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In rodents, the cholinomimetic convulsant pilocarpine is widely used to induce status epilepticus (SE), followed by hippocampal damage and spontaneous recurrent seizures, resembling temporal lobe epilepsy. This model has initially been described in rats, but is increasingly used in mice, including the C57BL/6 (B6) inbred strain. In the present study, we compared the effects of pilocarpine in three B6 substrains (B6JOla, B6NHsd and B6NCrl) that were previously reported to differ in several behavioral and genetic aspects. In B6JOla and B6NHsd, only a small percentage of mice developed SE independently of whether pilocarpine was administered at high bolus doses or with a ramping up dosing protocol, but mortality was high. The reverse was true in B6NCrl, in which a high percentage of mice developed SE, but mortality was much lower compared to the other substrains. However, in subsequent experiments with B6NCrl mice, striking differences in SE induction and mortality were found in sublines of this substrain coming from different barrier rooms of the same vendor. In B6NCrl from Barrier #8, administration of pilocarpine resulted in a high percentage of mice developing SE, but mortality was low, whereas the opposite was found in B6NCrl mice from four other barriers of the same vendor. The analysis of F1 mice from a cross of female Barrier 8 pilocarpine-susceptible mice with resistant male mice from another barrier (#9) revealed that F1 male mice were significantly more sensitive to pilocarpine than the resistant parental male mice whereas female F1 mice were not significantly different from resistant Barrier 9 females. These observations strongly indicate X-chromosome linked genetic variation as the cause of the observed phenotypic alterations. To our knowledge, this is the first report which demonstrates that not only the specific B6 substrain but also sublines derived from the same substrain may markedly differ in their response to convulsants such as pilocarpine. As the described differences have a genetic basis, they offer a unique opportunity to identify the genes and pathways involved and contribute to a better understanding of the underlying molecular mechanisms of seizure susceptibility.
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- Materials and methods
The three major findings of this study are that (1) a specific substrain of B6 mice, B6NCrl, is much more sensitive to SE induction by pilocarpine than two other B6 substrains, B6JOla and B6NHsd; (2) B6NCrl mice from different barrier rooms of the same supplier (Charles River) strikingly differ in their sensitivity to SE induction by pilocarpine; and (3) the high sensitivity of Barrier 8 B6NCrl mice is heritable, indicating that genetic mutation(s) represent the basis for these subline differences. To our knowledge, the only previous indication for substrain differences in sensitivity to SE induction by pilocarpine came from experiments of Borges et al. (2003) who reported that B6 mice from Charles River (B6NCrl) were more sensitive than B6 mice from Jackson Laboratories (B6J). Using single doses of pilocarpine in the range of 247–335 mg/kg i.p. in 99 B6J mice, only 12 mice exhibited SE and survived; total mortality was 64%, which is similar to the 56% mortality rate observed in the present experiments in B6JOla mice from Harlan. In contrast to B6J mice from the Jackson Laboratory, a larger percentage (58%) of B6NCrl mice from Charles River developed SE and survived (15 of 26 mice total) in the experiments reported by Borges et al. (2003). Neuro-pathological changes in the hippocampus examined in mice surviving SE were similar in the B6J and B6NCrl substrains and were characterized by the loss of pyramidal cells in CA1 and CA3 and the loss of neurons in the dentate hilus (Borges et al. 2003). Furthermore, spontaneous seizures were determined after SE in both B6 substrains (Borges et al. 2003).
The high susceptibility of B6NCrl mice to SE induction by pilocarpine found in the study of Borges et al. (2003), and the present study is substantiated by the study by Chen et al. (2005) in which >80% of B6NCrl mice developed SE after i.p. doses ranging from 272 to 340 mg/kg and 81% of the mice survived. Following SE, spontaneous recurrent seizures and hippocampal damage were observed in B6NCrl mice by Chen et al. (2005). Thus, overall, the B6NCrl substrain seems to be better suited for the pilocarpine model of temporal lobe epilepsy than other B6 substrains tested in this respect.
