Eradication of introduced fish allows successful recovery of a stream-dwelling amphibian.

Introduction of alien fish is a major problem for the conservation of amphibians inhabiting originally fishless mountain streams. While fish eradication programs in lakes and ponds have proven successful for the recovery of amphibian populations, there is no such information for stream-dwelling amphibians, possibly because fish removal from streams is difficult and costly. Here, we show the first case of successful recovery of a stream-dwelling amphibian (Rana iberica) in a mountain area of central Spain, following eradication of introduced brook trout (Salvelinus fontinalis) and native brown trout (Salmo trutta) translocated from downstream reaches by local anglers. Electrofishing for 12 consecutive years eradicated both fish species in the introduced area, and allowed the recovery of the R. iberica population as a result of natural recolonization from nearby streams and reintroduction of captive-reared individuals. Our results demonstrate how electrofishing can be a costly but effective method for the eradication of introduced fish and the conservation of stream-dwelling amphibians.

Itraconazole and thiophanate-methyl fail to clear tadpoles naturally infected with the hypervirulent lineage of Batrachochytrium dendrobatidis.

The emerging infectious disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, is a major driver pushing many amphibian species to the brink of extinction. Substantial efforts to develop effective protocols that use antifungal drugs have had notable success. Here, we used the antifungal agents itraconazole and thiophanate-methyl, singly and in combination, in an attempt to treat common midwife toad Alytes obstetricans larvae naturally infected with the globalized hypervirulent lineage of B. dendrobatidis. Despite the successful use of itraconazole in a closely related species (A. muletensis), our results show that these antifungal treatments are not always effective and that full clearance of animals cannot be assumed following treatment.

Multidimensional Genetic Analysis of Repeated Seizures in the Hybrid Mouse Diversity Panel Reveals a Novel Epileptogenesis Susceptibility Locus.

Epilepsy has many causes and comorbidities affecting as many as 4% of people in their lifetime. Both idiopathic and symptomatic epilepsies are highly heritable, but genetic factors are difficult to characterize among humans due to complex disease etiologies. Rodent genetic studies have been critical to the discovery of seizure susceptibility loci, including Kcnj10 mutations identified in both mouse and human cohorts. However, genetic analyses of epilepsy phenotypes in mice to date have been carried out as acute studies in seizure-naive animals or in Mendelian models of epilepsy, while humans with epilepsy have a history of recurrent seizures that also modify brain physiology. We have applied a repeated seizure model to a genetic reference population, following seizure susceptibility over a 36-d period. Initial differences in generalized seizure threshold among the Hybrid Mouse Diversity Panel (HMDP) were associated with a well-characterized seizure susceptibility locus found in mice: Seizure susceptibility 1 Remarkably, Szs1 influence diminished as subsequent induced seizures had diminishing latencies in certain HMDP strains. Administration of eight seizures, followed by an incubation period and an induced retest seizure, revealed novel associations within the calmodulin-binding transcription activator 1, Camta1 Using systems genetics, we have identified four candidate genes that are differentially expressed between seizure-sensitive and -resistant strains close to our novel Epileptogenesis susceptibility factor 1 (Esf1) locus that may act individually or as a coordinated response to the neuronal stress of seizures.

Eight Flurothyl-Induced Generalized Seizures Lead to the Rapid Evolution of Spontaneous Seizures in Mice: A Model of Epileptogenesis with Seizure Remission.

UNLABELLED: The occurrence of recurrent, unprovoked seizures is the hallmark of human epilepsy. Currently, only two-thirds of this patient population has adequate seizure control. New epilepsy models provide the potential for not only understanding the development of spontaneous seizures, but also for testing new strategies to treat this disorder. Here, we characterize a primary generalized seizure model of epilepsy following repeated exposure to the GABAA receptor antagonist, flurothyl, in which mice develop spontaneous seizures that remit within 1 month. In this model, we expose C57BL/6J mice to flurothyl until they experience a generalized seizure. Each of these generalized seizures typically lasts <30 s. We induce one seizure per day for 8 d followed by 24 h video-electroencephalographic recordings. Within 1 d following the last of eight flurothyl-induced seizures, ∼50% of mice have spontaneous seizures. Ninety-five percent of mice tested have seizures within the first week of the recording period. Of the spontaneous seizures recorded, the majority are generalized clonic seizures, with the remaining 7-12% comprising generalized clonic seizures that transition into brainstem seizures. Over the course of an 8 week recording period, spontaneous seizure episodes remit after ∼4 weeks. Overall, the repeated flurothyl paradigm is a model of epileptogenesis with spontaneous seizures that remit. This model provides an additional tool in our armamentarium for understanding the mechanisms underlying epileptogenesis and may provide insights into why spontaneous seizures remit without anticonvulsant treatment. Elucidating these processes could lead to the development of new epilepsy therapeutics. SIGNIFICANCE STATEMENT: Epilepsy is a chronic disorder characterized by the occurrence of recurrent, unprovoked seizures in which the individual seizure-ictal events are self-limiting. Remission of recurrent, unprovoked seizures can be achieved in two-thirds of cases by treatment with anticonvulsant medication, surgical resection, and/or nerve/brain electrode stimulation. However, there are examples in humans of epilepsy with recurrent, unprovoked seizures remitting without any intervention. While elucidating how recurrent, unprovoked seizures develop is critical for understanding epileptogenesis, an understanding of how and why recurrent, unprovoked seizures remit may further our understanding and treatment of epilepsy. Here, we describe a new model of recurrent, unprovoked spontaneous seizures in which the occurrence of spontaneous seizures naturally remits over time without any therapeutic intervention.

