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Suz mice exhibit physical and motor delays and heightened locomotor activity in response to novelty during development
Epilepsy & Behavior 6 (2005) 312–319 www.elsevier.com/locate/yebeh
Seizure-prone EL/Suz mice exhibit physical and motor delays and heightened locomotor activity in response to novelty during development Melanie P. McFadyen-Leussis *, Stephen C. Heinrichs Department of Psychology, Boston College, Chestnut Hill, MA 02467, USA Received 22 October 2004; revised 25 January 2005; accepted 26 January 2005
Abstract Seizure-prone EL/Suz mice have been studied as a model of multifactorial epilepsy for five decades. In prior behavioral studies, EL/Suz mice were shown to exhibit heightened locomotor activity, which implies a state of underlying hyperexcitability. The aim of the present study was to establish the premorbid behavioral development of basic motor skills and activity levels of EL/Suz mice, as compared with DDY mice, the control strain that is not seizure-prone. EL/Suz and DDY pups were monitored from Postnatal Day (PND) 3 to assess body weight, surface righting, negative geotaxis, forelimb grip strength, eye opening, habituation to a novel environment, and exploratory behavior in a two-compartment task. EL/Suz mice weighed less from PNDs 3 to 21 and exhibited delayed surface righting (PNDs 3, 5, 7) and negative geotaxis (PNDs 5, 7, 9) responses. EL/Suz and DDY mice differed in their habituation to a novel environment, with EL/Suz mice exhibiting higher activity, both within a single 10-minute session and across the 3 days of testing. EL/Suz and DDY mice also differed in the two-compartment task, with EL/Suz mice exhibiting increased locomotor activity and spending a greater amount of time in the light compartment. Thus, the present findings reveal that EL/Suz mice exhibit some developmental delays, altered habituation to a novel environment, and increased exploratory activity. Overall, the present results demonstrate that the behavioral and physiological phenotype of seizure-prone EL/Suz mice is deviant more than 2 months before the onset of seizure susceptibility. 2005 Elsevier Inc. All rights reserved. Keywords: Activity; Behavior; Development; Epilepsy; Habituation; Motor; Mouse
1. Introduction The EL/Suz mouse is a natural (i.e., noninduced) model of multifactorial, idiopathic epilepsy generated by selective breeding of seizure-prone mice [1]. EL/Suz mice have been registered internationally as a model of epilepsy [2]. Their function as an animal model of epilepsy has been validated through studies of the electroencephalographic patterns exhibited by these mice [3,4]. The major gene responsible for the EL/Suz seizure *
Corresponding author. Fax: +1 617 552 0523. E-mail address: [email protected] (M.P. McFadyen-Leussis).
1525-5050/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2005.01.010
disorder is located on chromosome 9, and is inherited in an autosomal recessive manner [5]. Susceptibility to seizures in EL/Suz mice increases with age, with onset at 70–90 days of age, and seizures are triggered by repeated sensory stimulation [6,7]. Given the natural ontogeny of seizure susceptibility in EL/Suz mice, this mouse strain appears to be an ideal animal model for longitudinal studies of epilepsy, as epileptogenesis appears to be mediated by both genetic and environmental factors [5,6]. To maximize their potential as an animal model of epilepsy, the phenotype of EL/Suz mice from infancy to seizure onset in adulthood should first be determined.
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There is a paucity of research regarding the behavioral phenotype of EL/Suz mice. It has been shown that EL/Suz mice, but not DDY mice, exhibit altered circadian rhythms, expressing periods of hyperactivity during the light phase of the light–dark cycle [8]. Further, it has been demonstrated that EL/Suz dams exhibit deficits in maternal behavior including decreased nursing/crouching and delays in pup retrieval relative to DDY mice [9]. Apart from descriptions of tonic–clonic seizures exhibited under the conditions described above [6], little else is presently known about the behavior of these mice. The behavioral phenotype of a mutant mouse depends not only on its genetic background, but also on the interaction of that background with the mutant genes and the interaction of either the background or the mutant genes with the environment [10]. It is important, therefore, to use a battery of tests aimed at thoroughly evaluating the behavioral phenotype of a mutant mouse, to fully assess any sensory or behavioral differences [11,12]. Despite the fact that seizure susceptibility in EL/Suz mice has a specific ontogeny, little research has focused on EL/Suz development, and existing studies focus overwhelmingly on biochemical measures or seizure development. Accordingly, the aim of this study was to assess the developmental phenotype of EL/Suz mice from a behavioral perspective, and as part of the overarching goal of assessing the behavioral phenotype of these mice in a systematic manner. In the past, batteries of developmental tests have been used to examine physical and behavioral development in inbred and mutant mice [11–16], following prenatal exposure to stress [17,18] or drugs such as methylphenidate [19] and cocaine [20] or following embryonic blockade of vasoactive intestinal peptide [21]. Thus, the present study evaluated the developmental phenotype of EL/Suz mice, as compared with the DDY control strain, in a series of tests examining motor development, habituation to a novel environment, and exploration in a two-compartment task. This strategy is consistent with screening methods developed by behavioral teratologists to detect perinatal and postnatal alterations in growth and development in immature mice [22]. We expected to find that EL/Suz mice would exhibit a number of developmental and behavioral differences when compared with DDY mice. Specifically, we predicted that EL/Suz mice would exhibit delayed motor development and an increased level of locomotor activity in the habituation and two-compartment tasks. These postulated differences represent the expression of a previously overlooked but nonetheless important portion of the overall phenotype in these mice for assessing the extent to which such premorbid functional differences constitute risk factors for seizure susceptibility.
