Dissociation of seizure traits in inbred strains of mice using the flurothyl kindling model of epileptogenesis
Introduction
Epilepsy is one of the most common neurological diseases, affecting approximately 1–4% of the total population by the age of 80 (Engel and Pedley, 1998). While many of the current treatments for epilepsy block the symptoms of the epilepsy through seizure suppression, there are no specific therapies that target the changes in the brain that render it hyperexcitable in the epileptic state. The alteration in the excitability of cellular activity in the brain is known as epileptogenesis (Schwartzkroin et al., 2004) and has been demonstrated both in vitro and in vivo (Dichter, 2006, Schwartzkroin, 1986, Schwartzkroin and Franck, 1986). Often these changes in brain excitability result in alterations in seizure threshold and in seizure phenotype (Leite et al., 2002, McNamara, 1986, Mhyre and Applegate, 2003, Onat et al., 2007, Wlaz et al., 1998). The presence of spontaneous/unprovoked seizures can occur in some experimental models (i.e., kainate- or pilocarpine-induced epileptogenesis), but not in others (i.e., electrical kindling)(reviewed in (Leite et al., 2002, Sharma et al., 2007)). Analyses in animal models of epilepsy have led to the identification of loci controlling seizure susceptibility, but the genetic factors controlling epileptogenesis remain uncharacterized. Therefore, elucidation of the molecular mechanisms responsible for epileptogenesis will be important, if we are to gain a better understanding of epilepsy, and if we are to identify new therapies that target epileptogenesis.
Epileptic disorders are complex, quantitative traits controlled by genetic and environmental factors. Genetic heterogeneity is evident in inbred strains of mice that demonstrate different generalized seizure thresholds (GST) induced by seizure-inducing stimuli such as convulsant drugs (i.e., kainic acid, pentylenetetrazol, or bicuculline) or non-pharmacological manipulations (i.e., maximal electroshock or audiogenic seizures) (Engstrom and Woodbury, 1988, Ferraro et al., 2002, Ferraro et al., 2001, Ferraro et al., 2004, Ferraro et al., 1997, Ferraro et al., 1999, Ferraro et al., 1998, Frankel et al., 2001, Frankel et al., 1995, Hain et al., 2000, Kosobud and Crabbe, 1990, Schauwecker et al., 2004). However, no systematic mouse studies have been performed to reveal the genetic determinants of the processes that contribute to epileptogenesis.
One of the most popular models of epileptogenesis is electrical kindling. Electrical kindling is defined as a process whereby repeated and focal administrations of an initially subconvulsive electrical current, through brain electrodes, produce clonic seizures that become progressively more severe with each trial (epileptogenesis) eventually resulting in the consistent and permanent expression of generalized clonic–forebrain seizures (Goddard et al., 1969, McNamara et al., 1993, Sutula et al., 1986). Thus, in kindling, a reorganization of the brain occurs, such that fully-kindled animals, when retested at a later date, show a seizure response identical to the one that was displayed when they were last stimulated (Goddard et al., 1969, McNamara et al., 1993). This indicates the presence of a sustained hyperexcitability of neuronal activity in the kindled brain. In addition, electrically-kindled animals do not develop spontaneous/unprovoked seizures in traditional electrical kindling paradigms; only after large numbers (> 100) of kindled seizures can spontaneous/unprovoked seizures occasionally be observed (Lothman et al., 1992, Michalakis et al., 1998, Pinel and Rovner, 1978a, Pinel and Rovner, 1978b, Sharma et al., 2007). However, electrical kindling is not amenable to high-throughput genetic studies of epileptogenesis, since it requires surgical implantation of electrodes. As an alternative, the flurothyl kindling model is a non-invasive protocol that allows the examination of baseline GST and epileptogenic processes.
The flurothyl kindling model was previously characterized in C57BL/6J mice that received daily exposures to a 10% flurothyl solution over eight days. These exposures resulted in the expression of generalized clonic–forebrain seizures (Ferland and Applegate, 1998a, Ferland and Applegate, 1998b, Samoriski and Applegate, 1997, Samoriski et al., 1998). Over the course of the 8-day induction period, the GST of C57BL/6J mice decreased and then plateaued. When the mice were retested after a 28-day rest period, flurothyl exposure produced a GST equal to the plateaued level. Upon this flurothyl re-exposure, C57BL/6J mice displayed a change in the behavioral seizure phenotype expressed, from a generalized clonic–forebrain seizure (as observed in the induction-phase) to a generalized clonic–forebrain seizure that rapidly progressed into a generalized brainstem seizure (another epileptogenic process, hereafter referred to as a forebrain→brainstem seizure)(Applegate et al., 1997, Ferland and Applegate, 1998a, Ferland and Applegate, 1998b, Samoriski and Applegate, 1997, Samoriski et al., 1998). These experiments demonstrated the occurrence of epileptogenic processes in the flurothyl kindling model: decreases in GST over trials (kindling) and a change in seizure phenotype.