Dynamin 1 isoform roles in a mouse model of severe childhood epileptic encephalopathy.

Dynamin 1 is a large neuron-specific GTPase involved in the endocytosis and recycling of pre-synaptic membranes and synaptic vesicles. Mutations in the gene encoding dynamin 1 (DNM1) underlie two epileptic encephalopathy syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Mice homozygous for the Dnm1 "fitful" mutation, a non-synonymous coding variant in an alternatively spliced exon of Dnm1 (exon 10a; isoform designation: Dnm1a(Ftfl)) have an epileptic encephalopathy-like disorder including lethal early onset seizures, locomotor and neurosensory deficits. Although fitful heterozygotes have milder recurrent seizures later in life, suggesting an additive or semi-dominant mechanism, the molecular etiology must also consider the fact that Dnm1a(Ftfl) exerts a dominant negative effect on endocytosis in vitro. Another complication is that the fitful mutation induces alterations in the relative abundance of Dnm1 splice variants; mutants have a downregulation of Dnm1a and an upregulation of Dnm1b, changes which may contribute to the epileptic pathology. To examine whether Dnm1a loss of function, Dnm1a(Ftfl) dominance or compensation by Dnm1b is the most critical for severe seizures, we studied alternate isoform-specific mutant mice. Mice lacking Dnm1 exon 10a or Dnm1 exon 10b have neither spontaneous seizures nor other overt abnormalities, suggesting that in normal conditions the major role of each isoform is redundant. However, in the presence of Dnm1a(Ftfl) only exon 10a deleted mice experience severe seizures. These results reveal functional differences between Dnm1a and Dnm1b isoforms in the presence of a challenge, i.e. toxic Dnm1(Ftfl), while reinforcing its effect explicitly in this model of severe pediatric epilepsy.

Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy.

The childhood epileptic encephalopathies (EE's) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1Ftfl or "fitful" mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE's. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1Ftfl mouse model of DNM1 EE. Observations of Dnm1Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE.

Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential ß-Defensin Expression.

Identification of genetic factors that modify complex traits is often complicated by gene-environment interactions that contribute to the observed phenotype. In model systems, the phenotypic outcomes quantified are typically traits that maximize observed variance, which in turn, should maximize the detection of quantitative trait loci (QTL) in subsequent mapping studies. However, when the observed trait is dependent on multiple interacting factors, it can complicate genetic analysis, reducing the likelihood that the modifying mutation will ultimately be found. Alternatively, by focusing on intermediate phenotypes of a larger condition, we can reduce a model's complexity, which will, in turn, limit the number of QTL that contribute to variance. We used a novel method to follow angiogenesis in mice that reduces environmental variance by measuring endothelial cell growth from culture of isolated skin biopsies that varies depending on the genetic source of the tissue. This method, in combination with a backcross breeding strategy, is intended to reduce genetic complexity and limit the phenotypic effects to fewer modifier loci. We determined that our approach was an efficient means to generate recombinant progeny and used this cohort to map a novel s.c. angiogenesis QTL to proximal mouse chromosome (Chr.) 8 with suggestive QTL on Chr. 2 and 7. Global mRNA expression analysis of samples from parental reference strains revealed ß-defensins as potential candidate genes for future study.

Unraveling genetic modifiers in the gria4 mouse model of absence epilepsy.

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype - making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era.

Loss of MeCP2 from forebrain excitatory neurons leads to cortical hyperexcitation and seizures.

