- Top of page
- Materials and Methods
Purpose: Autism is a multifactorial disorder that involves impairments in social interactions and communication, as well as restricted and repetitive behaviors. About 30% of individuals with autism develop epilepsy by adulthood. The EL mouse has long been studied as a natural model of multifactorial idiopathic generalized epilepsy with complex partial seizures. Because epilepsy is a comorbid trait of autism, we evaluated the EL mouse for behaviors associated with autism.
Methods: We compared the behavior of EL mice to age-matched control DDY mice, a genetically related nonepileptic strain. The mice were compared in the open field and in the light–dark compartment tests to measure activity, exploratory behavior, and restricted and repetitive behaviors. The social transmission of food preference test was employed to evaluate social communication. Home-cage behavior was also evaluated in EL and DDY mice as a measure of repetitive activity.
Key Findings: We found that EL mice displayed several behavioral abnormalities characteristic of autism. Impairments in social interaction and restricted patterns of interest were evident in EL mice. Activity, exploratory behavior, and restricted behavior were significantly greater in EL mice than in DDY mice. EL mice exhibited impairment in the social transmission of food preference assay. In addition, a stereotypic myoclonic jumping behavior was observed in EL mice, but was not seen in DDY mice. It is of interest to note that seizure activity within 24 h of testing exacerbated the autistic behavioral abnormalities found in EL mice.
Significance: These findings suggest that the EL mouse expresses behavioral abnormalities similar to those seen in persons with autism. We propose that the EL mouse can be utilized as a natural model of autism and epilepsy.
Autism involves a spectrum of multifactorial neurodevelopmental disorders affecting up to 1 in 110 children in the United States (Rutter, 2005; Centers for Disease Control and Prevention, 2009). Autism occurs in early childhood with symptoms that include impairments in social interactions and communication, as well as restricted and repetitive behaviors (Rapin & Tuchman, 2008). A strong interplay of genetics and environmental risk factors influence the autistic phenotype, as the concordance rate among monozygotic and dizygotic twins is 88% and 31%, respectively (Rosenberg et al., 2009).
Epilepsy is a comorbid disorder of autism (Malow, 2006; Seidenberg et al., 2009; Turk et al., 2009). Approximately 30% of individuals with autism develop epilepsy by adulthood (Tuchman & Rapin, 2002), and approximately 30% of individuals with epilepsy fit the criteria for being diagnosed with autism (Clarke et al., 2005). Complex partial seizures are often associated with individuals with autism and epilepsy (Rossi et al., 1995; Steffenburg et al., 1996), although most clinical seizure types are observed (Tuchman et al., 1991). Although mouse models of epilepsy have been useful in characterizing the mechanisms by which genetic and environmental factors affect seizure susceptibility, few mouse models of autism and epilepsy are known (Gilby, 2008).
The EL mouse has long been studied as a model of multifactorial idiopathic generalized epilepsy (Fueta et al., 1986; Rise et al., 1991; Seyfried et al., 1992; Frankel et al., 1995; Poderycki et al., 1998; Todorova et al., 1999, 2006). The EL mouse was discovered in 1954 and was electroencephalographically (EEG) validated as a model of epilepsy in 1976 (Imaizumi et al., 1959; Suzuki, 1976). The EL strain was originally derived from an outbred nonepileptic DDY mouse colony. The inbred EL strain and the parent DDY strain share approximately 70–75% of their genes (Frankel et al., 1995). EL mice experience complex partial seizures with secondary generalizations (Seyfried & Glaser, 1985; Brigande et al., 1989). The seizures in EL mice originate either in or near the parietal lobe (Suzuki et al., 1991; Kasamo et al., 1992; Ishida et al., 1993) or hippocampus (Mutoh et al., 1993) and generalize to other brain regions. The seizures are accompanied by abnormalities such as vocalizations; incontinence; loss of postural equilibrium (Straub tail); excessive salivation; and head, limb, swallowing, and chewing automatisms (Suzuki, 1976; Suzuki & Nakamoto, 1977; Sato, 1985; Ishida et al., 1993; Nakano et al., 1994; Seyfried et al., 1999; Todorova et al., 1999). Epileptic seizures in EL mice begin as early as 50–60 days, commensurate with the onset of sexual maturation (Todorova et al., 2000). Male EL mice are more susceptible to seizures than female EL mice at 60–90 days of age, although this disparity is transient and disappears by 120 days of age (Todorova et al., 1999). Therefore, EL mice have a well-defined epileptic phenotype.