One major epileptogenic process, observed in the flurothyl kindling model, is a kindling of GST due to repeated seizure stimulation (Applegate et al., 1997, Ferland and Applegate, 1998a, Ferland and Applegate, 1998b, Samoriski and Applegate, 1997, Samoriski et al., 1998). This permanent decrease in GST is one of the hallmarks of classical kindling (Barnes and Pinel, 2001). Although the stimulation in the flurothyl kindling model is not subconvulsive, Ferland and Applegate (1999) demonstrated bidirectional transfer of kindling between the flurothyl kindling model and classical electrical kindling in C57BL/6J mice. That is, after 8 flurothyl-induced seizures, the rate of electrical kindling was increased. Similarly, when animals were first electrically kindled, increases in flurothyl-induced seizure susceptibility were seen. Importantly, mice that were electrically kindled, given a 28-day rest period, and exposed to one (first) trial with flurothyl, demonstrated the same change in seizure phenotype to forebrain→brainstem seizures (Ferland and Applegate, 1999). Overall, these results illustrated that the decreases in GST and change in seizure phenotype were independent of the method of seizure induction, and further supports the idea that animals in the flurothyl kindling model undergo epileptogenic processes (Ferland and Applegate, 1999).
In the current study, we examine the seizure characteristics of C57BL/6J, DBA/2J, 129S1/SvImJ, BALB/cJ, and C3H/HeJ strains of mice in the flurothyl kindling model. Differences in behavioral seizure characteristics in the flurothyl kindling model were most pronounced between C57BL/6J and DBA/2J mice; therefore, we focused on those two strains for detailed comparisons. C57BL/6J mice had a high initial GST, demonstrated a decrease in GST across trials, and showed an alteration in seizure phenotype following flurothyl retest. Conversely, DBA/2J mice had a lower initial GST, did not demonstrate a decrease in GST across trials, and did not show an alteration in seizure phenotype upon flurothyl retest. [C57BL/6J × DBA/2J] F1 hybrid mice were similar to C57BL/6J mice, with respect to initial GST and decreases of GST across 8 trials (kindling), but they did not undergo the alteration in behavioral seizure phenotype that was observed in C57BL/6J mice. Our results suggest genetic dissociation of epileptogenic characteristics observed in the flurothyl kindling model.
Section snippets
Animals
Adult male DBA/2J (n = 28), C57BL/6J (n = 28), [C57BL/6J × DBA/2J] F1 hybrid (n = 28), 129S1/SvImJ (n = 12), BALB/cJ (n = 10), and C3H/HeJ (n = 10) mice (7 weeks of age) were used to examine genetic differences in the flurothyl kindling model using the standard 10%-flurothyl concentration (Applegate et al., 1997, Ferland and Applegate, 1999, Samoriski and Applegate, 1997). Additional adult male DBA/2J (n = 10), C57BL/6J (n = 10), and 129S1/SvImJ (n = 10) mice (7 weeks of age) were used to test flurothyl at a lower
Results
To examine the genetic effects on various phenotypic characteristics in the flurothyl paradigm, we evaluated several inbred strains of mice (C57BL/6J, 129S1/SvImJ, C3H/HeJ, BALB/cJ, and DBA/2J). Since many of the seizure characteristics examined in the flurothyl paradigm were found to be dissociable between C57BL/6J and DBA/2J mice, we tested [C57BL/6J × DBA/2J] F1 hybrid mice in the flurothyl kindling model.
Discussion
Differences in seizure threshold among inbred strains of mice have been established using a variety of seizure-inducing stimuli (Engstrom and Woodbury, 1988, Ferraro et al., 2002, Ferraro et al., 2001, Ferraro et al., 2004, Ferraro et al., 1997, Ferraro et al., 1999, Ferraro et al., 1998, Frankel et al., 2001, Frankel et al., 1995, Hain et al., 2000, Kosobud and Crabbe, 1990, Schauwecker et al., 2004). Using the flurothyl kindling model, we have found differences in seizure susceptibility and
Acknowledgments
This work was partly supported by NIH grants 1K01MH71801 (to RJF) and R01MH065400 (to BJH). We wish to thank members of the Ferland laboratory and Dr. Fern P. Finger (RPI) for critical review of the manuscript. The authors wish to thank Dr. Adriana Verschoor (Wadsworth Center) for critical reading and input on our manuscript.
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2017, Epilepsy and BehaviorCitation Excerpt :Surprisingly, B6J × DBA/2J F1-hybrids had an initial GST on the first flurothyl trial and a GST decrease across flurothyl exposures similar to what was observed in B6J mice, but these F1-hybrids did not undergo the alteration in behavioral seizure phenotype that is observed in B6J mice. These F1-hybrid data demonstrate that initial GST, decreases in GST across flurothyl trials (kindling), and the change in seizure phenotype after a 28-day incubation period differ from the parental strains, suggesting that these phenotypes are controlled by independent genetic loci [115]. In fact, we have now completed an inbred stain survey of over 70 mouse strains, further confirming that the phenotypes observed with repeated flurothyl exposures are influenced by genetic factors and have led to the identification of genetic loci controlling these seizure traits [119].
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2015, Epilepsy ResearchCitation Excerpt :All statistical analyses were performed using Statistica (StatSoft). Since flurothyl infusion rates were held constant, latencies to generalized seizures were used as a surrogate for generalized seizure thresholds (GST) (Fig. 1) as has been previously described (Ferland and Applegate, 1999; Franck et al., 1989; Hashimoto et al., 2006; Oommen et al., 2007; Papandrea et al., 2009a, 2009b; Samoriski and Applegate, 1997; Stafstrom et al., 1992); however, concentrations of flurothyl necessary for generalized seizure expression are also reported (Table 1). All C57BL/6J (B6) mice tested in the repeated-flurothyl model had a high initial latency to generalized seizure expression compared to DBA/2J (D2) mice, which significantly decreased over the course of eight repeated flurothyl exposures, plateauing on seizure trial 5 (Fig. 1; F7,161 = 142.65, P < 0.0001; Table 1).
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These authors contributed equally to this project.