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Keywords:

  • Autism;
  • exploration;
  • inbred strains;
  • locomotion;
  • mice;
  • olfaction;
  • rotarod;
  • sociability;
  • social interaction;
  • social preference

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Deficits in social interaction are important early markers for autism and related neurodevelopmental disorders with strong genetic components. Standardized behavioral assays that measure the preference of mice for initiating social interactions with novel conspecifics would be of great value for mutant mouse models of autism. We developed a new procedure to assess sociability and the preference for social novelty in mice. To quantitate sociability, each mouse was scored on measures of exploration in a central habituated area, a side chamber containing an unfamiliar conspecific (stranger 1) in a wire cage, or an empty side chamber. In a secondary test, preference for social novelty was quantitated by presenting the test mouse with a choice between the first, now-familiar, conspecific (stranger 1) in one side chamber, and a second unfamiliar mouse (stranger 2) in the other side chamber. Parameters scored included time spent in each chamber and number of entries into the chambers. Five inbred strains of mice were tested, C57BL/6J, DBA/2J, FVB/NJ, A/J and B6129PF2/J hybrids. Four strains showed significant levels of sociability (spend- ing more time in the chamber containing stranger 1 than in the empty chamber) and a preference for social novelty (spending more time in the chamber containing stranger 2 than in the chamber containing the now-familiar stranger 1). These social preferences were observed in both male and female mice, and in juveniles and adults. The exception was A/J, a strain that demonstrated a preference for the central chamber. Results are discussed in terms of potential applications of the new methods, and the proper controls for the interpretation of social behavior data, including assays for health, relevant sensory abilities and motor functions. This new standardized procedure to quantitate sociability and preference for social novelty in mice provides a method to assess tendencies for social avoidance in mouse models of autism.

Autism is a severe neurodevelopmental disorder defined by social and communication deficits and ritualistic-repetitive behaviors that are typically detectable in early childhood and continue throughout life (American Psychiatric Association 1994; see also Folstein & Rosen-Sheidley 2001; Kanner 1973; Lord et al. 2000a; Paul 2003; Piven et al. 1997; Piven 2001; Schloper & Mesibov 1987). The prevalence of autism is estimated at 1/1000 (with rates of 2.5/1000 when individuals with Asperger's syndrome and pervasive developmental disorder are included) (Fombonne 2003). A large body of evidence from twin and family studies supports the important role of genetic factors in the etiology of autism (Bailey et al. 1995; Ritvo et al. 1989; Szatmari et al. 1998). Linkage and candidate gene studies, aimed at identifying autism susceptibility genes, are currently the focus of research in numerous laboratories (Barrett et al. 1999; Folstein & Rosen-Sheidley 2001; Philippe et al. 2002; Shao et al. 2002). Neuroimaging and post-mortem studies have implicated a number of neural structures and circuits in autism (Bauman & Kemper 1985; Happéet al. 1996; Schultz et al. 2000). A non-human primate model, resulting from neonatal lesions to the medial temporal lobe, has been described (Bachevalier 1991). Given the complexity of this syndrome, multiple, complementary approaches will be necessary to identify the underlying neural and etiologic mechanisms, and gene-brain-behavior relationships, in this disorder. The study of genes, brain and behavior relationships in mice potentially provides an important addition to current approaches to studying the pathogenesis of autism. However, to date, behavioral models of autistic-like responses in mice have received little attention.

Social behavior is a complex construct. The particular set of behaviors that define the syndrome of autism are wide ranging in severity and type, and change in form with chronological development. So, for example, in the original descriptions of autism by Kanner (1943), autistic children were characterized by their dramatic lack of interest in others, whereas current DSM-IV criteria identify substantially more subtle deficits in reciprocal social interaction as sufficient for meeting criteria for this disorder (American Psychiatric Association 1994). Behavioral abnormalities in this domain in autism are varied and include such characteristics as deficits in non-verbal expression (e.g. deficits in eye-to-eye gaze and diminished expression of emotion as measured by lack of gesturing or facial expression), abnormalities in the social use or understanding of language, deficits in social approach (such as the absence of coming for comfort upon injury), or markedly diminished peer relationships.