Mutations of MECP2 cause Rett syndrome (RTT), a neurodevelopmental disorder leading to loss of motor and cognitive functions, impaired social interactions, and seizure at young ages. Defects of neuronal circuit development and function are thought to be responsible for the symptoms of RTT. The majority of RTT patients show recurrent seizures, indicating that neuronal hyperexcitation is a common feature of RTT. However, mechanisms underlying hyperexcitation in RTT are poorly understood. Here we show that deletion of Mecp2 from cortical excitatory neurons but not forebrain inhibitory neurons in the mouse leads to spontaneous seizures. Selective deletion of Mecp2 from excitatory but not inhibitory neurons in the forebrain reduces GABAergic transmission in layer 5 pyramidal neurons in the prefrontal and somatosensory cortices. Loss of MeCP2 from cortical excitatory neurons reduces the number of GABAergic synapses in the cortex, and enhances the excitability of layer 5 pyramidal neurons. Using single-cell deletion of Mecp2 in layer 2/3 pyramidal neurons, we show that GABAergic transmission is reduced in neurons without MeCP2, but is normal in neighboring neurons with MeCP2. Together, these results suggest that MeCP2 in cortical excitatory neurons plays a critical role in the regulation of GABAergic transmission and cortical excitability.

Overexpression of RCAN1 isoform 4 in mouse neurons leads to a moderate behavioral impairment.

OBJECTIVES: Recent evidence supports the involvement of RCAN1 in Down syndrome and Alzheimer's disease. To better assess this, we generated and analyzed transgenic mice overexpressing human RCAN1 isoform 4 in neurons. METHODS: Cognitive behavioral (Morris water maze, open field, zero maze, elevated plus maze assays); cognitive-associated proteins (CREB, ERK and Tau Western immunoblotting); motor coordination (Rotarod assay); structural abnormalities (immunohistological analyses), and proinflammatory cytokines (cytometric bead assay) were measured in young (2 month) and old (18 month) transgenics and compared with wild type controls. RESULTS: In old mice, male but not female transgenics exhibited a significant decrease in anxiety as compared with wild type controls, whereas female but not male transgenic mice exhibited significantly less motor coordination. No differences were observed in the Morris water maze (spatial learning). pERK levels were reduced in transgenic males but not females, while no differences were observed between genotypes for pCREB and pTau. In young mice, a modest learning and exploratory behavior was observed in transgenic mice using a limited number of mice, and at higher N values, pCREB and pERK (but not pTau) levels were reduced in transgenics. No macro- and micro-scopic structural abnormalities or proinflammatory cytokine level differences were observed. DISCUSSION: These results indicate that elevated RCAN1 isoform 4 in neurons leads to a modest cognition-related impairment that is overall stronger at 2 months, suggesting a compensatory adaptation over time. These RCAN1 isoform 4 effects may contribute to at least some of the observed phenotypes in individuals with Down syndrome and Alzheimer's.

Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice.

In a chemical mutagenesis screen, we identified the novel Scn8a(8J) allele of the gene encoding the neuronal voltage-gated sodium channel Na(v)1.6. The missense mutation V929F in this allele alters an evolutionarily conserved residue in the pore loop of domain 2 of Na(v)1.6. Electroencephalography (EEG) revealed well-defined spike-wave discharges (SWD), the hallmark of absence epilepsy, in Scn8a(8J) heterozygotes and in heterozygotes for two classical Scn8a alleles, Scn8a(med) (null) and Scn8a(med-jo) (missense). Mouse strain background had a significant effect on SWD, with mutants on the C3HeB/FeJ strain showing a higher incidence than on C57BL/6J. The abnormal EEG patterns in heterozygous mutant mice and the influence of genetic background on SWD make SCN8A an attractive candidate gene for common human absence epilepsy, a genetically complex disorder.

Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4.

Absence epilepsy, characterized by spike-wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses-consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures.

Severe epilepsy resulting from genetic interaction between Scn2a and Kcnq2.

A mutation in the voltage-gated sodium-channel Scn2a results in moderate epilepsy in transgenic Scn2a(Q54) mice maintained on a C57BL/6J strain background. The onset of progressive epilepsy begins in adults with short-duration partial seizures that originate in the hippocampus. The underlying abnormality is an increase in persistent sodium current in hippocampal neurons. The voltage-gated potassium channel Kcnq2 is responsible for generating M current (I(KM)) that is thought to control excitability and limit repetitive firing of hippocampal neurons. To determine whether impaired M current would exacerbate the seizure phenotype of Scn2a(Q54) mice, we carried out genetic crosses with two mutant alleles of Kcnq2. Szt1 mice carry a spontaneous deletion that removes the C-terminal domain of Kcnq2. A novel Kcnq2 missense mutation V182M was identified by screening the offspring of ENU-treated males for reduced threshold to electrically evoked minimal clonic seizures. Double mutant mice carrying the Scn2a(Q54) transgene together with either of the Kcnq2 mutations exhibited severe epilepsy with early onset, generalized tonic-clonic seizures and juvenile lethality by 3 weeks of age. This dramatic exacerbation of the sodium-channel mutant phenotype indicates that M current plays a critical role in preventing seizure initiation and spreading in this animal model. The genetic interaction between Scn2a and Kcnq2 demonstrates that combinations of mild alleles of monogenic epilepsy genes can result in severe disease and provides a model for complex inheritance of human epilepsy. The data suggest that interaction between these genes might contribute to the variable expressivity observed in human families with sodium-channel mutations. In a screen of 23 SMEI patients with missense mutations of SCN1A, no second-site mutations in KCNQ2 were identified.