Several neurochemical abnormalities have been characterized in EL mice. EL mice exhibit an increased expression of glial fibrillary acidic protein (GFAP) in the hippocampus compared to nonepileptic control C57BL/6J mice, although there is no increase in hippocampal GFAP-positive astrocytes prior to seizure onset (Brigande et al., 1992). Similar to individuals with autism (Vargas et al., 2005), EL mice exhibit increased microglial activation in the absence of obvious neuronal loss (Brigande et al., 1992; Drage et al., 2002). Ramified microglia appear in the cerebral cortex, hippocampus, cerebellum, as well as throughout the brain in EL mice. Brain gangliosides, sialic acid–containing glycosphingolipids, are also altered in EL mice. Ganglioside GD1a concentration is elevated in the cerebral cortex and hippocampus of EL mice (Brigande et al., 1992). EL mice have lowered levels of GT1b and GQ1b gangliosides and elevated levels of ganglioside GD3 in the cerebellum (Brigande et al., 1992). Because these gangliosides are enriched in neural membranes, these findings suggest that EL mice express altered neural membrane structures.
Along with a robust epileptic phenotype and defined neurochemical abnormalities, an abnormal behavioral profile of EL mice has also been well characterized. Motor abnormalities have been observed in EL mice as early as postnatal day 5 (McFadyen-Leussis & Heinrichs, 2005). EL mice have an increased latency in their surface-righting reflex (time to return from a supine to a pronated position) and their negative geotaxis reflex (time to orient head from downslope to upslope) at postnatal day 5 that persists through postnatal day 9. In addition to early motor abnormalities, EL mice also show an inability to habituate to a novel environment at postnatal days 25–30 (McFadyen-Leussis & Heinrichs, 2005). Therefore, EL mice express abnormal behavior by postnatal day 5.
The abnormal motor behavior of EL mice also extends to irregularities in social behavior. EL mice exhibit short-term memory deficits in social investigation with juvenile conspecific mice (Lim et al., 2007). EL mice also exhibit a deficit in interacting with and investigating an unfamiliar adult mouse in a novel cage environment (Turner et al., 2007). These findings indicate that EL mice exhibit impaired social interactions. The abnormal social behavior of EL mice is also connected to maternal deficiencies. For example, EL dams display nursing deficiencies and have an increased latency in initiating pup retrieval (Bond et al., 2003). EL dams also display a preponderance of nonmaternal activity between postpartum days 2 and 5. During this time, self-grooming and exploration is significantly elevated in EL dams. The maternal deficiencies support an impaired social interaction phenotype.
The present study aimed to further characterize the behavioral phenotype of the EL mouse with reference to novel tests that would evaluate the EL mouse as a natural model of autism and epilepsy. Behavioral tests were used to characterize the hyperactive and exploratory phenotype of the EL mouse. In addition, social communication testing and home-cage observation were used for the first time to evaluate potential deficits in communication and restricted and repetitive behaviors, both characteristics of autism (Bolton et al., 1994; Piven et al., 1997; Silverman et al., 2002). This strategy is consistent with the recommendations of evaluating autistic phenotypes in mice (Crawley, 2004; Murcia et al., 2005; Bernardet & Crusio, 2006; Klauck & Poustka, 2006; Moy et al., 2006; Crawley, 2007; Moy & Nadler, 2008; Halladay et al., 2009). We found a robust and abnormal phenotype in the EL mouse strain that characterizes the EL mouse as a model for autistic behaviors and autism. A preliminary report of this work has appeared (Mantis et al., 2009).