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2. Method 2.1. Subjects EL/Suz (N = 8 litters) and DDY (N = 7 litters) mice were bred and maintained at Boston College from stock originally donated by Dr. Thomas Seyfried. Each litter consisted of 6–10 pups, and included both males and females. All mice were housed in a reverse light–dark cycle colony (lights off 1000, lights on 2200), at a temperature of 71–73 F. Food (Lab Diet Prolab 5P00, PMI Nutrition International, Richmond, IN, USA) and water were available ad libitum. Sani chip bedding (PJ Murphy Forest Products, Montville, NJ, USA) was used in all cages. Experimental procedures described herein were approved by the Institutional Animal Care and Use Committee of Boston College. 2.2. Procedures Females were harem bred (two females per male) with a male of the same strain. Visibly pregnant females were singly housed around Gestational Days 17–19 and a cotton nest square (Old Mother Hubbard, Lowell, MA, USA) was provided as a source of nesting material. Females were observed daily for parturition, and this day was considered Postnatal Day 0 (PND 0). Litters were left undisturbed until PND 3, the first day of testing. At this time, the gender composition of the litter was recorded, and the pups were individually marked with a nontoxic marker and weighed. Body weight was recorded on each day on which testing took place (PNDs 3, 5, 7, 9, 11, 13), and at weaning (PND 21) (see Fig. 1 for a summary of developmental tests). Mice were observed daily, and the age at which eye opening occurred was recorded for each pup [17]. After weaning, mice were housed with same-sex littermates in groups of three to five mice per cage. All motor development and behavioral testing was conducted between the hours of 1030 and 1300. On days when multiple developmental tests were conducted, surface righting was always conducted first, followed by negative geotaxis and, finally, forelimb grip. 2.3. Tests of motor development 2.3.1. Surface righting task A pup in a supine position will attempt to right itself to a prone position. The surface righting task measures both motor coordination and vestibular function [23]. Surface righting was tested on PNDs 3, 5, 7, and 9 (Fig. 1). Pups were placed on a flat surface and the time taken to right, defined as all four limbs placed under the body, was recorded to a maximum of 60 seconds. Two trials were given on each test day, with a 60-second rest period between trials. If a pup failed to right, the maxi-
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Fig. 1. Timeline, from birth to Postnatal Day (PND) 31, of testing periods for each task included in the study. Body weight was recorded every other day from PND 3 through PND 13 and at weaning. Developmental measures were tested every second day over the period indicated. Habituation was tested over 3 consecutive days between PNDs 25 and 30.
mum allotted time (60 seconds) was recorded. The mean latency to right over the two daily trials was used for analysis. 2.3.2. Negative geotaxis task When placed in a downward direction on a slope, rodents tend to reorient themselves so that their head and body point upward (against the pull of gravity). Negative geotaxis assesses both vestibular function and motor coordination [24]. Negative geotaxis was assessed on PNDs 5, 7, 9, and 11 (Fig. 1). Pups were placed, head down, on a mesh-covered surface that was inclined 15. The time for the pup to turn 180 was recorded for two trials, to a maximum of 60 seconds per trial, with a 60second rest period between trials. The mean time to turn 180 over the two trials was used for analysis. 2.3.3. Forelimb grip strength task The forelimb grip strength task measures the strength and coordination of the forelimbs [25]. Forelimb grip strength was assessed on PNDs 7, 9, and 11 (Fig. 1). The forepaws of the pup were placed on a thin rubber band suspended 15–18 cm from the surface of the table. A container with soft bedding (pine shavings) was placed below the rubber band to catch the pup once it released the band. The time to fall was recorded to a maximum of 30 seconds. Two trials were administered on each test day, with a rest period of 60 seconds between trials. The longer (better) of the two scores was recorded and used for statistical analysis to minimize the effects of fatigue in this more demanding task. If the pup was able to hold onto the band for the maximum time on the first trial on a given test day, a second trial was not administered on that day. Pups were not tested on this task before PND 7 because they were not physically capable of gripping the band.
2.4. Habituation to a novel environment Habituation to a novel environment is commonly used in rodents as a paradigm for examining nonassociative learning and memory processes [26]. Habituation, defined as the waning of a response that is elicited by repeated exposure to a novel stimulus, is normally expressed in rodents as a decrease in exploratory activity when the animals are constantly or repeatedly exposed to a novel environment. Habituation can be separated into two components: within-session (intrasession) habituation and between-session (intersession) habituation. In the latter, memory or retention of the previous session is being tested by observing whether the animal reacts as if it is unfamiliar with the test environment, showing a high level of exploration roughly equivalent to its initial exposure to its open field, or whether it behaves as if it recalled the prior exposure, showing decreased exploration compared with earlier test sessions. In contrast, within-session habituation occurs as the animal becomes familiar with the environment in which it has been placed and refers to the decline in exploration that occurs specifically within the period of a single exposure to the novel environment. It has been proposed that within-session habituation measures adaptivity, whereas between-session habituation better reflects memory of the prior session [27]. For our habituation task, the young mice (PNDs 25– 30) were exposed to a novel environment for 10 minutes a day on three consecutive days (Fig. 1). The novel environment consisted of a polypropylene rat cage (45 · 24 · 21 cm) with no bedding or shavings, and a filter top but no wire top. Prior to the first test session each day, and after every animal, the cage was wiped down with a detergent (Quatricide germicidal detergent, Pharmacal Research Laboratories, Naugatuck, CT, USA).
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Horizontal locomotor activity was determined with a photocell-based monitoring system (San Diego Instruments, San Diego, CA, USA) which measured beam breaks that occurred over the course of testing. Activity was broken down by minute and recorded by a computer. The photocell arrays were adjusted to 3 cm above the surface of the shelf on which the cages were located. Daily locomotor activity was calculated as the mean activity for the 10-minute session each day, and this value was used to examine between-session habituation. Within-session habituation was examined by calculating the difference between the means of the first and last minute activity levels collapsed across the 3 test days. 2.4.1. Two-compartment task Mice were tested once between PNDs 30 and 32 for emotionality and exploratory behaviors using a twocompartment (light–dark) task (Fig. 1). The light–dark test is a naturalistic test of anxiety based on approach–avoidance conflict in mice [12]. The home cage was moved to a brightly lit testing room (420 lux). Animals were allowed to acclimate to the test environment for 15 minutes before testing. The home cage was placed under a cardboard box to create a dark environment, and this was joined to a ‘‘novel’’ clean cage by a PVC connecting tube that linked the two cages through holes drilled into their sides. The novel cage was not under the cardboard box and thus constituted the brightly lit environment. After the box was placed over the animalÕs home cage, the mouseÕs behavior on the light side of the two-compartment apparatus was videotaped for a period of 10 minutes. The video of each mouse was scored by a rater blind to mouse strain, who assessed the latency to enter the light side, the total time spent in the light side, and the total number of transitions between the two sides [12]. Mice were considered to have crossed into or out of the light side when all four of their feet crossed the threshold between the two cages. Mice that did not enter the light side at any point during the 10-minute trial were assigned the maximum score for latency (600 seconds) and a zero for the time spent in the light side and the number of transitions. 2.5. Statistical analyses Body weight, surface righting, negative geotaxis, forelimb grip, and habituation (both between- and within-session) were analyzed by repeated-measures analysis of variance. All behavioral measures in the two-compartment task were analyzed by one-way analysis of variance. The age at which eye opening occurred was analyzed by v2 test. Data for all measures were collapsed within a litter by gender to generate a litter mean for each gender, and it is these means that were used for analysis; thus strain and gender were considered as variables. Unless otherwise stated, the data were col-
Fig. 2. Mean body weight (±SEM) of EL/Suz and DDY pups from PNDs 3 to 21. Both strains consistently gained weight as they aged, but DDY pups weighed more than EL/Suz pups throughout the test period.
lapsed across gender as there were no significant gender effects.