Development of a new genetic model for absence epilepsy: spike-wave seizures in C3H/He and backcross mice.

To characterize the genetic basis of spike-wave discharges (SWDs) detected by electroencephalography (EEG) in C3H/He mice, substrains of C3H mice were evaluated by EEG and sensitivity to ethosuximide. Crosses with the SWD-negative strain C57BL/6J were performed to map the underlying gene(s). C3H/He substrains exhibited a modest incidence (average of 19 SWDs per hour) of 7-8 Hz SWDs when at rest, compared with the C3HeB/Fe subline (four SWDs per hour). In the mapping backcross, however, many mice showed a very high incidence (50-220 SWDs per hour) throughout the recording period. SWDs were first detected at 3.5 weeks of age, were associated with behavioral arrest, were suppressed by ethosuximide, and were strongest in the cerebral cortex and thalamus. The major C3H determinant of SWDs, spkw1 (spike-wave 1), mapped to chromosome (Chr 9), and together with a C57BL/6J determinant on Chr 8, spkw2, accounted for more than one-half of the phenotypic variation in the backcross mice. The modest SWD incidence in C3H/He mice and the high incidence in backcrosses implies that SWD could be a confounding variable for other behaviors. Because C3H/He mice have no other brain abnormalities, they are an attractive alternative for studying idiopathic absence epilepsy.

A targeted mutation in Cacng4 exacerbates spike-wave seizures in stargazer (Cacng2) mice.

The voltage-dependent calcium channel gamma4 subunit protein, CACNG4, is closely related to the gamma2 subunit, CACNG2. Both are expressed primarily in the brain and share 53% amino acid identity. The Cacng2 gene is disrupted in the stargazer mouse, with its distinctive phenotype including ataxia, frequent absence seizure episodes, and head elevation. A disruption within Cacng4 was engineered to assess its particular function. The homozygous Cacng4-targeted mutant mouse appeared normal with no ataxic gait or absence seizures, suggesting that other members of the gamma subunit family might functionally compensate for the absence of CACNG4. To test this hypothesis, the targeted Cacng4 mutation was combined with alleles of Cacng2. Absence seizures were observed in combination with the stargazer 3J mutation, which itself does not have seizures, and increased seizure activity was observed in combination with the waggler allele. Furthermore, within the corticothalamic loop, where absence seizures arise, CACNG4 expression is restricted to the thalamus. Our studies show that the CACNG4 protein has seizure suppressing activity, but this effect is revealed only when CACNG2 expression is also compromised, suggesting that CACNG subunits have in vivo overlapping functions.

Phenotypic heterogeneity in the stargazin allelic series.

The stargazer mutant mouse is characterized by its ataxic gait, head tossing, and absence seizures. The mutation was identified in the gamma 2 subunit gene of the high voltage-dependent calcium channel, Cacng2. Subsequently, two allelic variants of stargazer have arisen, waggler and stargazer 3J. In this study, we have compared these new alleles to the original stargazer allele. All three mutations affect the Cacng2 mRNA levels as they all arise from disruptions within the introns of this gene. Our results show that the mutations cause reduced Cacng2 mRNA and protein levels. Stargazer and waggler mice have the least amount of mRNA and undetectable protein, whereas stargazer 3J appears to be the mildest allele, both in terms of the phenotype and protein expression. Electroencephalographic (EEG) analysis confirmed that stargazer has frequent spike-wave discharges (SWDs); the average duration of each discharge burst is 5 seconds and recurs every minute. The waggler allele causes a greater variation in SWD activity depending on the individual mouse, and the stargazer 3J mouse has no SWDs. The preliminary characterization of this heterogeneous allelic series provides a basis to explore more biochemical and physiological parameters relating to the role of the Cacng2 product in calcium channel activity, AMPA receptor localization, and cerebellar disturbances.

Spontaneous deletion of epilepsy gene orthologs in a mutant mouse with a low electroconvulsive threshold.

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.