Mouse models may provide a useful research tool to advance the investigation of genes associated with autism (Insel 2001). In the present study, we sought to develop methods for characterizing some of the social behaviors in mice that could be viewed as qualitatively similar to social deficits that define autism. Mice are a highly social species (Blanchard et al. 2003; Grant & MacIntosh 1963; Laviola & Terranova 1998; Scott & Fredericson 1951; Valzelli 1973; Vandenbergh 1989; Winslow & Insel 2002). Social behaviors have been explored in mice with mutations in candidate genes such as oxytocin (Winslow & Insel 2002; Young et al. 2002), monoamine oxidase-A (Shih & Chen 1999), fragile X (Churchill et al. 2002; Mineur et al. 2002) and dishevelled-1 (Lijam et al. 1997). Good mouse models will require tasks specific for the types of social deficits that are considered the core symptoms of autism, including very low levels of social approach behaviors (Folstein & Rosen-Sheidley 2001; Lord et al. 2000a; Piven et al. 1997).

To quantitate sociability tendencies in mice, we developed a set of tasks that measure (a) time spent with a novel conspecific and (b) preference for a novel vs. a familiar conspecific. Conceptually similar to previous social interaction tests (Bales & Carter 2003; File & Seth 2003; Gheusi et al. 1994; Insel & Young 2001; Tang et al. 2003; Winslow 2003), the present behaviors focus on a more narrowly defined set of parameters. We reasoned that most autistic individuals demonstrate reduced or unusual social approach. Therefore, subject mice were given the choice between exploring an empty, novel chamber vs. exploring an identical novel chamber containing a mouse with which the subject had no previous interaction (stranger 1). Autistic individuals may further avoid unfamiliar social partners and display diminished interest in novelty (American Psychiatric Association 1994). Subject mice were given another choice between exploring the chamber containing the now-familiar stranger 1, vs. exploring an identical chamber containing a new mouse (stranger 2). Time spent in each chamber and number of entries into each chamber were scored by observation. These measures were subsequently automated, as described in a companion paper (Nadler et al. 2004).

Previous methods for testing social behavior in mice employed tethered novel conspecifics and other approaches to maximize the contribution of the subject mouse to the social interactions, and minimize the contributions of the target stranger mouse (Brodkin et al. 2004; Carter et al. 1995; Winslow 2003). The present method contains the target stranger within an inverted round wire cage that permits visual, tactile, auditory and olfactory communication. This modification focuses the task on social approach tendencies, relevant to the social approach deficit in autism, while avoiding potential confounds resulting from aggressive or sexual interactions.

To ensure that differences in social behaviors were not artifacts of other aspects of mouse behaviors, a series of independent control experiments was conducted to evaluate potential confounding factors. Measures of general health, home cage behavior and neurological reflexes were conducted to rule out gross physical abnormalities (Crawley 2000). Olfaction abilities are considered essential for mouse social interactions (Cheal & Sprott 1971; Liebenaur & Slotnick 1996; Wrenn et al. 2003). Retrieval of food buried in cage bedding was quantitated in a 15-minute olfactory test, to ensure that the subject mice were not grossly impaired in olfaction abilities. Further controls were designed to detect additional symptoms relevant to autism, such as abnormalities in locomotion or motor coordination (Hallett et al. 1993; Piven et al. 1997), that could directly affect performance of the social task.

Parameters scored within the test sessions in the social apparatus provided additional information on procedural abilities necessary for social behaviors. Before the start of the choice tests, subject mice were given a habituation session in a neutral central chamber. The number of times that the mouse exited from the habituated empty central chamber into the novel empty chamber provided a general measure of locomotor function. In addition, time spent in the habituated center chamber provided a preliminary measure of anxiety-like behaviors, analogous to the open field emergence test (Holmes et al. 2003; Smith et al. 1998).

