The relevance of inter- and intrastrain differences in mice and rats and their implications for models of seizures and epilepsy

Abstract

It is becoming increasingly clear that the genetic background of mice and rats, even in inbred strains, can have a profound influence on measures of seizure susceptibility and epilepsy. These differences can be capitalized upon through genetic mapping studies to reveal genes important for seizures and epilepsy. However, strain background and particularly mixed genetic backgrounds of transgenic animals need careful consideration in both the selection of strains and in the interpretation of results and conclusions. For instance, mice with targeted deletions of genes involved in epilepsy can have profoundly disparate phenotypes depending on the background strain. In this review, we discuss findings related to how this genetic heterogeneity has and can be utilized in the epilepsy field to reveal novel insights into seizures and epilepsy. Moreover, we discuss how caution is needed in regards to rodent strain or even animal vendor choice, and how this can significantly influence seizure and epilepsy parameters in unexpected ways. This is particularly critical in decisions regarding the strain of choice used in generating mice with targeted deletions of genes. Finally, we discuss the role of environment (at vendor and/or laboratory) and epigenetic factors for inter- and intrastrain differences and how such differences can affect the expression of seizures and the animals' performance in behavioral tests that often accompany acute and chronic seizure testing.

Keywords: Genetic background effects; Genetic heterogeneity; Genetic mapping; Inbred rodent strains.

Fig. 1

Differences in sensitivity to pilocarpine in various C57BL/6 (B6) substrains and sublines. All data were generated by a ramping-up dosing protocol with i.p. administration of 100 mg/kg pilocarpine every 20 min until onset of SE. All mice were pretreated with methylscopolamine to prevent peripheral cholinomimetic effects of pilocarpine. In case that no SE was induced, the maximum number of repeated injections was restricted to about 10, because mice died in individual convulsive seizures without reaching SE. “A” illustrates the percentage of mice per substrain or subline in which pilocarpine induced SE. Pilo-s F3 and B6NCrl#8 mice exhibited a significantly higher sensitivity to SE induction by pilocarpine than any other B6 substrain or subline. In “B” the SE-associated mortality (in % of mice per substrain) is correlated with percent SE induction in the respective B6 substrains and sublines, resulting in a significant negative correlation between the two endpoints. In A and B, the following substrains and sublines of B6 mice are shown: B6J, C57BL/6J mice from the Jackson Laboratory (JAX); B6NHsd, C57BL/6NHsd from Harlan (Harlan-Winkelmann; B6JOlaHsd, C57BL/6JOlaHsd mice from Harlan-Winkelmann; B6NCrl, C57BL/6NCrl mice from 4 different barriers (#4, #7, #8, and #9) from Charles River; Pilo-s F3, a pilocarpine-sensitive B6NCrl subline obtained by crossing female B6NCrl#8 mice with male F1 hybrids (B6NCrl#8 × B6NCrl); further sister-brother mating of the resulting F2 generation generated a highly susceptible F3 generation. Data are from Müller et al. [21] and Bankstahl et al. [32].

Fig. 2

C57BL substrain differences in flurothyl generalized seizure thresholds and in their final flurothyl-induced seizure phenotypes (following 8 seizures, a 28-day incubation phase, and a final flurothyl challenge). A) The latency to a generalized clonic-forebrain seizure (generalized seizure threshold (GST)) on each seizure trial was determined for 5 C57BL substrains by exposure to 10% flurothyl during eight induction trials followed by a 28-day rest period and a single flurothyl retest. B) While none of the 6NJ and KSJ mice expressed a more complex forebrain→brainstem seizure on flurothyl rechallenge, 25% of 10SNJ mice, 25% of 10J mice, and 80% of 6J mice did express a more complex forebrain→brainstem seizure. Forebrain→brainstem seizures are seizures in which the mouse has a generalized forebrain-clonic seizure that rapidly and uninterruptedly progresses into a seizure with tonic- brainstem components to the seizure. These differences in GST and seizure phenotype between these closely related strains of C57BL mice demonstrate the power of the genetic background on seizure measurements. Data are modified from Kadiyala et al. [116].

Fig. 3

Kindling rate in seven outbred and inbred rat strains. Rats were kindled by once daily stimulation of the basolateral amygdala with a stimulus intensity of 500 μA for 1 sec. A: Number of daily stimulations to first fully kindled stage 5 seizure. ANOVA on ranks indicated a highly significant differences between strains (P<0.001). B: Cumulative afterdischarge duration (ADD) to to first fully kindled stage 5 seizure. ANOVA on ranks indicated no significant differences between strains (P=0.09), although F344 and Lewis rats showed a trend for high ADD. C: Correlation between the two estimates of kindling rate shown in A and B. A significant linear correlation (r² = 0.6282, P = 0.0335) was calculated between the two estimates of kindling rate. Data are from Löscher et al. [136]. Abbreviations: BN, Brown Norway; SD, Sprague-Dawley; WK, Wistar Kyoto; F344, Fischer 344.

Fig. 4

Confounding variables at animal vendor and in the laboratory that may affect the experimental outcome of rodent studies on seizure expression, drug activities or behavior. Several of the environmental variables shown may induce epigenetic changes. For more details see text and Brown et al. [182] and Schellinck et al. [6].

Fig. 5

Effect of strain and sex on different types of seizure threshold in mice. Seizure thresholds were determined either in NMRI outbred mice from MØllegard (Ejby, Denmark), Wiga (Sulzfeld, Germany), or Charles River (Crl; Sulzfeld, Germany), or in Swiss mice from Mus Rattus (Brunnthal, Germany). In A, the threshold for maximal electroshock seizures (max EST) is shown. The threshold was determined at the same time in the morning in groups of 20 mice per strain and sex and is indicated as the voltage inducing hind limb extension in 50% of the animals with confidence limits (CI) for 95% probability. Statistical analysis of data by ANOVA indicated significant differences between groups for both male (P = 0.009) and female (P = 0.0094) mice. In B, the threshold for minimal electroshock seizures (min EST) is shown; this type of seizure threshold was not determined in NMRI mice from Crl. The threshold was determined in groups of 20 mice per strain and sex and is indicated as the current inducing clonic activity of the facial musculature, ears, or forelimbs for several seconds in 50% of the animals with CI for 95% probability. Statistical analysis of data by ANOVA indicated significant differences between groups for both male (P<0.0001) and female (P<0.0001) mice. In C, the threshold for clonic seizures induced by PTZ is shown. The threshold was determined in groups of 10 mice and is given as the mean dose (± S.D.) of PTZ causing clonic seizures by i.v. infusion in freely moving animals. Statistical analysis of data by ANOVA indicated significant differences between groups for female (P = 0.0027) but not male mice. In all graphs, significant sex differences within each strain are indicated by circles (^oP<0.05; ^oooP<0.001), whereas significant differences between strains within each sex are indicated by asterisks (*P<0.05; **P<0.01; ***P<0.001). Data are from Löscher et al. [91] and unpublished experiments of W. Löscher.