3. Results 3.1. Body weight There was a main effect of age as both strains of mice significantly increased in body weight from 3 to 21 days of age [F (6, 60) = 698.22, P < 0.001]. There was also a significant effect between strains, with EL/Suz pups exhibiting lower body weight than DDY pups across the testing period [F (1, 10) = 1229.79, P < 0.001]. Finally, there was a significant strain · age interaction [F (6, 60) = 12.25, P < 0.001], denoting the fact that DDY pups gained weight more quickly than EL/Suz pups across this period of development (Fig. 2). 3.2. Eye opening There was no difference between the two strains in the age at which eye opening was observed (v2 (1) = 2.637, NS). Eye opening occurred at PND 13 in both strains. 3.3. Tests of neuromotor development 3.3.1. Surface righting reflex There was a significant decrease in the latency to right from a supine position over the 4 test days in both strains [F (3, 39) = 21.61, P < 0.001]. There was also a significant between-subjects effect of strain, with EL/ Suz mice exhibiting a longer latency to show the righting reflex than DDY mice [F (1, 13) = 6.10, P < 0.03]; however, there was no strain · age interaction for this neuromotor reflex (Fig. 3).
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Fig. 3. Mean latency to right (±SEM) of EL/Suz and DDY pups in the surface righting reflex task. Both strains improved in the task from PNDs 3 to 9 (latency to right decreased), although in this task DDY pups exhibited lower latencies than EL/Suz pups across the testing period.
3.3.2. Negative geotaxis reflex There was a significant decrease in negative geotaxis latency (turning 180 on the incline) over the 4 test days [F (3, 39) = 55.65, P < 0.001]. There was a significant difference between strains [F (1, 13) = 284.44, P < 0.001], with EL/Suz pups exhibiting a longer latency to respond than DDY pups, and a significant interaction between strain and age at testing [F (3, 39) = 4.53, P < 0.01] as performance of both strains reached asymptote at the conclusion of testing (Fig. 4). 3.3.3. Forelimb grip strength There was a significant increase in forelimb grip strength, as measured by the latency to fall, over the 3 test days [F (2, 26) = 11.14, P < 0.001] but there was no
Fig. 5. Mean latency to fall (±SEM) for EL/Suz and DDY pups in the forelimb grip strength task. Both strains exhibited similar performance at PND 7, but EL/Suz pups showed greater improvement (longer latency to fall) than DDY mice at PNDs 9 and 11.
main effect between strains. There was an interaction between strain and age at testing [F (2, 26) = 3.52, P < 0.05], with both strains exhibiting similar performance at PND 7, but EL/Suz pups showing a significant improvement (longer latency to fall) at PNDs 9 and 11 relative to DDY pups (Fig. 5). 3.4. Habituation to a novel environment There was a significant strain difference in activity exhibited across the 3 days of habituation testing, with EL/Suz mice exhibiting consistently higher activity on all 3 days and DDY mice exhibiting a decrease in activity across the test period [F (2, 44) = 9.65, P < 0.001]. Similarly, there was a significant between-subject effect of strain for between-session habituation [F (1, 22) = 123.71, P < 0.001], with EL/Suz mice showing an increase in activity and DDY mice showing a decrease in activity across the 3 test days (Fig. 6A). Within-session habituation also differed between the two strains from Minute 1 of testing to Minute 10 of testing, with EL/Suz mice once more exhibiting higher activity at both time points compared with DDY mice [F (1, 22) = 10.28, P < 0.005]. The between-subject effect of strain was also significant for within-session habituation [F (1, 22) = 101.36, P < 0.001]. EL/Suz mice exhibited a decrease in activity between Minutes 1 and 10 of each test day, while DDY mice showed an increase in activity between the first and last minute of each test day (Fig. 6B). 3.5. Light–dark (two-compartment) test
Fig. 4. Mean latency to turn 180 (±SEM) of EL/Suz and DDY pups in the negative geotaxis reflex task. The latency to turn decreased (improved) from PNDs 5 to 11 in both strains; however, the DDY pups performed better than EL/Suz pups across the testing period.
EL/Suz mice entered the light side of the two-compartment apparatus in 55.6 ± 5.9 seconds, whereas DDY mice entered after 215.5 ± 24.1 seconds. Further,
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4. Discussion
Fig. 6. (A) Between-session habituation was evaluated as the mean activity (±SEM) of young EL/Suz and DDY mice, measured by beam breaks, as the animals habituated to a novel environment for 10 minutes per day, over 3 consecutive days. EL/Suz mice exhibited significantly higher activity overall, and there was no evidence of habituation in this strain as their activity increased across days. In contrast, the activity of DDY mice decreased between Days 1 and 3 of testing; thus, between-session habituation did occur in this strain. (B) Within-session habituation was evaluated as the mean activity (±SEM) of the first and the last minute of the test session averaged over the 3 consecutive days of testing. Activity of EL/Suz mice was significantly higher than activity of DDY mice at both Minutes 1 and 10. EL/Suz mice exhibited within-session habituation, as seen by the decrease in activity from Minutes 1 to 10 of testing, while DDY mice did not exhibit within-session habituation as their activity increased from Minutes 1 to 10.
EL/Suz mice spent 200.3 ± 15.3 seconds in the light side of the compartment, whereas DDY mice spent only 75.9 ± 14.6 seconds on the light side. Thus, EL/Suz mouse juveniles entered the light side of the two-compartment box more quickly [F (1, 12) = 48.02, P < 0.001] and spent significantly more time in the light side of the two-compartment box [F (1, 12) = 34.00, P < 0.001] than DDY mice. EL/Suz mice exhibited 18.8 ± 0.9 transitions between the light and dark compartments of the testing apparatus, which was significantly greater than the 8.2 ± 1.7 transitions exhibited by DDY mice [F (1, 12) = 34.43, P < 0.001]. All data points are given as the means ± SEM.
