeble mutant mice have severe locomotor instability and significant neuronal loss in the cerebellum and in the hippocampal CA1 field. Genetic mapping was used to localize the mutation to the gene encoding inositol polyphosphate 4-phosphatase type I (Inpp4a), where a single nucleotide deletion results in a likely null allele. The substrates of INPP4A are intermediates in a pathway affecting intracellular Ca(2+) release but are also involved in cell cycle regulation through binding the Akt protooncogene; dysfunction in either may account for the neuronal loss of weeble mice. Although other mutations in phosphoinositide enzymes are associated with synaptic defects without neuronal loss, weeble shows that Inpp4a is critical for the survival of a subset of neurons during postnatal development in mice.

Of mice and genome sequence.

Availability of the mouse genome sequence will have a major impact on the study of vertebrate evolution, mammalian biology, and animal models of human disease. Resources to explore genome biology in mice will maximize the effect of this watershed event.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.

Electroconvulsive thresholds of inbred mouse strains.

The electroconvulsive threshold (ECT) test is used commonly in the screening of anti-epileptic drugs in rodent models, but little is known about its genetic or mechanistic basis. Thresholds for minimal clonic, maximal tonic, or psychomotor (partial) seizures were determined in 16 different inbred mouse strains in two different laboratories. A wide range of thresholds was observed, suggesting that a variety of neuroexcitability alleles exist in inbred strains. Although there was generally good cross-strain correlation between the three seizure types, several outlier strains were detected, showing that genetically encoded differences can affect the ability of a particular seizure type to spread through the brain. Furthermore, the relative seizure susceptibility of a strain was comparable between the two laboratories, suggesting that despite different test sites, instrumentation, and personnel, the ECT assay is portable and that common inbred strains can often be relied upon as calibration standards. Last, the ECT paradigm was also sensitive enough to detect single locus differences, laying the groundwork for mutation screens for new neuroexcitability models.

High-resolution genetic and physical mapping of modifier-of-deafwaddler (mdfw) and Waltzer (Cdh23v).

Modifier-of-deafwaddler (mdfw) and waltzer (Cdh23v) are loci on mouse chromosome 10 encoding factors that are essential for the function of auditory hair cells. The BALB/cByJ-specific mdfw allele encodes a necessary and sufficient modifier that induces progressive early onset hearing loss in CBy-dfw2J heterozygotes. Recessive mutations in the waltzer locus result in circling behavior and congenital deafness. In this report we present a high-resolution integrated genetic and physical map of mdfw and Cdh23(v). Our genetic analyses localize mdfw between markers D10Mit60 and 148M13T7 within a 1.01-cM region. The Cdh23v critical interval is fully contained within the mdfw region and localizes between markers 146O23T7 and 148M13T7 within a 0.35-cM interval that is represented in an approximately 500-kb BAC contig. Our data suggest that mdfw and Cdh23v are allelic.

Mouse models for the Wolf-Hirschhorn deletion syndrome.

Wolf-Hirschhorn syndrome (WHS) is a deletion syndrome caused by segmental haploidy of chromosome 4p16.3. Its hallmark features include a 'Greek warrior helmet' facial appearance, mental retardation, various midline defects and seizures. The WHS critical region (WHSCR) lies between the Huntington's disease gene, HD, and FGFR3. In mice, the homologs of these genes map to chromosome 5 in a region of conserved synteny with human 4p16.3. To derive mouse models of WHS and map genes responsible for subphenotypes of the syndrome, five mouse lines bearing radiation-induced deletions spanning the WHSCR syntenic region were generated and characterized. Similar to WHS patients, these animals were growth-retarded, were susceptible to seizures and showed midline (palate closure, tail kinks), craniofacial and ocular anomalies (colobomas, corneal opacities). Other phenotypes included cerebellar hypoplasia and a shortened cerebral cortex. Expression of WHS-like traits was variable and influenced by strain background and deletion size. These mice represent the first animal models for WHS. This collection of nested chromosomal deletions will be useful for mapping and identifying loci responsible for the various subphenotypes of WHS, and provides a paradigm for the dissection of other deletion syndromes using the mouse.

Multiple seizure susceptibility genes on chromosome 7 in SWXL-4 congenic mouse strains.

The SWXL-4 recombinant inbred mouse strain is unusually sensitive to recurrent tonic-clonic seizures upon routine handling and to seizures induced by chemoconvulsants. In a conventional intercross with the ABP/Le strain, we previously mapped a SWXL-4-derived quantitative trait locus called Szf1 (seizure frequency 1) to Chromosome 7. In the present study, we confirm the existence of Szf1 in both an independent cross and a congenic strain. However, derivative congenic recombinant strains show that an interaction between at least two genes on Chromosome 7-each of which has a very small effect on its own-account for Szf1.

The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

A new spontaneous mouse mutation in the Kcne1 gene.

A new mouse mutant, punk rocker (allele symbol Kcne1(pkr)), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1(pkr) mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21.

The roads from phenotypic variation to gene discovery: mutagenesis versus QTLs.

In model organisms, chemical mutagenesis provides a powerful alternative to natural, polygenic variation (for example, quantitative trait loci (QTLs)) for identifying functional pathways and complex disease genes. Despite recent progress in QTLs, we expect that mutagenesis is will ultimately prove more effective because the prospects of gene identification are high and every gene affecting a trait is potentially a target.