Research reportDevelopment of a mouse test for repetitive, restricted behaviors: Relevance to autism
Introduction
The diagnostic criteria for autism include impaired social interaction, communication deficits, and repetitive, restricted interests and behaviors [2]. Recent work examining autistic-like traits in twin pairs has provided evidence that these three core symptoms of autism, while all highly heritable, are genetically heterogeneous [68] (see also [69]). The findings suggest that the genes that mediate the non-social component of autism are different from the genes underlying abnormalities in social interaction or communication. The non-social component, the domain of repetitive behavior, encompasses a broad range of clinical indices, including motor stereotypy, self-injury, obsessions and compulsions, an insistence on sameness, and other signs of inflexible, ritualistic responses. Studies using a factor analysis approach to examine repetitive behavior in autism have identified different dimensions within the domain, including the factors “repetitive sensory motor actions and resistance to change” [15] and “repetitive sensory and motor behaviours and insistence on sameness” [74].
In a recent review, Lewis et al. [35] noted that, overall, the many different forms of repetitive behavior may fall within two clusters, one composed of overt, “lower order” motoric stereotypy and self-injury, and the other containing more complex, “higher order” signs of cognitive rigidity, such as obsessions, repeated ritualistic acts, and an insistence on sameness in the environment (see also [80]). Repetitive behaviors from both clusters tend to co-occur in autistic populations with mental retardation, as well as in high-functioning autistic children or those with Asperger's syndrome [7], [11], [47], [73]. The complexity of the repetitive behavior domain poses particular challenges for the development of animal models relevant to the autism phenotype [35].
Our research group has proposed a set of mouse behavioral tasks for modeling the core symptoms of autism [48], [49], [51]. For the lower order repetitive behavior domain, periodic home cage observations and automated measures for activity are used to detect motor stereotypy, self-injurious responses, and other overt signs of aberrant repetitive movements. It is notable that, in mice, cage-related stereotypies can include remarkably high rates of repeated jumping, backward flipping, or cage-top “twirling” [63], [64], [65], [66], [79]. One concern with the use of measures of stereotyped motor actions to model autistic-like behavior in mice is that this may not provide a valid measure of the more cognitively-oriented or higher order repetitive behaviors seen clinically in autism.
Pierce and Courchesne [60] used an environmental exploration task to assess another dimension of repetitive behavior, restricted interests, in normal and autistic children. In this study, subjects were instructed to play in a large room with many different containers, such as tins, boxes, and bags, holding stuffed animals, balls, “magic wands,” and many other items. The researchers found that the autistic children showed significantly less exploration than the normal subjects. In particular, the autistic children spent much less time opening the various containers or examining the novel contents. These findings suggest that restricted interests characteristic of children with autism may interfere with their ability to adaptively explore novel environments. Based on this premise, we reasoned that exploration tasks may be a reasonable way to model higher order repetitive behaviors like restricted interests in mice. Thus, pairing exploration tasks with established procedures for measuring stereotyped motor behavior would, together, allow researchers to model both lower order (stereotyped motor) and higher order (restricted interest) features of the repetitive behavior phenotype of autism.
In the present studies, we have used an automated 16-hole nose poke task in mice to model the repetitive behavior and restricted interests observed in autism spectrum disorders. Previous work has shown that nose pokes or head dipping in a hole-board test provide measures of directed exploration [14], [24], [36], [37], [54]. In this procedure, mice are placed in a standard open field, with a hole-board on the floor, and measures are taken of the number of nose pokes into each hole. Similar to the novel items that were used by Pierce and Courchesne [60], different types of novel olfactory stimuli may be placed in the holes, with screen covers to prevent the mice from touching the stimuli.
Studies of locomotor patterns during exploration in an open field have shown that mice tend to remain in the corner regions or near the walls of the chamber, with less activity in the center region [53], [58], [67]. However, patterns of locomotion during exploration can show significant variations across inbred mouse strains [58], [67]. Therefore, we predicted that mice tested in the nose poke exploration task would also demonstrate strain-dependent patterns of hole preference, with some strains showing the highest rates of nose pokes for holes in the corner regions, and the lowest rates of nose pokes in the center regions. Further, these patterns could be modified by the placement of novel olfactory stimuli in the less-preferred center holes. A lack of hole preference might indicate a resistance to modify nose poke responses in regards to environmental factors, such as hole location or olfactory stimuli. Overall, we conceptualized that a deficit in selective hole preference or persistent responses to one particular olfactory stimulus would reflect the resistance to change and restricted interests observed in the autism spectrum disorders.