The present study provides additional evidence for altered physiological and behavioral phenotypes in seizure-prone EL/Suz mice on PNDs 3–30, more than 2 months prior to the onset of seizure susceptibility. Although all testing occurred prior to PNDs 70–90, when human handling can trigger seizures in EL/Suz mice, it is important to note that in the present studies no seizures were observed at any point, including during testing and weekly cage changes. EL/Suz mice exhibited a number of motor reflex delays including slower onset of the surface righting and negative geotaxis reflexes. However, the strain difference in the development of these two motor reflexes was not long-lasting as EL/ Suz and DDY mice performed equally well in these two tasks on their respective final days of testing. EL/ Suz and DDY mice performed equally well in the forelimb grip strength task at 7 days of age; however, on PNDs 9 and 11, EL/Suz pups performed better than DDY pups. The delay in the acquisition of the surface righting reflex, which relies mainly on vestibular and proprioceptive stimuli [28], suggests that the EL/Suz mice may harbor an underlying functional change in the vestibular system, as vestibular stimuli such as tail suspension are known to elicit seizures in older, seizure-susceptible EL/Suz mice [6]. EL/Suz mice weighed less than DDY mice from 3 days of age through weaning at 21 days. The decreased body weight of EL/Suz mice observed from PND 3 until weaning might be the result of a decrease in suckling due to a reduced level of maternal care, as a prior study indicated that EL/Suz dams spend less time nursing than DDY dams [9]. The observed differences in weight do not likely mediate the differences in behavior observed between EL/Suz and DDY pups, as previous findings in mice suggest that factors other than weight, but related in some way to litter size, appear to be critical for developmental behaviors such as surface righting and negative geotaxis reflexes [29]. Moreover, the underweight EL/Suz mice exhibited comparable or superior righting, negative geotaxis, and grasping performance by PND 11, suggesting that low body weight is not a liability for performance of these tasks. As litters were matched for size across the two strains, litter size should not be a confounding factor either. As seizures in our dams were not recorded, we cannot rule out the possibility that seizures occurring in EL/Suz dams during pregnancy may affect the offspring in a manner similar to other prenatal stressors, such as restraint stress [30], handling, increased temperatures, light and noise [17,31] and the presence of a cat [18]. These prenatal stressors resulted in maturational delays of motor reflexes such as negative geotaxis and surface righting [17,18,30,31], decreased weight during the first postnatal week [17], and increased exploratory behavior
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[17,32]. These studies do not address whether the changes in development resulting from prenatal stress are mediated by genetic factors, environmental factors, or by an interaction of these two factors. The present findings are consistent with those previously reported, as EL/Suz mice exhibit delayed surface righting and negative geotaxis reflexes, decreased body weight, and increased exploratory behavior in the two-compartment task. Further studies are needed to determine whether seizures during gestation are responsible for the strain differences observed in this study. Behavior of juvenile mice was examined using a nonassociative learning task, habituation to a novel environment, and the two-compartment task, which examines activity and emotionality. EL/Suz mice exhibited significantly greater activity than DDY mice in both of these paradigms, which is consistent with previous findings of hyperactivity in the EL/Suz strain [8,33]. Given that habituation is defined as a decrease in activity with repeated or prolonged exposure to a novel environment, we found that EL/Suz mice did not exhibit any between-session habituation, although there was some evidence for within-session habituation in these animals. In contrast, DDY mice exhibited between-session habituation, but did not exhibit within-session habituation. This could suggest that DDY mice better recall their prior exposures to the novel environment than the EL/ Suz mice, although the hyperactivity exhibited by the EL/Suz strain may mask any decrease in activity that would be mediated by learning in this paradigm. The increases in between-session activity of EL/Suz mice could also reflect a behavioral sensitization in which a constant stimulus intensity evokes a progressively increased response with repeated presentation [34,35]. A state of behavioral sensitization could certainly be perilous for seizure-prone EL/Suz mice known to be hypersensitive to vestibular stimuli that trigger seizures in these mice. In the light–dark paradigm, EL/Suz mice took significantly less time to enter the light side of the two-compartment box, and spent much more time in the light side than DDY mice. Further, the EL/Suz mice exhibited a much larger number of transitions between the two sides of the light–dark box. Given that EL/Suz mice are reported to be hyperactive in a familiar environment, these results suggest EL/Suz mice also exhibit increased activity in an unfamiliar environment. Thus, the present findings provide further support of the notion that EL/ Suz mice exhibit a generalized hyperexcitability that is observed predominantly as an increase in spontaneous locomotor activity. The aim of the present study was to establish the physical and behavioral phenotype of EL/Suz and DDY mice during development using a battery of tests. This approach was used to provide a more thorough evaluation of the early physical, motor, and behavioral differences between seizure-prone EL/Suz mice and
DDY controls, which has largely been ignored in EL/ Suz research to date. Our hypothesis that EL/Suz mice would exhibit delayed motor development and increased locomotor activity was supported by the present findings. This study contributes significantly to our knowledge of the seizure-prone EL/Suz mouse, with a focus on the phenotype prior to the onset of seizure susceptibility. It is important to note that the mice in the present study were not followed to 90 days of age; thus the onset of seizure susceptibility was never verified. This study was also constrained by the time frame of observation and the tests that were chosen, both of which limited the physical, motor, and behavioral differences that could be detected. Studies examining other aspects of development, such as learning and memory, should also be conducted. More importantly, future research should be geared toward differentiating genetic and environmental contributions to the differences between these two strains, as reported in the present study. The present examination of the early developmental phenotype of EL/Suz mice revealed a number of differences from that of the non-seizure-prone DDY strain. Distinctive characteristics of EL/Suz mice include persistently lower body weight, delays in the surface righting and negative geotaxis reflexes, and a marked increase in activity in both the habituation and two-compartment tasks. These findings suggest that the phenotype of EL/Suz mice is not limited to an abrupt increase in seizure susceptibility at PNDs 70–90, but includes numerous physiological and behavioral warning signs of predisposition that can be observed on PNDs 3–30, well before the onset of seizure susceptibility. It is therefore possible that early motor and behavioral performance is predictive of adult pathology in epilepsy as well as in other animal models. Acknowledgments Many thanks to Dr. Thomas Seyfried for his generous donation of EL and DDY mice. Thanks also to Karen Rusak and Samantha Korbey for their help in scoring the light–dark behaviors.
