Delayed myelination and neurodevelopment in male seizure-prone versus seizure-resistant rats.

OBJECTIVE: Aberrant myelination and developmental delay have been reported in epilepsy. However, it is unclear whether these are linked to intrinsic mechanisms that support a predisposition toward seizures and the development of epilepsy. Thus, we compared rates of myelination and neurodevelopment in male rats selectively bred for enhanced susceptibility to kindling epileptogenesis (FAST) with male rats bred for resistance (SLOW). METHODS: Myelin-specific gene expression was compared in the brainstem, cerebellum, and cerebral hemisphere of FAST and SLOW rats on postnatal days (PNDs) 5, 11, 17, 23, and 90 to determine strain-specific myelination rates. Myelin protein levels were also compared at PNDs 5 and 23 in the brainstem. Relative rates of neurodevelopment were evaluated between PNDs 5 and 21 using physical growth landmarks and neuromotor tests including righting reflex, cliff avoidance, negative geotaxis, and locomotor activity. RESULTS: Myelin-specific mRNA expression was significantly down-regulated in FAST rats on PNDs 5 and 11 in all 3 brain structures, indicating relatively delayed myelination. Likewise, corresponding protein levels were significantly lower in FAST brainstem on PND 5. Developmental delay was evident in the FAST strain such that only 9% of FAST pups, compared to 81% of SLOW, had open eyes by PND 13, locomotor activity was significantly reduced between PNDs 12 and 16, and neuromotor task acquisition was delayed between PNDs 5 and 10. SIGNIFICANCE: Relative delays in myelination and neurodevelopment co-occurred in the seizure-prone FAST strain in the absence of seizures. These findings suggest these symptoms are not seizure-induced and may be mechanistically linked to an underlying pathophysiology supporting a predisposition toward developing epilepsy.

Differences in white matter structure between seizure prone (FAST) and seizure resistant (SLOW) rat strains.

Alterations in white matter integrity have been well documented in chronic epilepsy and during epileptogenesis. However, the relationship between white matter integrity and a predisposition towards epileptogenesis has been understudied. The FAST rat strain exhibit heightened susceptibility towards kindling epileptogenesis whereas SLOW rats are highly resistant. FAST rats also display behavioral phenotypes reminiscent of those observed in neurodevelopmental disorders that commonly comorbid with epilepsy. In this study, we aim to identify differences in white matter integrity that may contribute to a predisposition towards epileptogenesis and its associated comorbidities in 6month old FAST (n=10) and SLOW (n=10) male rats. Open field and water consumption tests were conducted to confirm the behavioral phenotype difference between FAST and SLOW rats followed by ex-vivo diffusion-weighted magnetic resonance imaging to identify differences in white matter integrity. Diffusion tensor imaging scalar values namely fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity were compared in the anterior commissure, corpus callosum, external capsule, internal capsule, fimbria and optic tract. Electron microscopy was used to evaluate microstructural alterations in myelinated axons. Behavioral phenotyping confirmed higher activity levels (distance moved on days 2-4, p<0.001; number of rearings on days 2 and 4, p<0.05 at both days) and polydipsia (p<0.001) in FAST rats. Comparative analysis of diffusion tensor imaging scalars found a significant decrease in fractional anisotropy in the corpus callosum (p<0.05) of FAST versus SLOW rats. Using electron microscopy, alterations in myelinated axons including increased axon diameter (p<0.001) and reduced g-ratio (p<0.001) in the midline of the corpus callosum in 6month old FAST (n=3) versus SLOW (n=4) male rats. These findings suggest that differences in white matter integrity between FAST and SLOW rats could be a contributing factor to the differential seizure susceptibility and behavioral phenotypes observed in these strains.

Neuroanatomical differences in FAST and SLOW rat strains with differential vulnerability to kindling and behavioral comorbidities.