In order to validate the test as relevant to mouse models of the autism phenotype, inbred mouse strains were chosen that had been characterized with varying levels of social approach or reversal learning in spatial tests [48], [49], [51]. These behavioral domains reflect the impaired social interaction and resistance to change learned patterns of behavior observed in autism [2]. C57BL/6J and FVB/NJ were selected as strains with behavioral profiles that include moderate to high levels of social approach, exploration, and reversal learning. C57BL/6J was of particular interest, since this strain provides the genetic background for many mouse models of neuropsychiatric disorders. Performance in this strain could indicate the patterns of exploration and olfactory preference that might be typical of wildtype groups for mutant line comparisons. Both young adult and older adult C57BL/6J mice were tested, providing information on the use of the assay in mice of differing ages. BALB/cByJ was chosen because the BALB substrains have been characterized as high in anxiety-like behavior and neophobia (dependent upon the behavioral measure [16], [26], [27], [34], [49], [78]), and with good reversal learning in spatial tasks [49]. Responses of the BALB/cByJ strain would provide information on the effects of anxiety on exploration and olfactory preference in the nose poke task. The fourth inbred mouse strain, BTBR T+tf/J, has a behavioral profile that reflects some components of autism, including low social preference and a selective deficit in reversal learning, without high levels of anxiety-like behavior [49] (see also [8]). Given the initial findings of an autism-like phenotype, we predicted that BTBR T+tf/J mice would also show low levels of general exploration and demonstrate preference for only one or two olfactory stimuli, similar to the low levels of exploration and restricted interests observed in autistic children [60].
The present studies also included Grin1neo/neo (NR1neo/neo) mice, which have reduced levels of the NR1-NMDA receptor subunit. This mutant line is characterized by an aberrant behavioral phenotype, including marked deficiencies in social behavior [21], [46] and a tendency for self-injurious responses (present studies), suggesting that the NR1neo/neo mice might demonstrate repetitive behavior and restricted interests in the nose poke assay. In addition to the autism-like behavioral profile, the deficiency in glutamate function found in the NR1neo/neo mice may be relevant to the syndrome in humans. Carlsson [12] has proposed a hypoglutamatergic hypothesis for autism, based on the neuropathology observed in autistic patients. In particular, the reduced size of the hippocampus and amygdala observed in autism [5], [71] might be associated with deficiencies in the glutamatergic neuronal projections that originate in these regions. Abnormal concentrations of glutamine/glutamate in the amygdala and hippocampus have been reported in adults diagnosed with autism spectrum disorders, in comparison to healthy subjects [55]. Changes in glutamatergic neurotransmission might also be relevant to the abnormal synaptic function found in many genetic mouse models for autism and related syndromes [4], [29], [30], [42], [84].
Section snippets
Inbred mouse strains
Male mice from four inbred strains, C57BL/6J (B6), BALB/cByJ (BALB), BTBR T+tf/J (BTBR), and FVB/NJ (FVB), were purchased from The Jackson Laboratory, Bar Harbor, ME (JAX). Mice were 3–4 weeks of age upon arrival at the University of North Carolina animal facility in Chapel Hill, NC. Mice were 6–9 weeks of age at the start of hole-board testing, except for one group of B6 (n = 8) and the BALB mice (n = 10), which were 7 months of age at the start of testing. Animals were housed separately by
B6 and BALB
The first mice tested for general patterns of exploration in the hole-board assay were young B6 (8–9 weeks in age; previous tests: elevated plus maze and activity test), and older groups of B6 and BALB, which were 7 months in age (previous tests, completed by 3 months in age: rotarod, activity, social approach test, olfactory test). For this study, mice were given two 1-h tests, with one week between each test. The distributions of percent total nose pokes for the first and second tests are
Discussion
The purpose of the present studies was to determine if the hole-board test could be used to measure repetitive behaviors and restricted interests in mice, as part of an on-going initiative to develop mouse behavioral tasks relevant to the autism phenotype [48], [49], [51]. The strategy for this study was to characterize two strains (B6 and FVB) that, based on previous work in our laboratory [49], would not be expected to show any autism-like profiles of behavior. The patterns of exploration and
Acknowledgements
Behavioral tests were conducted by the Mouse Behavioral Phenotyping Laboratory of the Neurodevelopmental Disorders Research Center, University of North Carolina. This work was supported by NICHD grant P30 HD03110, NIMH grants R01 MH73402 and MH063398, and NIH STAART grant U54 MH66418.
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