- Top of page
- Materials and Methods
Two separate mouse cohorts were tested. The first cohort consisted of juvenile female and male EL and DDY mice (30–40 days of age). Testing of these mice was completed at or before 40 days. In addition, adult female (>180 days of age) EL and DDY mice were tested to assess the effects of seizures on the autistic phenotype. Of the 12 adult EL mice used in the first cohort, 7 experienced a seizure within 24 h prior to testing and 5 had not experienced a seizure within 1 week prior to testing. To control for litter effects, we used juvenile EL and DDY mice from five and four litters, respectively. Adult EL and DDY mice were derived from five and four litters, respectively. These mice were used in the open field test, light–dark compartment test, and the myoclonic jumping assessment. Myoclonic jumping was evaluated first followed randomly on the same day by evaluation in the open field test or the light–dark compartment test. The remaining test (open field or light–dark compartment) was performed on the following day. The second cohort consisted of juvenile and adult female EL and DDY mice. The EL and DDY mice used were derived from seven litters. The second mouse cohort was tested for social transmission of food preference. Adult EL male mice were excluded from the study, as adult EL males die sporadically from acute uremia poisoning due to urinary retention (Todorova et al., 2003).
Open field test
The open field test examines abnormal behavior in locomotor/exploratory activity and assesses the level of fear-related response in mice (File, 1980; Crawley & Paylor, 1997; Paylor et al., 1998). Restricted and repetitive behavior was measured by fine movement in the open field. Fine movement specifically measures self-grooming and head weaving, which are both considered forms of restricted and repetitive behavior when performed excessively (Devaud, 2003; Lewis et al., 2007). Significant differences in locomotor activity in the open field were observed between EL and DDY mice (Fig. 1). Basic movement (whole body position changes) in the open field was greater in EL mice than in DDY mice (Fig. 1A). EL mice spent significantly less time resting in the periphery of the open field than DDY mice, indicating a more active phenotype (Fig. 1B). EL mice exhibited a profile of increased and sustained rearing compared to DDY mice (Fig. 1C,D).
Figure 1. Locomotor activity of juvenile DDY and EL mice in the open field test. EL mice exhibited greater locomotor activity than DDY mice in the parameters of (A) basic movement, (B) rest time in periphery, (C) rearing, and (D) time rearing. The data are expressed as mean ± standard error of the mean (SEM) (n, number of mice tested). The * indicates that the difference between the DDY and EL mice was significant at the level p < 0.01.
Download figure to PowerPoint
Exploratory behavior, along with the level of fear-related response, was measured by the activity of the mice in the center of the open field. Mice normally fear and avoid the center of open fields (Parks et al., 1998). The center of the open field was defined as a 20 × 10 cm area within the 40 × 20 cm open field. Exploratory behavior was greater and the level of fear-related response in the open field was less in EL mice than in DDY mice (Fig. 2). EL mice entered (Fig. 2A), perambulated (Fig. 2B), and remained in the center of the open field significantly more than DDY mice (Fig. 2C), as illustrated by the open field hot spots (Fig. 3). DDY mice displayed normal thigmotaxic behavior in the open field, as evidenced by their preponderance of activity near the corners and sides of the cage, whereas EL mice displayed indiscriminate activity throughout the open field (Fig. 3). Repetitive and restricted behavior, as measured by fine movement, was also significantly greater in EL mice than in DDY mice (Fig. 4A). Therefore, the open field test indicated that EL mice express abnormally high locomotor activity, have high levels of exploratory behavior and decreased fear-related responses, and engage in restricted and repetitive behaviors.
Figure 2. Exploratory behavior and level of fear-related response of juvenile DDY and EL mice in the open field test. EL mice exhibited greater exploratory behavior and less of a fear-related response than DDY mice in the parameters of (A) entries in center, (B) distance in center, and (C) time in center. The data are expressed as mean ± standard error of the mean (SEM) (n, number of mice tested). The * indicates that the difference between the DDY and EL mice was significant at the level p < 0.01.
Download figure to PowerPoint
Figure 3. Hot spots in the open field of representative juvenile DDY and EL mice. The relative time that a mouse spent in each section of the open field was quantified. DDY mice tend to remain quiescent in the periphery, whereas EL mice exhibit hyperactive behavior and spend more time in the center of the open field.
Download figure to PowerPoint
Figure 4. Repetitive and restricted behaviors in juvenile DDY and EL mice. (A) Fine movement in the open field. Fine movement involves repetitive behavior (i.e., self-grooming and head weaving). (B) Myoclonic jumping. Myoclonic jumping involves a restricted and repetitive behavior, observed in the home-cage environment. No DDY mice exhibited myoclonic jumping. The data are expressed as mean ± standard error of the mean (SEM) (n, number of mice tested). The * indicates that the difference between the DDY and EL mice was significant at the level p < 0.01.