OBJECTIVE: The neurobiological factors underlying a predisposition towards developing epilepsy and its common behavioral comorbidities are poorly understood. FAST rats are a strain that has been selectively bred for enhanced vulnerability to kindling, while the SLOW strain has been bred to be resistant to kindling. FAST rats also exhibit behavioral traits reminiscent of those observed in neurodevelopmental disorders (autism spectrum disorder (ASD)/attention-deficit/hyperactivity disorder (ADHD)) commonly comorbid with epilepsy. In this study, we aimed to investigate neuroanatomical differences between these strains that may be associated with a differential vulnerability towards these interrelated disorders. METHODS: Ex vivo high-resolution magnetic resonance imaging on adult male FAST and SLOW rat brains was performed to identify morphological differences in regions of interest between the two strains. Behavioral examination using open-field, water consumption, and restraint tests was also conducted on a subgroup of these rats to document their differential ASD/ADHD-like behavior phenotype. Using optical stereological methods, the volume of cerebellar granule, white matter, and molecular layer and number of Purkinje cells were compared in a separate cohort of adult FAST and SLOW rats. RESULTS: Behavioral testing demonstrated hyperactivity, impulsivity, and polydipsia in FAST versus SLOW rats, consistent with an ASD/ADHD-like phenotype. Magnetic resonance imaging analysis identified brain structural differences in FAST compared with SLOW rats, including increased volume of the cerebrum, corpus callosum, third ventricle, and posterior inferior cerebellum, while decreased volume of the anterior cerebellar vermis. Stereological measurements on histological slices indicated significantly larger white matter layer volume, reduced number of Purkinje cells, and smaller molecular layer volume in the cerebellum in FAST versus SLOW rats. SIGNIFICANCE: These findings provide evidence of structural differences between the brains of FAST and SLOW rats that may be mechanistically related to their differential vulnerability to kindling and associated comorbid ASD/ADHD-like behaviors.

WONOEP appraisal: new genetic approaches to study epilepsy.

New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type-specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy.

Epilepsy, autism, and neurodevelopment: kindling a shared vulnerability?

Epilepsy and autism spectrum disorder (ASD) share many primary and comorbid symptoms. The degree of clinical overlap is believed to signify a 'spectrum of vulnerability' that arises out of an early common dysfunction in central nervous system development. However, research into the underlying, and potentially shared, etiopathological mechanisms is challenging given the extensive comorbidity profiles. Adding to the degree of difficulty is the frequently evolving recompartmentalization of diagnostic criteria within each disorder. This review discusses potential preclinical strategies that, through the use of animal models, are designed to gain insight into the biological basis of the overlap between epilepsy and autism and to foster a rapid clinical translation of the insights gained.

Postnatal epigenetic influences on seizure susceptibility in seizure-prone versus seizure-resistant rat strains.

The creation of seizure-prone (Fast) and seizure-resistant (Slow) rat strains via selective breeding implies genetic control of relative seizure vulnerability, yet ample data also advocates an environmental contribution. To investigate potential environmental underpinnings to the differential seizure sensitivities in these strains, the authors compared amygdala kindling profiles in adult male Fast and Slow rats raised by (a) their own mother, (b) a foster mother from the same strain, or (c) a foster mother from the opposing strain. Ultimately, strain-specific kindling profiles were not normalized by cross-fostering. Instead, both strains became more seizure-prone regardless of maternal affiliation (i.e., cross-fostered groups from both strains kindled faster than uncrossed controls). Interhemispheric seizure spread was also facilitated in cross-fostered Slow rat groups and was associated with increased commissural cross-sectional areas, giving them a Fast-like profile. It is important to note, however, that all Fast groups remained significantly more seizure-prone than Slow groups, suggesting that although the postnatal environment strongly influenced seizure disposition in both strains, it did not wholly account for their relative dispositions. Investigation into mechanisms fundamental to cross-fostering-induced seizure facilitation should help prevent postnatal worsening of pathology in already seizure-prone individuals.

Investigating epigenetic influences on seizure disposition.

Rats selectively bred to be seizure-prone versus resistant naturally exhibit behaviors, neuroanatomy and physiology that are reminiscent of attention deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD) in humans. Evidence suggests these characteristics evolve via genetic/epigenetic mechanisms acting prior to birth that most likely involve aberrant lipid handling.

Kindling as a model of human epilepsy.

The evidence supporting the suggestion that kindling is a good model of human temporal lobe epilepsy is briefly reviewed. Parallels between the human condition, involving both partial and secondarily generalized seizures, and kindling in rats and other animals are drawn and contrasted.

A new rat model for vulnerability to epilepsy and autism spectrum disorders.