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Seizure-prone EL/Suz mice exhibit physical and motor delays and heightened locomotor activity in response to novelty during development Melanie P. McFadyen-Leussis *, Stephen C. Heinrichs Department of Psychology, Boston College, Chestnut Hill, MA 02467, USA Received 22 October 2004; revised 25 January 2005; accepted 26 January 2005
Abstract Seizure-prone EL/Suz mice have been studied as a model of multifactorial epilepsy for five decades. In prior behavioral studies, EL/Suz mice were shown to exhibit heightened locomotor activity, which implies a state of underlying hyperexcitability. The aim of the present study was to establish the premorbid behavioral development of basic motor skills and activity levels of EL/Suz mice, as compared with DDY mice, the control strain that is not seizure-prone. EL/Suz and DDY pups were monitored from Postnatal Day (PND) 3 to assess body weight, surface righting, negative geotaxis, forelimb grip strength, eye opening, habituation to a novel environment, and exploratory behavior in a two-compartment task. EL/Suz mice weighed less from PNDs 3 to 21 and exhibited delayed surface righting (PNDs 3, 5, 7) and negative geotaxis (PNDs 5, 7, 9) responses. EL/Suz and DDY mice differed in their habituation to a novel environment, with EL/Suz mice exhibiting higher activity, both within a single 10-minute session and across the 3 days of testing. EL/Suz and DDY mice also differed in the two-compartment task, with EL/Suz mice exhibiting increased locomotor activity and spending a greater amount of time in the light compartment. Thus, the present findings reveal that EL/Suz mice exhibit some developmental delays, altered habituation to a novel environment, and increased exploratory activity. Overall, the present results demonstrate that the behavioral and physiological phenotype of seizure-prone EL/Suz mice is deviant more than 2 months before the onset of seizure susceptibility. 2005 Elsevier Inc. All rights reserved. Keywords: Activity; Behavior; Development; Epilepsy; Habituation; Motor; Mouse
1. Introduction The EL/Suz mouse is a natural (i.e., noninduced) model of multifactorial, idiopathic epilepsy generated by selective breeding of seizure-prone mice [1]. EL/Suz mice have been registered internationally as a model of epilepsy [2]. Their function as an animal model of epilepsy has been validated through studies of the electroencephalographic patterns exhibited by these mice [3,4]. The major gene responsible for the EL/Suz seizure *
Corresponding author. Fax: +1 617 552 0523. E-mail address: [email protected] (M.P. McFadyen-Leussis).
1525-5050/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2005.01.010
disorder is located on chromosome 9, and is inherited in an autosomal recessive manner [5]. Susceptibility to seizures in EL/Suz mice increases with age, with onset at 70–90 days of age, and seizures are triggered by repeated sensory stimulation [6,7]. Given the natural ontogeny of seizure susceptibility in EL/Suz mice, this mouse strain appears to be an ideal animal model for longitudinal studies of epilepsy, as epileptogenesis appears to be mediated by both genetic and environmental factors [5,6]. To maximize their potential as an animal model of epilepsy, the phenotype of EL/Suz mice from infancy to seizure onset in adulthood should first be determined.
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There is a paucity of research regarding the behavioral phenotype of EL/Suz mice. It has been shown that EL/Suz mice, but not DDY mice, exhibit altered circadian rhythms, expressing periods of hyperactivity during the light phase of the light–dark cycle [8]. Further, it has been demonstrated that EL/Suz dams exhibit deficits in maternal behavior including decreased nursing/crouching and delays in pup retrieval relative to DDY mice [9]. Apart from descriptions of tonic–clonic seizures exhibited under the conditions described above [6], little else is presently known about the behavior of these mice. The behavioral phenotype of a mutant mouse depends not only on its genetic background, but also on the interaction of that background with the mutant genes and the interaction of either the background or the mutant genes with the environment [10]. It is important, therefore, to use a battery of tests aimed at thoroughly evaluating the behavioral phenotype of a mutant mouse, to fully assess any sensory or behavioral differences [11,12]. Despite the fact that seizure susceptibility in EL/Suz mice has a specific ontogeny, little research has focused on EL/Suz development, and existing studies focus overwhelmingly on biochemical measures or seizure development. Accordingly, the aim of this study was to assess the developmental phenotype of EL/Suz mice from a behavioral perspective, and as part of the overarching goal of assessing the behavioral phenotype of these mice in a systematic manner. In the past, batteries of developmental tests have been used to examine physical and behavioral development in inbred and mutant mice [11–16], following prenatal exposure to stress [17,18] or drugs such as methylphenidate [19] and cocaine [20] or following embryonic blockade of vasoactive intestinal peptide [21]. Thus, the present study evaluated the developmental phenotype of EL/Suz mice, as compared with the DDY control strain, in a series of tests examining motor development, habituation to a novel environment, and exploration in a two-compartment task. This strategy is consistent with screening methods developed by behavioral teratologists to detect perinatal and postnatal alterations in growth and development in immature mice [22]. We expected to find that EL/Suz mice would exhibit a number of developmental and behavioral differences when compared with DDY mice. Specifically, we predicted that EL/Suz mice would exhibit delayed motor development and an increased level of locomotor activity in the habituation and two-compartment tasks. These postulated differences represent the expression of a previously overlooked but nonetheless important portion of the overall phenotype in these mice for assessing the extent to which such premorbid functional differences constitute risk factors for seizure susceptibility.