Download figure to PowerPoint
Light–dark compartment test
The light–dark compartment test examines the propensity of mice to explore a novel environment (Crawley et al., 1997; Crawley, 1999; Bourin & Hascoet, 2003). Exploratory behavior was significantly greater in EL mice than in DDY mice (Fig. 5). Latency for emergence from the dark compartment was significantly less in EL mice than in DDY mice (Fig. 5A). EL mice also spent a significantly greater amount of time in the light compartment than DDY mice (Fig. 5B). The light–dark compartment test confirmed findings in the open field test that EL mice engage in high levels of exploratory activity.
Figure 5. Locomotor activity and exploratory behavior of juvenile DDY and EL mice in the light–dark compartment test. (A) Dark emergence latency. (B) Total time spent in the light. The data are expressed as mean ± standard error of the mean (SEM) (n, number of mice tested). The * indicates that the difference between the DDY and EL mice was significant at the level p < 0.01.
Download figure to PowerPoint
Myoclonic jumping assessment
Social transmission of food preference
The social transmission of food preference test examines the ability of mice to communicate through social interaction (Galef & Wigmore, 1983; Wrenn, 2004; McFarlane et al., 2008; Ryan et al., 2010). EL mice showed significantly less preference for the cued food compared to DDY mice (Fig. 6). Cued food consumption relative to total food consumption was significantly less in EL mice (61 ± 6%) than in DDY mice (79 ± 3%).
Figure 6. Social transmission of food preference in EL and DDY mice. EL mice consumed a significantly lower percentage of cued food to total (cued and novel) food compared to DDY mice. Juvenile and adult female EL mice that were seizure-free for at least 1 week prior to and during testing were compared to juvenile and adult female DDY mice for this test. The data are expressed as mean ± standard error of the mean (SEM) (n, number of mice tested). The * indicates that the difference between the DDY and EL mice was significant at the level p < 0.05.
Download figure to PowerPoint
Seizure activity influence on autistic behavior
Seizures occur in EL mice due to fear and stress, and routine handling induces seizure activity in EL mice (Todorova et al., 1999). Seven EL mice had seizures induced within 24 h prior to testing (seizing EL mice) and were compared to five EL mice that had not seized within 7 days prior to or during testing. Significant differences in behavior were observed between nonseizing and seizing EL mice at adult ages (Table 1). In the open field, EL mice that had seized exhibited greater basic movements and spent less time resting in the periphery than nonseizing EL mice. In addition, entrance and perambulation in the open field center was significantly greater in EL mice that seized than in EL mice that did not seize. EL mice that seized also had a tendency to spend more time in the center of the open field than EL mice that did not seize, although this difference in behavior did not reach statistical significance (p = 0.081). A greater number of fine movements were recorded from the EL mice that seized compared to nonseizing EL mice. In the light–dark compartment test, EL mice that seized spent significantly more time in the light compartment than nonseizing EL mice. EL mice that seized also had a tendency to emerge from the dark compartment quicker than nonseizing EL mice, although this difference was not statistically significant (p = 0.546). The myoclonic jumping behavior was observed significantly less in the seizing EL mice compared to seizure-free EL mice. These results suggest that seizure activity exacerbated the hyperactivity and exploratory behavior in EL mice.