Tremendous concern has arisen in response to the recent diagnostic outbreak of childhood developmental disorders, particularly involving attention deficit hyperactivity disorder (ADHD) and the autism spectrum disorders (ASD). Interestingly, similarities in clinical presentation across these disorders may suggest common predisposing factors. For instance, though not widely recognized, an increased predisposition toward seizure is a symptom that is very often associated with ADHD and ASD. Accordingly, a rat strain naturally bred to be seizure-prone simultaneously developed behavioral and physical characteristics analogous to those observed in ADHD/ASD patients. These rats also show early signs of aberrant lipid handling, which is another symptom common to human patients with these disorders. As such, this rat strain could serve as an excellent model system through which to identify common pathophysiological events that constitute a"spectrum of vulnerability"toward ADHD/ASD and epilepsy.

Ruling out postnatal origins to attention-deficit/hyperactivity disorder (ADHD)-like behaviors in a seizure-prone rat strain.

Adult Fast (seizure-prone) and Slow (seizure-resistant) kindling rat strains exhibit divergent behaviors in paradigms relevant to attention-deficit/hyperactivity disorder (ADHD) in humans. Similar dissociations in rodent behavior have been linked to disparities in early life experience, suggesting that differential maternal care or postnatal interactions may underlie these behaviors. Consequently, the authors compared maternal behavior and preweaning pup weights in these 2 strains under control and cross-fostered conditions and examined its effects on subsequent adult offspring behavior. Ultimately, several distinct maternal behaviors were apparent between the 2 strains under control conditions, and some of those behaviors were then malleable by pup condition. Yet, in spite of the resultant complex maternal patterns across groups, all offspring showed behavioral phenotypes akin to their genetic strain. Thus, a specific postnatal environment is unlikely to underwrite ADHD-like behaviors in the seizure-prone Fast rats, which implicates a genetic or prenatal origin for the ADHD phenotype.

Parahippocampal networks, intractability, and the chronic epilepsy of kindling.

Clearly, the root cause of intractability in epilepsy is currently unknown. Whereas the aforementioned findings may shed light on putative underpinnings, they are by no means an exhaustive list of possibilities. However, new and more effective animal models are continually being created or discovered that take into account genetic predisposition for seizure. At the moment, amygdala kindling appears to be the best choice of the intact animal models. In this vein, the genetically predisposed seizure-prone (Fast kindling) and seizure-resistant (Slow kindling) strains may help speak to many important remaining questions in human epilepsy. Hopefully, these models, to some degree, target correct human subpopulations that are prone or resistant to epilepsy and, when used appropriately, could expedite epilepsy research and future discoveries leading to pharmacoresistance and intractability.

Studying autism in rodent models: reconciling endophenotypes with comorbidities.

Autism spectrum disorder (ASD) patients commonly exhibit a variety of comorbid traits including seizures, anxiety, aggressive behavior, gastrointestinal problems, motor deficits, abnormal sensory processing, and sleep disturbances for which the cause is unknown. These features impact negatively on daily life and can exaggerate the effects of the core diagnostic traits (social communication deficits and repetitive behaviors). Studying endophenotypes relevant to both core and comorbid features of ASD in rodent models can provide insight into biological mechanisms underlying these disorders. Here we review the characterization of endophenotypes in a selection of environmental, genetic, and behavioral rodent models of ASD. In addition to exhibiting core ASD-like behaviors, each of these animal models display one or more endophenotypes relevant to comorbid features including altered sensory processing, seizure susceptibility, anxiety-like behavior, and disturbed motor functions, suggesting that these traits are indicators of altered biological pathways in ASD. However, the study of behaviors paralleling comorbid traits in animal models of ASD is an emerging field and further research is needed to assess altered gastrointestinal function, aggression, and disorders of sleep onset across models. Future studies should include investigation of these endophenotypes in order to advance our understanding of the etiology of this complex disorder.

Relation between startle reactivity and sucrose avidity in two rat strains bred for differential seizure susceptibility.