Five inbred strains of mouse were chosen for the present evaluation of autistic-like social behaviors in mice: C57BL/6J, DBA/2J, FVB/NJ, A/J and the hybrid B6129PF2/J. Many approaches to studying mouse models of human disease have focused on mice with specific targeted mutations in single genes. Available evidence from family and twin studies, and the results to date from linkage studies, suggest that, for most cases, the simplest model of autism involves the interaction of multiple genes (Risch et al. 1999). Given data from family genetic studies showing a broad range of the behavioral expression of the underlying genetic liability for autism in nonautistic relatives (Piven et al. 1997), the possibility must also be considered that variation in the social and cognitive behaviors manifested by autistic individuals represents one end of the extreme of normal genetically-controlled variation. As an initial effort to characterize social behaviors in mice that might be relevant to the study of autism, we therefore focused on comparing and contrasting the continuum of social behaviors that could be observed across several ‘normal’ strains of inbred mice.

The present study presents the first results using the three-chambered apparatus to score social approach behaviors relevant to autism. For the purposes of this study, ‘sociability’ is defined as a significant propensity to spend time with another mouse, as compared to time spent in an identical but empty chamber. ‘Preference for social novelty’ is defined as a significant propensity to spend more time with a new mouse than with a familiar mouse. Most experimental groups consisted of male mice, for consistency with the 4:1 male:female difference in the diagnosis of autism (Folstein & Rosen-Sheidley 2001). Mice were tested at both juvenile and adult ages, consistent with the neurodevelopmental aspects of autism. The choice of inbred strains was designed to represent those commonly used in behavioral genetics, and to provide baseline scores that could later be compared to lines of targeted gene mutations bred into these background strains.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Animals

Male mice from four inbred strains, C57BL/6J, DBA/2J, FVB/NJ, A/J and the hybrid B6129PF2/J, were purchased from The Jackson Laboratory (Bar Harbor, ME). Male DBA/2 mice were purchased from Harlan (Indianapolis, IN). Mice ranged in age from 3 to 10 weeks upon arrival at the University of North Carolina animal facility in Chapel Hill, NC. An additional group of male and female C57BL/6J mice, used in only one experiment (data shown in Figs 5 and 6), were the first generation derived from stock purchased from The Jackson Laboratory. All animals were housed in group cages by strain and gender, three to four per plastic tub cage, and provided with food and water ad libitum. The housing room was maintained at 23 °C on a 12-h light/dark cycle (lights off at 19:00). All procedures were conducted in strict compliance with the policies on animal welfare of the National Institutes of Health and the University of North Carolina (stated in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, 1996 edition), and approved by the University of North Carolina Institutional Animal Care and Use Committee.

Test procedures

The first series of behavioral assays was conducted with 20 male mice from each of three strains, C57BL/6J, DBA/2J and FVB/NJ, aged 3–4 weeks at the time of arrival into the animal facility. Mice were evaluated on measures of home cage behavior, neurological reflexes, locomotor activity, motor coordination and balance, olfaction and social behavior.

Home cage behavior

During the first week in the animal facility, assessments of home cage behavior were conducted by investigator observations at three different time points: 8:00, 12:00 and 18:50. Observations were taken across two to three days, for 20 min at each time point, for a total of 60 min of home cage observation. Two hours before the noon observation, one white cotton nestlet square (Ancare Corp., Bellmore, NY) was added to each cage, in order to assess nest-building behavior. The evening observation was conducted 10 min before lights off, and then for another 10 min after the lights had gone off, using red light illumination. Records were taken for nestlet shredding, nest building, sleeping in huddles, activity, fighting and any aberrant behaviors, such as tremor or seizures.

General health and neurological reflexes

Behavioral testing began one week after arrival into the animal facility. The mice were first evaluated for general health, using several measures of overall appearance and behavior, as previously described (Crawley 2000; Holmes et al. 2003). The measures included general observations on the appearance of the fur and whiskers, body posture and normality of gait. Normal reflexive reactions to a gentle touch from a cotton swab to the whiskers on each side of the face, the approach of the cotton swab to the eyes, and the sound from a metal clicker (Preyer reflex) were assessed. Each animal was placed in a small, empty plastic cage, and its ability to remain upright when the cage was moved from side-to-side or up-and-down was noted. Animals were observed for the visual placing reflex (forepaw extension when lowered toward a visible surface), and for ability to grasp a metal grid with forepaws and hindpaws.