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2. Method 2.1. Subjects EL/Suz (N = 8 litters) and DDY (N = 7 litters) mice were bred and maintained at Boston College from stock originally donated by Dr. Thomas Seyfried. Each litter consisted of 6–10 pups, and included both males and females. All mice were housed in a reverse light–dark cycle colony (lights off 1000, lights on 2200), at a temperature of 71–73 F. Food (Lab Diet Prolab 5P00, PMI Nutrition International, Richmond, IN, USA) and water were available ad libitum. Sani chip bedding (PJ Murphy Forest Products, Montville, NJ, USA) was used in all cages. Experimental procedures described herein were approved by the Institutional Animal Care and Use Committee of Boston College. 2.2. Procedures Females were harem bred (two females per male) with a male of the same strain. Visibly pregnant females were singly housed around Gestational Days 17–19 and a cotton nest square (Old Mother Hubbard, Lowell, MA, USA) was provided as a source of nesting material. Females were observed daily for parturition, and this day was considered Postnatal Day 0 (PND 0). Litters were left undisturbed until PND 3, the first day of testing. At this time, the gender composition of the litter was recorded, and the pups were individually marked with a nontoxic marker and weighed. Body weight was recorded on each day on which testing took place (PNDs 3, 5, 7, 9, 11, 13), and at weaning (PND 21) (see Fig. 1 for a summary of developmental tests). Mice were observed daily, and the age at which eye opening occurred was recorded for each pup [17]. After weaning, mice were housed with same-sex littermates in groups of three to five mice per cage. All motor development and behavioral testing was conducted between the hours of 1030 and 1300. On days when multiple developmental tests were conducted, surface righting was always conducted first, followed by negative geotaxis and, finally, forelimb grip. 2.3. Tests of motor development 2.3.1. Surface righting task A pup in a supine position will attempt to right itself to a prone position. The surface righting task measures both motor coordination and vestibular function [23]. Surface righting was tested on PNDs 3, 5, 7, and 9 (Fig. 1). Pups were placed on a flat surface and the time taken to right, defined as all four limbs placed under the body, was recorded to a maximum of 60 seconds. Two trials were given on each test day, with a 60-second rest period between trials. If a pup failed to right, the maxi-
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Fig. 1. Timeline, from birth to Postnatal Day (PND) 31, of testing periods for each task included in the study. Body weight was recorded every other day from PND 3 through PND 13 and at weaning. Developmental measures were tested every second day over the period indicated. Habituation was tested over 3 consecutive days between PNDs 25 and 30.
mum allotted time (60 seconds) was recorded. The mean latency to right over the two daily trials was used for analysis. 2.3.2. Negative geotaxis task When placed in a downward direction on a slope, rodents tend to reorient themselves so that their head and body point upward (against the pull of gravity). Negative geotaxis assesses both vestibular function and motor coordination [24]. Negative geotaxis was assessed on PNDs 5, 7, 9, and 11 (Fig. 1). Pups were placed, head down, on a mesh-covered surface that was inclined 15. The time for the pup to turn 180 was recorded for two trials, to a maximum of 60 seconds per trial, with a 60second rest period between trials. The mean time to turn 180 over the two trials was used for analysis. 2.3.3. Forelimb grip strength task The forelimb grip strength task measures the strength and coordination of the forelimbs [25]. Forelimb grip strength was assessed on PNDs 7, 9, and 11 (Fig. 1). The forepaws of the pup were placed on a thin rubber band suspended 15–18 cm from the surface of the table. A container with soft bedding (pine shavings) was placed below the rubber band to catch the pup once it released the band. The time to fall was recorded to a maximum of 30 seconds. Two trials were administered on each test day, with a rest period of 60 seconds between trials. The longer (better) of the two scores was recorded and used for statistical analysis to minimize the effects of fatigue in this more demanding task. If the pup was able to hold onto the band for the maximum time on the first trial on a given test day, a second trial was not administered on that day. Pups were not tested on this task before PND 7 because they were not physically capable of gripping the band.
2.4. Habituation to a novel environment Habituation to a novel environment is commonly used in rodents as a paradigm for examining nonassociative learning and memory processes [26]. Habituation, defined as the waning of a response that is elicited by repeated exposure to a novel stimulus, is normally expressed in rodents as a decrease in exploratory activity when the animals are constantly or repeatedly exposed to a novel environment. Habituation can be separated into two components: within-session (intrasession) habituation and between-session (intersession) habituation. In the latter, memory or retention of the previous session is being tested by observing whether the animal reacts as if it is unfamiliar with the test environment, showing a high level of exploration roughly equivalent to its initial exposure to its open field, or whether it behaves as if it recalled the prior exposure, showing decreased exploration compared with earlier test sessions. In contrast, within-session habituation occurs as the animal becomes familiar with the environment in which it has been placed and refers to the decline in exploration that occurs specifically within the period of a single exposure to the novel environment. It has been proposed that within-session habituation measures adaptivity, whereas between-session habituation better reflects memory of the prior session [27]. For our habituation task, the young mice (PNDs 25– 30) were exposed to a novel environment for 10 minutes a day on three consecutive days (Fig. 1). The novel environment consisted of a polypropylene rat cage (45 · 24 · 21 cm) with no bedding or shavings, and a filter top but no wire top. Prior to the first test session each day, and after every animal, the cage was wiped down with a detergent (Quatricide germicidal detergent, Pharmacal Research Laboratories, Naugatuck, CT, USA).
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Horizontal locomotor activity was determined with a photocell-based monitoring system (San Diego Instruments, San Diego, CA, USA) which measured beam breaks that occurred over the course of testing. Activity was broken down by minute and recorded by a computer. The photocell arrays were adjusted to 3 cm above the surface of the shelf on which the cages were located. Daily locomotor activity was calculated as the mean activity for the 10-minute session each day, and this value was used to examine between-session habituation. Within-session habituation was examined by calculating the difference between the means of the first and last minute activity levels collapsed across the 3 test days. 2.4.1. Two-compartment task Mice were tested once between PNDs 30 and 32 for emotionality and exploratory behaviors using a twocompartment (light–dark) task (Fig. 1). The light–dark test is a naturalistic test of anxiety based on approach–avoidance conflict in mice [12]. The home cage was moved to a brightly lit testing room (420 lux). Animals were allowed to acclimate to the test environment for 15 minutes before testing. The home cage was placed under a cardboard box to create a dark environment, and this was joined to a ‘‘novel’’ clean cage by a PVC connecting tube that linked the two cages through holes drilled into their sides. The novel cage was not under the cardboard box and thus constituted the brightly lit environment. After the box was placed over the animalÕs home cage, the mouseÕs behavior on the light side of the two-compartment apparatus was videotaped for a period of 10 minutes. The video of each mouse was scored by a rater blind to mouse strain, who assessed the latency to enter the light side, the total time spent in the light side, and the total number of transitions between the two sides [12]. Mice were considered to have crossed into or out of the light side when all four of their feet crossed the threshold between the two cages. Mice that did not enter the light side at any point during the 10-minute trial were assigned the maximum score for latency (600 seconds) and a zero for the time spent in the light side and the number of transitions. 2.5. Statistical analyses Body weight, surface righting, negative geotaxis, forelimb grip, and habituation (both between- and within-session) were analyzed by repeated-measures analysis of variance. All behavioral measures in the two-compartment task were analyzed by one-way analysis of variance. The age at which eye opening occurred was analyzed by v2 test. Data for all measures were collapsed within a litter by gender to generate a litter mean for each gender, and it is these means that were used for analysis; thus strain and gender were considered as variables. Unless otherwise stated, the data were col-
Fig. 2. Mean body weight (±SEM) of EL/Suz and DDY pups from PNDs 3 to 21. Both strains consistently gained weight as they aged, but DDY pups weighed more than EL/Suz pups throughout the test period.
lapsed across gender as there were no significant gender effects.