Rat strains selectively bred to be seizure-prone (Fast) versus seizure-resistant (Slow) show differing levels of anxiety, with Slow rats displaying relatively enhanced anxiety-like behaviors to aversive stimuli. Ample data has suggested that highly anxious rodents exhibit reduced avidity for sucrose and greater startle responses than rodents with relatively low anxiety levels. Thus, it was hypothesized that the Slow rats would have lower appetitive (sucrose consumption) and greater defensive (startle response) behaviors than Fast rats. Results confirmed that Slow rats consumed significantly less sucrose and exhibited greater acoustic startle responses than Fast rats. Startle response magnitude was not associated with water consumption, food consumption or body weight but was negatively correlated with sucrose consumption. These observations attest to the link between sucrose avidity and startle reactivity and further reveal that genetic selection for amygdala excitability lead to strain differences in appetitive and defensive behaviors. Thus, Fast and Slow rats may be two unique strains with which to further elucidate the genetic and neurobiological mechanisms underlying appetitive and defensive behaviors and their relation to anxiety and seizure sensitivity.

Caloric restriction alters seizure disposition and behavioral profiles in seizure-prone (fast) versus seizure-resistant (slow) rats.

Caloric restriction (CR), primarily known for extending life span, has proven anticonvulsant in several seizure models and antiepileptogenic in a strain of inherently seizure susceptible mice. Our animal model consisted of a seizure-prone (Fast) strain that naturally exhibits attention-deficit/hyperactivity disorder (ADHD)-like behaviors and a comparison seizure-resistant (Slow) strain; we evaluated CR's effect on the typical seizure sensitivities and behavioral profiles of each strain. Fast and Slow rats were fed ad libitum or were calorically restricted to 80% of free-feeding body weight. Rats were then tested in the open field (hyperactivity), Morris water maze (learning and attention), and restraint (impulsivity) paradigms and finally kindled from the amygdala. Ultimately, CR abolished signs of abnormal hyperactivity in the Fast strain and retarded their kindling rates, making it the first manipulation to demonstrate an antiepileptogenic effect in this animal model. CR also shortened seizure durations in fully kindled Slow rats but had no effect on their kindling rates, implying a differential effect of CR on genotype. These results clearly endorse further investigation into the potential benefits of CR for both epilepsy and ADHD.

Mapping seizure pathways in the temporal lobe.

Interest in temporal lobe seizure pathways has a long history based initially on the human condition of temporal lobe epilepsy (TLE). This interest in TLE has extended more recently into explorations of experimental models. In this review, the network structures in the temporal lobe that are recruited in animal models during various forms of limbic seizures and status epilepticus are described. Common to all of the various models is recruitment of the parahippocampal cortices, including the piriform, perirhinal, and entorhinal areas. This cortical involvement is seen in in vitro and in vivo electrophysiological recordings throughout the network, in trans-synaptic neuroplastic changes in associated network structures manifest at the molecular level, in network energy utilization visualized by 14C2-deoxyglucose uptake, and finally, in the behavioral consequences of network lesions. The conclusions of the animal models reviewed here are very similar to those described for the human condition presented recently in the 2006 Lennox lecture by Warren Blume, and addressed 53 years ago in the quadrennial meeting of the ILAE in 1953 by Henri Gastaut.

Genetically seizure-prone or seizure-resistant phenotypes and their associated behavioral comorbidities.

It was questioned whether amygdala kindling, a model of temporal lobe epilepsy, is under genetic control, and is associated with comorbid behavioral features. Initially, rats were selectively bred for speed of amygdala kindling, and, in subsequent generations, were assessed in behavioral paradigms to measure activity, emotionality, impulsivity, and learning. Clearly kindling was under genetic control, as two strains were developed to be either Fast or Slow to kindle, and each was associated with different neurological, electrophysiological and behavioral features. Behaviorally, the Fast rats appear much like humans with attention deficit hyperactivity disorder (ADHD), showing easy distraction, hyperactivity and impulsivity, compared to Slow rats.

Effect of focal low-frequency stimulation on amygdala-kindled afterdischarge thresholds and seizure profiles in fast- and slow-kindling rat strains.