Locomotion

One day after the neurobehavioral screen, exploratory activity in a novel environment was assessed by a five-minute session in an open field chamber (40 cm L × 30 cm W × 40 cm H), constructed of clear Plexiglas. A grid of squares (10 × 6) was drawn on the floor of the chamber, and counts were taken of number of squares crossed and rears during the session.

Olfactory test

A simple test for olfaction was conducted two days following the activity test. One or two days before the olfactory test, an unfamiliar food high in carbohydrates (Froot Loops, Kellogg Co., Battle Creek, MI) was placed overnight in the home cages of the subject mice. Observations of consumption were taken to ensure that the novel food was palatable to the mice. On the day of the test, each mouse was placed in a large, clean tub cage (46 cm L × 23.5 W cm × 20 H cm), containing 3 cm deep paper chip bedding (Canbrands Product, Moncton NB, Canada), and allowed to explore for five minutes. The animal was removed from the cage, and one Froot Loop was buried in the cage bedding. The animal was then returned to the cage and given 15 minutes to locate the buried food. Measures were taken of latency to find the Froot Loop and whether it was consumed. In addition, observations were taken of behavioral responses (sniffing, digging, locomotion) during the test session.

Rotarod performance

One day following the olfactory test, the mice were assessed for balance and motor coordination on an accelerating rotarod (IITC Inc., Woodland Hills, CA). Mice were placed on a cylinder which slowly accelerated to a constant rotating speed. The task required the mouse to walk steadily forward in order to remain on top of the barrel. Revolutions per minute (r.p.m.) were set at an initial value of 3, with a progressive increase to a maximum of 30 r.p.m. across five minutes, the maximum trial length. Each animal was given a single test session consisting of two trials, a practice trial and a test trial, with 45 seconds between each trial. Latency to fall from the top of the rotating barrel was recorded by the rotarod timer.

Tests for sociability and social novelty preference

The initial set of 60 mice was tested in the social task at 5–6 weeks of age, after completion of the control tasks described above. Methods were adapted from established procedures for testing pair-bonding in voles (Dean, S.M. & Vandenbergh, J.G. (2001) Prenatal exposure to antiandrogenic or estrogenic compounds alters monogamous behavior in pine voles (Microtus pinetorum). Presented at the Society for Behavioral Neuroendocrinology Annual Meeting, Tempe, Arizona; Winslow 2003). The social testing apparatus, illustrated in Fig. 1, was a rectangular, three-chambered box fabricated by Philip L. Thompson and Steve Medlin, University of North Carolina Physics Instrument Shop. Each chamber was 20 cm L × 40.5 cm W × 22 cm H. Dividing walls were made from clear Plexiglas, with small circular openings (3.5 cm in diameter) allowing access into each chamber. The chambers of the social apparatus were cleaned and fresh paper chip bedding was added between trials.

image

Figure 1. The social test chamber. Mice were habituated in the center compartment. During testing, mice could enter adjacent chambers through the doorways in the dividing walls. In the test for sociability (a), the test mouse is given a choice between spending time in the side with an unfamiliar mouse, vs. an empty side. In the test of preference for social novelty (b), the test mouse is given a choice between the first unfamiliar mouse (stranger 1), and a newly introduced unfamiliar mouse (stranger 2).

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The test mouse was first placed in the middle chamber and allowed to explore for five minutes. The doorways into the two side chambers were obstructed by plastic boxes during this habituation phase. After the habituation period, an unfamiliar C57BL/6J male (stranger 1), that had no prior contact with the subject mice, was placed in one of the side chambers. The location of stranger 1 in the left vs. right side chamber was systematically alternated between trials. The stranger mouse was enclosed in a small, round wire cage (Galaxy Cup, Spectrum Diversified Designs, Inc., Streetsboro, OH), which allowed nose contact between the bars, but prevented fighting. The cage was 11 cm in height, with a bottom diameter of 10.5 cm and bars spaced 1 cm apart. A weighted cup was placed on the top of the cage to prevent climbing by the test mice. The animals serving as strangers were male C57BL/6J mice that had previously been habituated to placement in the small cage. Both doors to the side chambers were then unblocked and the subject was allowed to explore the entire social test box for a 10-minute session. Measures were taken of the amount of time spent in each chamber and the number of entries into each chamber, by a human observer seated five feet from the apparatus. An entry was defined as all four paws in one chamber. All data were entered via an event keyboard connected to data collection software on a PC. Interrater reliability was greater than 95% when results were compared between two trained observers scoring the same subject mice simultaneously.