Table 1. Comparative analysis of adult DDY and EL mice and influence of seizures on behavioral tasksa
|DDYb||Nonseizing ELc||Seizing ELd|
| Basic movements (#)||689 ± 74||1783 ± 250||2513 ± 164*|
| Total rest time in periphery (s)||1334 ± 42||1085 ± 98||747 ± 45*|
| Rearing events (#)||32 ± 6||485 ± 99||501 ± 57|
| Time rearing (s)||37 ± 8||653 ± 100||675 ± 92|
| Entries in center (#)||13 ± 4||41 ± 7||101 ± 12*|
| Total distance in center (cm)||146 ± 44||294 ± 47||733 ± 76*|
| Total time in center (s)||45 ± 13||129 ± 28||209 ± 31|
| Fine movements (#)||307 ± 23||344 ± 19||435 ± 29*|
| Emergence latency (s)||380 ± 68||122 ± 22||66 ± 7|
| Time in light (s)||15 ± 6||145 ± 7||245 ± 18*|
| Mice jumping (%)||0 ± 0||80 ± 20||29 ± 18*|
- Top of page
- Materials and Methods
The EL mouse is a widely recognized model of idiopathic epilepsy. Treatments, such as antiepileptic drugs and dietary therapies, have been utilized in EL mice and in the clinic for seizure management (Matsumoto et al., 1983; Honda, 1984; Mizoule et al., 1985; Mori et al., 1985; Sugaya et al., 1986; Nagatomo et al., 1996, 2000; Todorova et al., 2000; Greene et al., 2001). The present study further analyzed the behavioral characteristics of the EL mouse to discern the fitness of the EL mouse as a model of autism and epilepsy. No previous studies have evaluated restricted and repetitive behaviors and social communication in EL or DDY mice. Novel methods were employed to build upon and provide further analysis of irregular behaviors in EL mice. Because autism is generally identified in young children (Baroh-Cohen et al., 1996; Robins et al., 2001; Lord et al., 2006), we tested and compared EL mice at juvenile ages (prior to seizure onset). We also tested adult EL mice (post seizure onset) to determine the influence of seizures on the autistic phenotype.
Results from the open field and the light–dark compartment tests showed that the EL mice were hyperactive. Hyperactivity is a symptom prevalent in children with autism (Ando & Yoshimura, 1979). In addition, attention-deficit hyperactivity disorder has been suggested as a comorbid trait of autism (Spencer et al., 1999; Frazier et al., 2001; Leyfer et al., 2006). EL mice also expressed abnormal ambulatory behavior in the open field, and decreased fear-related response in the open field and in light–dark compartment tests. Mice are normally fearful of and avoid lighted areas and open space, as this exposes mice to predators (Archer, 1973; Walsh & Cummins, 1976; Powell & Banks, 2004). It is known that individuals with autism have a deficit in perception and attention to environmental stimuli (Courchesne et al., 1994). Similar deficits in EL mice may prevent focus on environmental cues that would normally evoke a fear-related response. It is also possible that EL mice have inhibitory deficits in their locomotor activity and exploratory behavior, even in the presence of a threatening situation. A lack of inhibition leading to hyperexcitability has been well-established with regard to seizure generation (Balcar et al., 1978; Ribak et al., 1979; Ribak & Reiffenstein, 1982; Ribak et al., 1989). Inhibition deficits may exist in neural circuits responsible for controlling locomotion and exploratory behavior in EL mice. This, coupled with their hyperactivity, may lead to the observed high exploratory behavior in EL mice. Further behavioral tests will be needed to discern if an inability to perceive environmental stimuli or inhibit behaviors actually exists in the EL mouse.
Restricted and repetitive behavioral abnormalities, part of the diagnostic criteria for autism (American Psychiatric Association, 2000), were also observed in EL mice. This was seen in the open field as fine movements in the form of excessive self-grooming and head weaving. Repetitive and restricted behavior was also exhibited as a whole-body movement in the form of myoclonic jumping. The myoclonic activity observed in EL mice is reflective of an autistic phenotype and not a seizure phenotype, since juvenile mice exhibited the behavior prior to seizure onset. Hence, a robust phenotype consisting of restricted and repetitive behaviors is present in EL mice.
We used the social transmission of food preference assay, as it is considered the standard test for measuring communication in mice (Boylan et al., 2007; McFarlane et al., 2008; Ryan et al., 2010). It is important to note that EL mice do not have a deficit in investigating novel odors (Pascual & Heinrichs, 2007). The results of the social transmission of food preference assay show that EL mice exhibit a deficit in olfactory communication, an important mode of social communication in mice (Hurst, 1989). This deficit validates the autism diagnostic criteria of impaired communication (Ryan et al., 2008).
Our results of stereotypic behaviors and impaired communication, together with previous reports of social deficits in EL mice (Lim et al., 2007; Turner et al., 2007), are the first to demonstrate salient abnormalities relevant to all three diagnostic components of autism in EL mice. This, along with other abnormalities relevant to autism (Table 2), highlights the value of the EL mouse as a model of autism and epilepsy.