Several previous studies have found behavioral and genetic differences between B6J and B6N substrains of B6 mice. Stiedl et al. (1999) reported that B6JOla and B6NCrl differed in their course of extinction of conditioned fear, with B6NCrl showing a slower decline of fear in response to a conditioned stimulus than B6JOla. In B6JOla from Harlan, Specht & Schoepfer (2001) found a spontaneous chromosomal deletion of the gene that encodes the presynaptic protein alpha-synuclein, which has been implicated in synaptic transmission and the etiology of a range of neurodegenerative disorders (Windisch et al. 2007), whereas the alpha-synuclein gene is present in B6NCrl mice and B6JCrl mice (Siegmund et al. 2005). This prompted Siegmund et al. (2005) to study whether this inter-substrain difference in alpha-synuclein expression is involved in the different extinction of conditioned fear in B6 substrains, but no relationship was found. Grottick et al. (2005) reported dramatic differences in prepulse inhibition of the startle response (PPI) in B6J and B6NHsd mice and sought to identify the molecular mechanisms underlying these differences in sensorimotor gating by a microarray-based approach. In the two substrains, differences in glutamatergic and GABAergic signaling were found that may explain the different PPI but may also be involved in the different response to pilocarpine determined in the experiments of Borges et al. (2003) and in the present study. In a study in which the relationship of 102 mouse strains, including B6 substrains, was assessed by using a panel of 1638 single nucleotide polymorphisms (SNPs), substrains derived from the B6 colony at the NIH differed in only five SNPs from the B6J strain at Jackson Laboratory (Petkov et al. 2004), suggesting that the genetic drift between B6J and B6J-derived substrains is minor.
Determination of genotypes for 342 microsatellite markers in B6J (obtained from Jackson Laboratory) and B6N (obtained from Taconic Farms, Germantown, NY) identified only 12 microsatellite differences between the two B6 substrains and no substrain difference was found in three behavioral tests, i.e. rotarod, open field and fear conditioning (Bothe et al. 2004). However, significant differences between the B6J and B6NCrl substrains were found in ethanol drinking and dependence (Khisti et al. 2006). The difference in ethanol preference between the two substrains was inversely correlated with ethanol-induced dopamine release in the ventral striatum (Ramachandra et al. 2007). B6J mice exhibited significantly greater ethanol preference and less ethanol-stimulated dopamine release compared to B6NCrl mice (Ramachandra et al. 2007).
To our knowledge, the experiments presented here describe for the first time that behaviors studied in B6 mice are not only affected by the B6 substrain used but that different responses may also occur in the same substrain obtained from different barriers of the same vendor. As demonstrated by the experiment on SE induction by pilocarpine, the differences in SE induction and mortality determined between B6NCrl mice from Barriers 8 and 9 of the same vendor (Charles River) were as pronounced as the differences determined between B6J and B6N substrains from different vendors. In other words, if we would have compared only B6NCrl mice from Barrier 9 with the B6JOla and B6NHsd substrains, our conclusion would have been that all three substrains are resistant to SE induction by pilocarpine, whereas the B6NCrl mice from Barrier 8 were much more sensitive to SE induction by pilocarpine than any of the other substrains.
There are three possibilities to explain the differences found between B6NCrl mice from the two barriers of the same vendor. First, a spontaneous genetic alteration that might have arisen in one colony or the other may explain the difference. Second, the difference could be a consequence of an environmental effect initiated at the vendor, or, third, the difference could be a consequence of a complex gene-environment interaction that may involve multiple factors both genetic and from the environment. Therefore, we performed several experiments to directly address the possibility of a genetic variation. First, B6NCrl mice from three additional barriers of the same vendor (Charles River) were tested. They all exhibited similar resistance to SE induction by pilocarpine as B6NCrl mice from Barrier 9, demonstrating that Barrier 8 mice differed from the other four barriers of this vendor. This strongly indicates that the high sensitivity to SE induction of Barrier 8 mice was due to a genetic variation that has arisen only in this subline of B6NCrl mice. To further test this hypothesis, we generated F1 mice from matings of female pilocarpine-sensitive Barrier 8 mice with male resistant Barrier 9 mice. In the F1 generation, a significant difference was observed between male F1 hybrid and the resistant Barrier 9 parental subline. On the other hand, female F1 mice were not significantly different compared to Barrier 9 resistant female mice. These results strongly indicate that the phenotypic differences between Barrier 8 and Barrier 9 mice are caused by a recessive genetic variation which occurred in the Barrier 8 subpopulation, and that the allele which causes pilocarpine-sensitivity resides on the X-chromosome. It should be noted that for the described phenotype assay, mice had to be tested in groups. Thus, to further prove genetic inheritance and to map the underlying genetic alterations, it will be necessary to develop phenotype assays which can distinguish between individual susceptible and resistant carriers. As the breeder has discontinued the Barrier 8 breeding, we have started to establish an independent breeding colony of Barrier 8 mice at our department. This should allow us to test the above hypotheses further in the future.