At the end of the first 10 minutes, each mouse was tested in a second 10-minute session to quantitate social preference for a new stranger. A second, unfamiliar mouse was placed in the chamber that had been empty during the first 10-minute session. This second stranger was also enclosed in an identical small wire cage. The test mouse had a choice between the first, already-investigated unfamiliar mouse (stranger 1), and the novel unfamiliar mouse (stranger 2). As described above, measures were taken of the amount of time spent in each chamber and the number of transitions between chambers of the apparatus during the second 10-minute session.

Critical elements of the sociability and social novelty preference tests

Results from the initial group of mice indicated significant sociability and a preference for social novelty in young male mice from three different inbred strains. Potential modifier variables concerning sociability were then evaluated, including assessment of two additional strains, a comparison between male and female mice, replicability with repeated testing of the same subjects and a comparison between juvenile and adult mice.

Social tests in hybrid male mice

A group of 10 male B6129PF2/J mice, age 6 weeks at the time of testing, were assessed with the same social test procedures as used with the initial group of mice. This hybrid mouse strain was of particular interest, because it has been used as the genetic background control for transgenic lines maintained on a mixed C57BL/6J × 129P3/J background (e.g. Waite et al. 2002; Zhu et al. 2003), including mutant lines phenotyped by our laboratory (unpublished results).

Social tests in male and female mice

Sex differences were investigated using a group of 12 male and 8 female C57BL/6J mice, age 6 weeks at the time of testing. These animals were assessed with the same procedure as used with the initial group of mice. In this case, the males and females were tested in separate social test boxes. For both sexes, C57BL/6J males served as the unfamiliar mice (stranger 1 and stranger 2). There were no confounding sexual or aggressive behaviors, because the strangers were contained within the wire cages.

Test for sociability in adult male mice

In comparison to the juvenile male mice tested in the initial experiment, adult male mice were tested at three months of age. Three inbred strains were analyzed for social behavior in adult males: C57BL/6J, DBA/2 and A/J, 10 mice per inbred strain. In this case, the mice were each given a five-minute habituation period in the social test box with the doors open, and then a 10-minute choice task between the chamber containing an unfamiliar mouse and the empty chamber (the social novelty test was not conducted).

Because the A/J mice showed markedly low levels of entry into the different chambers, concern was raised that the small openings between chambers presented a physical barrier to exploratory locomotion in this strain. Low levels of exploration throughout the three chambers represent a potential artifact in the interpretation of social behavior deficits. Therefore, the A/J mice were given a second test one week later, with the dividing walls between the chamber sides partially retracted to facilitate exploration.

Replicability within subjects

A subset of the adult C57BL/6J mice (n = 5) and the DBA/2 mice (n = 6) was retested with the sociability procedure 11–12 days following the first test, to determine similarity of results across repeated testing of the same mice.

Statistical analysis

One-way Analysis of Variance (anova) was used to compare data for the inbred strains on measures of body weight, activity, rotarod performance and latency to find buried food. Social data were first analyzed using repeated measures anovas, with the factors of strain (or sex), test condition (sociability or social novelty) and chamber side (e.g. stranger 1 side or the opposite side). Within-group repeated measures anovas were used to determine side preferences, and to compare results from retesting in the sociability procedure. Fisher's protected least-significant difference (PLSD) tests were used to compare group means only when a significant F-value was determined. For all comparisons, significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

General health, home cage behavior, neurophysiological reflexes and sensory and motor function