Table 2. Summary of the fidelity of the EL mouse as a natural model of autism
|Component||Characteristics of persons with autism and EL mice|
|Social interactiona||Failure to develop peer relationships appropriate to developmental level||Impairment in exploring conspecific, juvenile mice (Lim et al., 2007)|
|Lack of social or emotional reciprocity||Impairment in interacting with conspecific, age-matched mice (Turner et al., 2007)|
|Communicationa||Impairment in language development and communication||Impairment in social communication (Fig. 6)|
|Restricted and repetitive behaviora||Stereotypic and repetitive motor mannerisms||Myoclonic Jumping (Fig. 4)|
|Preoccupation with a restricted pattern of interest||Excessive self-grooming and head weaving (Fig. 4)|
|Genetics||Multifactorial (Rutter, 2005)||Multifactorial (Todorova et al., 1999)|
|Neuroanatomic||Microglial activation (Vargas et al., 2005)||Microglial activation (Drage et al., 2002)|
|Cellular maldevelopment in brain without destructive lesions (Kemper & Bauman, 1993; Casanova et al., 2002)||Gliosis observed in hippocampus, but no obvious neuronal loss (Brigande et al., 1992)|
|Neurochemical||Change in gangliosides (Nordin et al., 1998)||Change in gangliosides (Brigande et al., 1992)|
|Comorbid traits||Hyperactivity (Ando & Yoshimura, 1979)||Increased locomotor activity and exploratory behavior (Figs. 1 and 5)|
|Epilepsy (Malow, 2006)||Model of epilepsy|
The behavioral abnormalities associated with autism generally occur prior to the onset of seizures in children with autism and epilepsy (Volkmar & Nelson, 1990; Tuchman & Rapin, 2002). This developmental relationship between autism and epilepsy was also observed in EL mice, as seizures do not occur in juvenile mice, but rather at sexual maturity (Todorova et al., 1999). Interestingly, we found that seizures exacerbated the autistic behavior in EL mice. The EL mice that had not seized within 24 h prior to testing were confirmed to be seizure-free for this period, since seizures do not occur spontaneously in EL mice, and must be induced through handling (Todorova et al., 1999). Seizures increased hyperactivity, exploratory behavior, the stereotypic behaviors of self-grooming, and head weaving, but reduced the incidence of myoclonic jumping. To our knowledge, the influence of clinical seizures on autistic behavior has not been well-characterized in children (Di Martino & Tuchman, 2001; Canitano, 2007). Previous reviews suggest that autistic-like behaviors could be improved following treatment of epileptiform discharges (Binnie, 1994; Tuchman, 2000). A previous study showed that the behavior in autistic children with EEG abnormalities, but who never experienced clinical seizures, was improved following the administration of valproic acid (Plioplys, 1994). We suggest that seizure management might reduce some autistic behaviors in individuals with autism and epilepsy.
The utility of the EL mouse as a model of autism and epilepsy is seen in the robustness of the model’s phenotype, along with the ability of the model to replicate several behavioral abnormalities relevant to the major diagnostic criteria of autism. Previous models of autism have been generated through targeted gene disruption or through surgical brain lesions (Murcia et al., 2005; Klauck & Poustka, 2006; Moy et al., 2006). Other models of autistic behaviors have been developed through a forward genetics approach (Gilby, 2008; Ryan et al., 2010). The EL mouse is the first model in which all animals consistently replicate multiple behavioral abnormalities relevant to autism while sharing the comorbid disorder of epilepsy. Given the multifactorial nature of autism and the lack of consistent neurologic abnormalities found in autism, research has trended toward identifying autistic subpopulations that share neurologic abnormalities. The EL mouse can be an important model to parse the underlying etiology responsible for the codevelopment of autism and epilepsy. The EL mouse will also be integral in studying the unique neurologic challenges that affect individuals with autism and epilepsy.
- Top of page
- Materials and Methods
We thank Dr. Xandra Breakefield and her staff (Molecular Neurogenetics Unit, Neuroscience Center, Massachusetts General Hospital, and Neurology Department, Harvard Medical School, Boston, MA, U.S.A.) for kindly supplying the open field apparatus and Motor Monitor software for our use during the study. We also thank Dr. Stephen C. Heinrichs (VA Medical Center, Research 151–Neuropharmacology, Boston, MA, U.S.A.) whose helpful comments improved the scope and focus of the manuscript. This research was supported by the Boston College Research Fund, and NIH grant (NS055195).