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.

Generation of quality-controlled SARS-CoV-2 variant stocks.

One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research-for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controlled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5-8 d are required when virus is isolated from clinical swab material, and another 6-7 d is needed for sucrose-purifying the stocks.

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.

Spatiotemporal differences in the c-fos pathway between C57BL/6J and DBA/2J mice following flurothyl-induced seizures: A dissociation of hippocampal Fos from seizure activity.

Significant differences in seizure characteristics between inbred mouse strains highlight the importance of genetic predisposition to epilepsy. Here, we examined the genetic differences between the seizure-resistant C57BL/6J (B6) mouse strain and the seizure-susceptible DBA/2J (D2) strain in the phospho-Erk and Fos pathways to examine seizure-induced neuronal activity to uncover potential mechanistic correlates to these disparate seizure responsivities. Expression of neural activity markers was examined following 1, 5, or 8 seizures, or after 8 seizures, a 28 day rest period, and a final flurothyl rechallenge. Two brain regions, the hippocampus and ventromedial nucleus of the hypothalamus (VMH), had significantly different Fos expression profiles following seizures. Fos expression was highly robust in B6 hippocampus following one seizure and remained elevated following multiple seizures. Conversely, there was an absence of Fos (and phospho-Erk) expression in D2 hippocampus following one generalized seizure that increased with multiple seizures. This lack of Fos expression occurred despite intracranial electroencephalographic recordings indicating that the D2 hippocampus propagated ictal discharge during the first flurothyl seizure suggesting a dissociation of seizure discharge from Fos and phospho-Erk expression. Global transcriptional analysis confirmed a dysregulation of the c-fos pathway in D2 mice following 1 seizure. Moreover, global analysis of RNA expression differences between B6 and D2 hippocampus revealed a unique pattern of transcripts that were co-regulated with Fos in D2 hippocampus following 1 seizure. These expression differences could, in part, account for D2's seizure susceptibility phenotype. Following 8 seizures, a 28 day rest period, and a final flurothyl rechallenge, ∼85% of B6 mice develop a more complex seizure phenotype consisting of a clonic-forebrain seizure that uninterruptedly progresses into a brainstem seizure. This seizure phenotype in B6 mice is highly correlated with bilateral Fos expression in the VMH and was not observed in D2 mice, which always express clonic-forebrain seizures upon flurothyl retest. Overall, these results illustrate specific differences in protein and RNA expression in different inbred strains following seizures that precede the reorganizational events that affect seizure susceptibility and changes in seizure semiology over time.

Segregation of seizure traits in C57 black mouse substrains using the repeated-flurothyl model.

Identifying the genetic basis of epilepsy in humans is difficult due to its complexity, thereby underlying the need for preclinical models with specific aspects of seizure susceptibility that are tractable to genetic analyses. In the repeated-flurothyl model, mice are given 8 flurothyl-induced seizures, once per day (the induction phase), followed by a 28-day rest period (incubation phase) and final flurothyl challenge. This paradigm allows for the tracking of multiple phenotypes including: initial generalized seizure threshold, decreases in generalized seizure threshold with repeated flurothyl exposures, and changes in the complexity of seizures over time. Given the responses we previously reported in C57BL/6J mice, we analyzed substrains of the C57BL lineage to determine if any of these phenotypes segregated in these substrains. We found that the generalized seizure thresholds of C57BL/10SNJ and C57BL/10J mice were similar to C57BL/6J mice, whereas C57BL/6NJ and C57BLKS/J mice showed lower generalized seizure thresholds. In addition, C57BL/6J mice had the largest decreases in generalized seizure thresholds over the induction phase, while the other substrains were less pronounced. Notably, we observed only clonic seizures during the induction phase in all substrains, but when rechallenged with flurothyl after a 28-day incubation phase, ∼80% of C57BL/6J and 25% of C57BL/10SNJ and C57BL/10J mice expressed more complex seizures with tonic manifestations with none of the C57BL/6NJ and C57BLKS/J mice having complex seizures with tonic manifestations. These data indicate that while closely related, the C57BL lineage has significant diversity in aspects of epilepsy that are genetically controlled. Such differences further highlight the importance of genetic background in assessing the effects of targeted deletions of genes in preclinical epilepsy models.