3. Results 3.1. Body weight There was a main effect of age as both strains of mice significantly increased in body weight from 3 to 21 days of age [F (6, 60) = 698.22, P < 0.001]. There was also a significant effect between strains, with EL/Suz pups exhibiting lower body weight than DDY pups across the testing period [F (1, 10) = 1229.79, P < 0.001]. Finally, there was a significant strain · age interaction [F (6, 60) = 12.25, P < 0.001], denoting the fact that DDY pups gained weight more quickly than EL/Suz pups across this period of development (Fig. 2). 3.2. Eye opening There was no difference between the two strains in the age at which eye opening was observed (v2 (1) = 2.637, NS). Eye opening occurred at PND 13 in both strains. 3.3. Tests of neuromotor development 3.3.1. Surface righting reflex There was a significant decrease in the latency to right from a supine position over the 4 test days in both strains [F (3, 39) = 21.61, P < 0.001]. There was also a significant between-subjects effect of strain, with EL/ Suz mice exhibiting a longer latency to show the righting reflex than DDY mice [F (1, 13) = 6.10, P < 0.03]; however, there was no strain · age interaction for this neuromotor reflex (Fig. 3).
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Fig. 3. Mean latency to right (±SEM) of EL/Suz and DDY pups in the surface righting reflex task. Both strains improved in the task from PNDs 3 to 9 (latency to right decreased), although in this task DDY pups exhibited lower latencies than EL/Suz pups across the testing period.
3.3.2. Negative geotaxis reflex There was a significant decrease in negative geotaxis latency (turning 180 on the incline) over the 4 test days [F (3, 39) = 55.65, P < 0.001]. There was a significant difference between strains [F (1, 13) = 284.44, P < 0.001], with EL/Suz pups exhibiting a longer latency to respond than DDY pups, and a significant interaction between strain and age at testing [F (3, 39) = 4.53, P < 0.01] as performance of both strains reached asymptote at the conclusion of testing (Fig. 4). 3.3.3. Forelimb grip strength There was a significant increase in forelimb grip strength, as measured by the latency to fall, over the 3 test days [F (2, 26) = 11.14, P < 0.001] but there was no
Fig. 5. Mean latency to fall (±SEM) for EL/Suz and DDY pups in the forelimb grip strength task. Both strains exhibited similar performance at PND 7, but EL/Suz pups showed greater improvement (longer latency to fall) than DDY mice at PNDs 9 and 11.
main effect between strains. There was an interaction between strain and age at testing [F (2, 26) = 3.52, P < 0.05], with both strains exhibiting similar performance at PND 7, but EL/Suz pups showing a significant improvement (longer latency to fall) at PNDs 9 and 11 relative to DDY pups (Fig. 5). 3.4. Habituation to a novel environment There was a significant strain difference in activity exhibited across the 3 days of habituation testing, with EL/Suz mice exhibiting consistently higher activity on all 3 days and DDY mice exhibiting a decrease in activity across the test period [F (2, 44) = 9.65, P < 0.001]. Similarly, there was a significant between-subject effect of strain for between-session habituation [F (1, 22) = 123.71, P < 0.001], with EL/Suz mice showing an increase in activity and DDY mice showing a decrease in activity across the 3 test days (Fig. 6A). Within-session habituation also differed between the two strains from Minute 1 of testing to Minute 10 of testing, with EL/Suz mice once more exhibiting higher activity at both time points compared with DDY mice [F (1, 22) = 10.28, P < 0.005]. The between-subject effect of strain was also significant for within-session habituation [F (1, 22) = 101.36, P < 0.001]. EL/Suz mice exhibited a decrease in activity between Minutes 1 and 10 of each test day, while DDY mice showed an increase in activity between the first and last minute of each test day (Fig. 6B). 3.5. Light–dark (two-compartment) test
Fig. 4. Mean latency to turn 180 (±SEM) of EL/Suz and DDY pups in the negative geotaxis reflex task. The latency to turn decreased (improved) from PNDs 5 to 11 in both strains; however, the DDY pups performed better than EL/Suz pups across the testing period.
EL/Suz mice entered the light side of the two-compartment apparatus in 55.6 ± 5.9 seconds, whereas DDY mice entered after 215.5 ± 24.1 seconds. Further,
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4. Discussion
Fig. 6. (A) Between-session habituation was evaluated as the mean activity (±SEM) of young EL/Suz and DDY mice, measured by beam breaks, as the animals habituated to a novel environment for 10 minutes per day, over 3 consecutive days. EL/Suz mice exhibited significantly higher activity overall, and there was no evidence of habituation in this strain as their activity increased across days. In contrast, the activity of DDY mice decreased between Days 1 and 3 of testing; thus, between-session habituation did occur in this strain. (B) Within-session habituation was evaluated as the mean activity (±SEM) of the first and the last minute of the test session averaged over the 3 consecutive days of testing. Activity of EL/Suz mice was significantly higher than activity of DDY mice at both Minutes 1 and 10. EL/Suz mice exhibited within-session habituation, as seen by the decrease in activity from Minutes 1 to 10 of testing, while DDY mice did not exhibit within-session habituation as their activity increased from Minutes 1 to 10.
EL/Suz mice spent 200.3 ± 15.3 seconds in the light side of the compartment, whereas DDY mice spent only 75.9 ± 14.6 seconds on the light side. Thus, EL/Suz mouse juveniles entered the light side of the two-compartment box more quickly [F (1, 12) = 48.02, P < 0.001] and spent significantly more time in the light side of the two-compartment box [F (1, 12) = 34.00, P < 0.001] than DDY mice. EL/Suz mice exhibited 18.8 ± 0.9 transitions between the light and dark compartments of the testing apparatus, which was significantly greater than the 8.2 ± 1.7 transitions exhibited by DDY mice [F (1, 12) = 34.43, P < 0.001]. All data points are given as the means ± SEM.
