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

Abstract

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.

Dissociation of spontaneous seizures and brainstem seizure thresholds in mice exposed to eight flurothyl‐induced generalized seizures

“…For determination of forebrain and brainstem seizure thresholds throughout the repeated‐flurothyl paradigm, a second group of male mice (C57BL/6J [n = 24] and DBA/2J [n = 24]) were utilized, and their generalized forebrain and brainstem seizure thresholds were determined at three time points in the repeated‐flurothyl model: after one or eight flurothyl induction trials, or following eight flurothyl seizures, 28‐day incubation, and final flurothyl rechallenge (n = 8/time‐point/strain). Mice were ~8 weeks old when seizure testing began, consistent with previous studies utilizing the repeated‐flurothyl model …”

“…Previously, we have shown that the ventromedial nucleus of hypothalamus (VMH) displays increased bilateral neuronal activity, as determined by Fos immunolabeling, in C57BL/6J mice having forebrain→brainstem seizures . Functionally, bilateral ibotenic acid lesions of the VMH are able to block the expression of the brainstem seizure component of forebrain→brainstem seizures .…”

“…Following a 1‐month incubation period and a flurothyl rechallenge, C57BL/6J mice subsequently express a clonic‐forebrain seizure that rapidly transitions uninterruptedly into a tonic‐brainstem seizure . We refer to these seizures as forebrain→brainstem seizures, denoting the ictal progression from the forebrain seizure network to the brainstem seizure network . In fact, there is a loss of ictal discharge in hippocampal electrodes when the behavioral seizure transitions from the clonic phase to the brainstem phase .…”

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

“…Studies are currently underway attempting to elucidate what these genetic differences could be and the signaling pathways that may be involved. Last, gene expression changes have been observed in C57BL/6J mice over the course of the flurothyl induction period (Kadiyala et al, 2015), which could indicate the possibility of flurothyl-induced and/or seizure-induced changes in gene expression contributing to the transient nature of spontaneous seizures in C57BL/6J mice. Genes that were shown to change with flurothyl-induced seizures were similar to what has been observed in electrical kindling models (Christensen et al, 2010;Kadiyala et al, 2015).…”

The Repeated Flurothyl Seizure Model in Mice

“…Following a one month incubation period and a rechallenge with flurothyl, C57BL/6J mice express a clonic-forebrain seizure that rapidly and uninterruptedly transitions into a tonic-brainstem seizure (Samoriski and Applegate, 1997; Ferland and Applegate, 1998b). We refer to these seizures as forebrain→brainstem seizures denoting the ictal progression from the forebrain seizure network to the brainstem seizure network (Papandrea et al ., 2009; Kadiyala et al ., 2015). Lastly, C57BL6/J mice exposed to the repeated flurothyl seizure model rapidly develop spontaneous seizures that appear to remit without treatment following 1 month (Kadiyala et al ., 2016), in contrast to DBA2/J mice which also rapidly develop spontaneous seizures that do not remit (Kadiyala and Ferland, 2017).…”