Dissociation of seizure traits in inbred strains of mice using the flurothyl kindling model of epileptogenesis.

Previous seizure models have demonstrated genetic differences in generalized seizure threshold (GST) in inbred mice, but the genetic control of epileptogenesis is relatively unexplored. The present study examined, through analysis of inbred strains of mice, whether the seizure characteristics observed in the flurothyl kindling model are under genetic control. Eight consecutive, daily generalized seizures were induced by flurothyl in mice from five inbred strains. Following a 28-day rest period, mice were retested with flurothyl. The five strains of mice demonstrated inter-strain differences in GST, decreases in GST across seizure trials, and differences in the behavioral seizure phenotypes expressed. Since many of the seizure characteristics that we examined in the flurothyl kindling model were dissociable between C57BL/6J and DBA/2J mice, we analyzed these strains in detail. Unlike C57BL/6J mice, DBA/2J mice had a lower GST on trial 1, did not demonstrate a decrease in GST across trials, nor did they show an alteration in seizure phenotype upon flurothyl retest. Surprisingly, [C57BL/6JxDBA/2J] F1-hybrids had initial GST on trial 1 and GST decreases across trials similar to what was found for C57BL/6J, but they did not undergo the alteration in behavioral seizure phenotype that had been observed for C57BL/6J mice. Our data establish the significance of the genetic background in flurothyl-induced epileptogenesis. The [C57BL/6JxDBA/2J] F1-hybrid data demonstrate that initial GST, the decrease in GST across trials, and the change in seizure phenotype differ from the characteristics of the parental strains, suggesting that these phenotypes are controlled by independent genetic loci.

Analysis of flurothyl-induced myoclonus in inbred strains of mice.

Myoclonus is often observed in epilepsy. It is characterized by sudden involuntary shock-like movements of the body (myoclonic jerks, MJs). This study examined whether epileptic myoclonus was under genetic control. Inbred strains of mice were administered eight daily flurothyl exposures, a 28-day rest period, and a final flurothyl retest. For all trials, the latency to the first MJ (threshold) and the number of MJs (MJ#) were recorded. The inbred strains that we examined exhibited significant variability in initial myoclonic response, and myoclonus across the eight flurothyl exposures. C57BL/6J and DBA/2J mice displayed significantly different initial latencies to a MJ, MJ# preceding a generalized seizure (GS), and changes in MJ threshold and MJ# across the eight seizure trials. [C57BL/6J x DBA/2J] F1-hybrid mice showed an initial MJ threshold and decreases in MJ threshold over the eight trials, which were similar to C57BL/6J; however, F1-hybrids had an initial MJ# and trend in MJ# over the eight trials that were similar to DBA/2J. Decreases in MJ threshold and MJ# following multiple seizure trials, observed in C57BL/6J mice, were dependent on the expression of GSs and not on MJ occurrence. Our study is the first to document the potential for genetic heterogeneity of myoclonus in mice; we show that significant alterations in myoclonic behavior occur after GSs. These results indicate that multiple GSs affect MJ thresholds. An understanding of the genetics of myoclonus will be important for determination of the brain areas responsible for myoclonus as well as for identification of candidate genes.

Effects of aging and D-glucose on locomotor activity, spontaneous alternation, and plasma glucose levels in preweanling Sprague-Dawley rats.

Previous research has shown that developmentally related memory impairments in immature rodents are malleable and may be attenuated using a variety of pharmacological and behavioral treatments. This experiment examined the effects of glucose (10, 100, 250 or 500 mg/kg) or saline on blood glucose level, locomotor activity, and spontaneous alternation (SA) in the T-maze. Studies were conducted with 20-, 22-, and 24-day-old preweanling Sprague-Dawley rats. Results indicated a general decline in blood glucose level with age and size of glucose injection. The largest overall decline in blood glucose was found following the 500 mg/kg dose in 24-day-old animals. An increase in T-maze arm entries with age indicated increased exploratory activity. SA generally improved with age, but glucose had no reliable effect on behavior. The ontogenetic state of the nervous system, including changes in cholinergic system activity, glucoregulation, and proliferation of glucose transporters, is discussed.