The present study provides additional evidence for altered physiological and behavioral phenotypes in seizure-prone EL/Suz mice on PNDs 3–30, more than 2 months prior to the onset of seizure susceptibility. Although all testing occurred prior to PNDs 70–90, when human handling can trigger seizures in EL/Suz mice, it is important to note that in the present studies no seizures were observed at any point, including during testing and weekly cage changes. EL/Suz mice exhibited a number of motor reflex delays including slower onset of the surface righting and negative geotaxis reflexes. However, the strain difference in the development of these two motor reflexes was not long-lasting as EL/ Suz and DDY mice performed equally well in these two tasks on their respective final days of testing. EL/ Suz and DDY mice performed equally well in the forelimb grip strength task at 7 days of age; however, on PNDs 9 and 11, EL/Suz pups performed better than DDY pups. The delay in the acquisition of the surface righting reflex, which relies mainly on vestibular and proprioceptive stimuli [28], suggests that the EL/Suz mice may harbor an underlying functional change in the vestibular system, as vestibular stimuli such as tail suspension are known to elicit seizures in older, seizure-susceptible EL/Suz mice [6]. EL/Suz mice weighed less than DDY mice from 3 days of age through weaning at 21 days. The decreased body weight of EL/Suz mice observed from PND 3 until weaning might be the result of a decrease in suckling due to a reduced level of maternal care, as a prior study indicated that EL/Suz dams spend less time nursing than DDY dams [9]. The observed differences in weight do not likely mediate the differences in behavior observed between EL/Suz and DDY pups, as previous findings in mice suggest that factors other than weight, but related in some way to litter size, appear to be critical for developmental behaviors such as surface righting and negative geotaxis reflexes [29]. Moreover, the underweight EL/Suz mice exhibited comparable or superior righting, negative geotaxis, and grasping performance by PND 11, suggesting that low body weight is not a liability for performance of these tasks. As litters were matched for size across the two strains, litter size should not be a confounding factor either. As seizures in our dams were not recorded, we cannot rule out the possibility that seizures occurring in EL/Suz dams during pregnancy may affect the offspring in a manner similar to other prenatal stressors, such as restraint stress [30], handling, increased temperatures, light and noise [17,31] and the presence of a cat [18]. These prenatal stressors resulted in maturational delays of motor reflexes such as negative geotaxis and surface righting [17,18,30,31], decreased weight during the first postnatal week [17], and increased exploratory behavior
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[17,32]. These studies do not address whether the changes in development resulting from prenatal stress are mediated by genetic factors, environmental factors, or by an interaction of these two factors. The present findings are consistent with those previously reported, as EL/Suz mice exhibit delayed surface righting and negative geotaxis reflexes, decreased body weight, and increased exploratory behavior in the two-compartment task. Further studies are needed to determine whether seizures during gestation are responsible for the strain differences observed in this study. Behavior of juvenile mice was examined using a nonassociative learning task, habituation to a novel environment, and the two-compartment task, which examines activity and emotionality. EL/Suz mice exhibited significantly greater activity than DDY mice in both of these paradigms, which is consistent with previous findings of hyperactivity in the EL/Suz strain [8,33]. Given that habituation is defined as a decrease in activity with repeated or prolonged exposure to a novel environment, we found that EL/Suz mice did not exhibit any between-session habituation, although there was some evidence for within-session habituation in these animals. In contrast, DDY mice exhibited between-session habituation, but did not exhibit within-session habituation. This could suggest that DDY mice better recall their prior exposures to the novel environment than the EL/ Suz mice, although the hyperactivity exhibited by the EL/Suz strain may mask any decrease in activity that would be mediated by learning in this paradigm. The increases in between-session activity of EL/Suz mice could also reflect a behavioral sensitization in which a constant stimulus intensity evokes a progressively increased response with repeated presentation [34,35]. A state of behavioral sensitization could certainly be perilous for seizure-prone EL/Suz mice known to be hypersensitive to vestibular stimuli that trigger seizures in these mice. In the light–dark paradigm, EL/Suz mice took significantly less time to enter the light side of the two-compartment box, and spent much more time in the light side than DDY mice. Further, the EL/Suz mice exhibited a much larger number of transitions between the two sides of the light–dark box. Given that EL/Suz mice are reported to be hyperactive in a familiar environment, these results suggest EL/Suz mice also exhibit increased activity in an unfamiliar environment. Thus, the present findings provide further support of the notion that EL/ Suz mice exhibit a generalized hyperexcitability that is observed predominantly as an increase in spontaneous locomotor activity. The aim of the present study was to establish the physical and behavioral phenotype of EL/Suz and DDY mice during development using a battery of tests. This approach was used to provide a more thorough evaluation of the early physical, motor, and behavioral differences between seizure-prone EL/Suz mice and
DDY controls, which has largely been ignored in EL/ Suz research to date. Our hypothesis that EL/Suz mice would exhibit delayed motor development and increased locomotor activity was supported by the present findings. This study contributes significantly to our knowledge of the seizure-prone EL/Suz mouse, with a focus on the phenotype prior to the onset of seizure susceptibility. It is important to note that the mice in the present study were not followed to 90 days of age; thus the onset of seizure susceptibility was never verified. This study was also constrained by the time frame of observation and the tests that were chosen, both of which limited the physical, motor, and behavioral differences that could be detected. Studies examining other aspects of development, such as learning and memory, should also be conducted. More importantly, future research should be geared toward differentiating genetic and environmental contributions to the differences between these two strains, as reported in the present study. The present examination of the early developmental phenotype of EL/Suz mice revealed a number of differences from that of the non-seizure-prone DDY strain. Distinctive characteristics of EL/Suz mice include persistently lower body weight, delays in the surface righting and negative geotaxis reflexes, and a marked increase in activity in both the habituation and two-compartment tasks. These findings suggest that the phenotype of EL/Suz mice is not limited to an abrupt increase in seizure susceptibility at PNDs 70–90, but includes numerous physiological and behavioral warning signs of predisposition that can be observed on PNDs 3–30, well before the onset of seizure susceptibility. It is therefore possible that early motor and behavioral performance is predictive of adult pathology in epilepsy as well as in other animal models. Acknowledgments Many thanks to Dr. Thomas Seyfried for his generous donation of EL and DDY mice. Thanks also to Karen Rusak and Samantha Korbey for their help in scoring the light–dark behaviors.
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