New mutations in sublines of inbred mice have been described recently for epilepsy, for example, within the B6J substrain of B6 mice (Yang et al. 2003) and in the C3H/HeJ strain (Beyer et al. 2008). Large breeding colonies at vendors are historically organized in the form of subcolonies. Here, the possibility exists that a spontaneous mutation occurs in a founder animal and becomes fixed in the resulting subcolony. Although this may be a rare event, once discovered, it offers a unique possibility to identify new susceptibility genes.
Compared to a variety of other inbred and outbred mouse strains, B6 mice are generally considered seizure resistant in that higher doses of various convulsants are needed to induce seizures in this strain (Engstrom & Woodbury 1988; Ferraro et al. 1997, 1998, 1999, 2002, 2004, 2007; Kosobud & Crabbe 1990). By mapping murine loci for seizure response, Ferraro and colleagues reported that the difference in seizure susceptibility between seizure-resistant B6 and seizure-sensitive DBA/2J mice is a polygenetic phenomenon with loci of significant effects on chromosomes 1, 5, 7 and 15 (Ferraro et al. 1997, 1999, 2004, 2007). All these studies were performed in B6J mice from the Jackson Laboratory. The present and previous studies (Borges et al. 2003; Chen et al. 2005) suggest that B6NCrl mice are more sensitive to convulsants than B6J mice. In this respect, it is also interesting to note that the MES threshold of B6NCrl mice was low in the present study and not different from that of seizure-sensitive NMRI mice (unpublished data).
It is important to note that the main difference between B6 substrains and sublines determined in the present study was not related to the dose of pilocarpine inducing convulsions, but to the consequences of convulsive doses of pilocarpine, i.e. induction of SE and mortality. In experiments on mapping genome loci for seizure response to kainate in B6J and DBA/2 J mice, two types of genetic influence upon the seizure phenotype were identified: one that increases susceptibility to kainate-seizures and another that increases severity of the seizure syndrome once it has been initiated (Ferraro et al. 1997). Schauwecker & Steward (1997) reported that kainate, 30 mg/kgs.c., induced comparable seizures in B6 mice (obtained from Hilltop Labs, Philadelphia) and the FVB/n and 129/SvEMS inbred strains, but that, in contrast to these other mouse strains, B6 mice did not exhibit excitotoxic cell death in the hippocampus after kainate-induced seizures. These results indicated that B6 mice carry genes that convey protection from glutamate-induced excitotoxicity (Schauwecker & Steward, 1997). However, this protection does not extend to pilocarpine-induced excitotoxicity, because pilocarpine-induced SE results in hippocampal damage and spontaneous seizures in B6 mice (Borges et al. 2003; Chen et al. 2005; Peng et al. 2004; Shibley & Smith 2002).
In addition to substrain differences in SE induction by pilocarpine, we also found significant differences in seizure induction. Thus, B6JOla mice were significantly more resistant to seizure induction than B6NHsd and B6NCrl#8 mice. In contrast, B6NCrl from the different barriers did not differ in seizure induction but only in SE induction by pilocarpine. Thus, these B6 substrains and sublines can be used both to explore mechanisms of seizure susceptibility and mechanisms of SE induction.
In conclusion, the present study demonstrates that substrains of B6 mice from different vendors markedly differ in their response to the convulsant pilocarpine. In studies using B6 mice, the exact nomenclature of the B6 substrain is often not indicated, which makes it difficult to interpret the results of such studies, considering the various behavioral and genetic differences between B6 substrains that have been reported (see above). Thus, although B6 substrains are closely related mouse lines with limited genetic differences, such differences may critically affect the phenotype in behavioral studies and interact with targeted mutations that are based on a B6 background. This situation is getting even more complex by the present finding that sublines of the same B6 substrain may differ between different barriers of the same vendor. On the other hand, B6 substrain or subline differences as obtained in the present study with pilocarpine offer the possibility of identifying genetic loci of critical relevance to seizure phenotypes in mice and may further our understanding of basic processes that are involved in the evolution of single seizures to a self-sustaining SE.