PURPOSE: To determine whether low-frequency, 1-Hz sine-wave stimulation (LFS) applied to a fully kindled amygdala focus would show antiepileptic properties in rats that were either naturally seizure prone (Fast) or seizure resistant (Slow). METHODS: Normal twisted and/or "spanning" bipolar electrode configurations were implanted in the amygdalae of adult male Fast and Slow rats. In experiment one, rats were kindled daily to stage-5 levels through one electrode type until stable afterdischarge thresholds (ADTs) were obtained. Next, LFS was applied through the kindled electrode, and ADTs were redetermined 1 min later, and daily for a week, without reapplying the LFS. In experiment two, a single, normal bipolar kindling electrode was implanted in the amygdala and centered between two poles of a spanning electrode. After stable kindled ADTs were obtained, LFS was applied to the amygdala "area" through the spanning electrode. ADTs were redetermined at the kindled electrode as earlier. RESULTS: LFS through the kindling electrode had no effect on ADTs 1 min later, but the ADTs increased dramatically 24 h later and then slowly returned to baseline over days. In experiment two, LFS applied through the nonkindled spanning electrode also showed a small but significant threshold elevation at the interposing kindled electrode. Importantly, no obvious neuropathology was associated with these LFS treatments. CONCLUSIONS: LFS applied directly to the kindled network has significant threshold-elevating properties that are less evident when applied to the "general area"; here LFS must be delivered through a larger surface area and/or at higher intensity.

Neurodevelopment in seizure-prone and seizure-resistant rat strains: recognizing conflicts in management.

Cytoarchitectural alterations during central nervous system (CNS) development are believed to underlie aberrations in brain morphology that lead to epilepsy. We have recently reported marked reductions in hippocampal and white matter volumes along with relative ventriculomegaly in a rat strain bred to be seizure-prone (FAST) compared to a strain bred to be seizure-resistant (SLOW) (Gilby et al., 2002, American Epilepsy Society 56th Annual Meeting). This study was designed to investigate deviations in gene expression during late-phase embryogenesis within the brains of FAST and SLOW rats. In this way, we hoped to identify molecular mechanisms operating differentially during neurodevelopment that might ultimately create the observed differences in brain morphology and/or seizure susceptibility. Using Superarray technology, we compared the expression level of 112 genes, known to play a role in neurodevelopment, within whole brains of embryonic day 21 (E21) FAST and SLOW rats. Results revealed that while most genes investigated showed near equivalent expression levels, both Apolipoprotein E (APOE) and the beta2 subunit of the voltage-gated sodium channel (SCN2beta) were significantly underexpressed in brains of the seizure-prone embryos. Currently, these transcripts have no known interactions during embryogenesis; however, they have both been independently linked to seizure disposition and/or neurodevelopmental aberrations leading to epilepsy. Thus, alterations in the timing and/or degree of expression for APOE and SCN2beta may be important to developmental cascades that ultimately give rise to the differing brain morphologies, behaviors, and/or seizure vulnerabilities that characterize these strains.

Differential GABA(A) subunit expression following status epilepticus in seizure-prone and seizure-resistant rats: a putative mechanism for refractory drug response.

PURPOSE: Two rat strains were selectively bred to be prone (Fast) or resistant (Slow) to amygdala kindling. The first objective of this experiment was to determine whether that selection was specific to kindling or was sensitive more broadly to another seizure induction agent, kainic acid (KA). Second, we investigated whether these strains exhibit distinct molecular responses to KA with respect to GABA(A) receptor subunit expression. METHODS: Development of status epilepticus (SE) was profiled in Fast and Slow rats injected with 20 mg/kg KA (i.p.). Two hours post-SE onset, rats received a sedative dose of sodium pentobarbital. Behavioral profiles included latency to SE, number of wet dog shakes (WDS), and number and duration of stage 3-5 generalized seizures. Rats were killed 24 h post-SE, and alpha(1) and alpha(4) mRNA levels were compared in the hippocampus and amygdala using QPCR. RESULTS: Slow rats exhibited a much greater latency to SE onset (p < 0.01) and many more WDS (p < 0.01) than Fast rats. During SE, Fast rats spent more time in and exhibited more repeated bouts of generalized stage 3-5 seizures (p < 0.01) than Slow rats. Constitutive levels of alpha1 and alpha4 were not different between the strains in either structure and equivalent reductions in alpha4 were evident 24 h post-SE. However, while Fast rats showed KA-induced reductions in alpha1 in both structures, Slow rats showed significant elevations. CONCLUSIONS: Genetic selection for temporal lobe excitability, manifested as differential amygdala kindling rates, is paralleled by vulnerability to KA-induced SE. Further, these strains exhibited at least one opposing molecular response to SE, namely alpha1 expression. This finding may offer a putative mechanism through which seemingly similar epilepsies can be intractable in some patients but treatable in others.