As shown in Table 1, preliminary observations of the inbred strains provided evidence that the mice were in good general health, without overt signs of impairment or aberrant behaviors. In the home cages, all groups of mice made nests and, in almost all cases, were observed sleeping together in huddles. There were no significant differences between the strains in body weight or rotarod performance (see Table 1 for mean latency to fall on the second trial). Overall, neurological reflexes in response to visual, tactile or auditory stimuli were normal. For the olfactory test, the majority of the subjects successfully located and retrieved the buried food. The FVB/NJ mice were particularly adept at this task, with significantly lower latency scores to find the buried food, in comparison to the other two groups (post hoc tests following significant effect of strain; F2,56 = 4.399, P = 0.0168). Behavioral observations recorded during the olfactory task confirmed that all of the subjects showed exploration of the novel test cage. On measures of activity, the FVB/NJ mice had higher levels of locomotion in an open field than the other two groups, and the DBA/2J mice showed less locomotion (post hoc tests following significant effect of strain; F2,57 = 44.664, P < 0.0001). The FVB/NJ mice also showed more rearing responses in the open-field, in comparison to the other two strains (post hoc tests following significant effect of strain; F2,57 = 86.234, P < 0.0001).

Table 1. : General health, home cage behaviors, neurological reflexes and motor function
 C57BL/6JDBA/2JFVB/NJ
  1. Results for home cage behavior were compiled across three different observation periods, in most cases. Observation of lights-off locomotion was taken for 10 min following the initiation of the dark cycle in the animal housing room, under red light illumination. Neurological reflexes included reaction to a sterile cotton swab approaching the eye (corneal) or touching whiskers (vibrissae orienting). For visual placing, mice were lowered towards a mesh grid and observed for forepaw-reaching. Preyer reflex was elicited by a loud metal clicker. Rotarod data are the group means for the second of two trials. Data are expressed as percentage of the total number of mice tested, or as mean ± standard error of the mean (SEM). n = 20 for each strain. Data were lost from one DBA/2J mouse for olfaction test. # P < 0.05, strain different from other two strains on comparable measures.

Physical characteristics
 Body weight (g)19.9 ± 0.318.8 ± 0.519.6 ± 0.3
 Poor coat condition0%0%0%
 Piloerection0%0%35%
Home cage behaviors
 Type of nestsphericalpartial, flatspherical/flat
 Huddling together (% cages)100%100%80%
 Sleep during day (% subjects)95%100%80%
 Lights-off locomotion (% subjects)25%100%20%
 Aberrant responses (% subjects)000
Neurological reflexes (% normal)
 Corneal90%80%100%
 Visual placing100%100%100%
 Vibrissae orientating100%100%100%
 Preyer reflex100%100%100%
Olfaction
 Uncovered buried food (% subjects)75%68%100%
 Latency to uncover buried food (seconds)422.5 ± 74.8459.5 ± 78198.4 ± 46.2#
Motor/muscular abilities
 Locomotor score297.0 ± 19.2198.2 ± 9.3#418.1 ± 19.0#
 Rears29.0 ± 1.928.3 ± 1.964.0 ± 2.7#
 Rotarod latency (seconds)157.5 ± 21.4130.0 ± 16.1128.7 ± 15.6

Social behavior

Test for sociability and social novelty in three inbred mouse strains

C57BL/6J, DBA/2J and FVB/NJ male juvenile mice demonstrated a significant preference for spending time in the chamber containing stranger 1, vs. time spent exploring the empty chamber (Fig. 2a; post hoc tests following significant main effect of side; F1,59 = 39.29, P < 0.0001). When the choice was between the first stranger mouse and a second stranger mouse (Fig. 2b), all three inbred strains demonstrated a preference for social novelty (post hoc tests following significant main effect of side; F1,59 = 42.867, P < 0.0001). An overall repeated measures anova was performed, looking at the effects of strain, side of chamber (stranger 1 side or opposite side) and test condition (sociability or social novelty). A significant interaction was found between side of chamber and test condition (F1,57 = 67.634, P < 0.0001), reflecting the finding that preference was for the stranger 1 side during the first half of the session (the sociability condition), but switched to the stranger 2 side during the last part of the session (the social novelty condition). There were no significant differences between the three inbred strains (C57BL/6J, DBA/2J and FVB/NJ) on measures of duration (no significant effect of strain, F2,57 = 2.369, P = 0.1027).

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Figure 2. Social behavior in juvenile C57BL/6J, DBA/2J and FVB/NJ mice. Duration measures were taken for (a) sociability, i.e. preference for a stranger mouse vs. an empty chamber, and (b) preference for a novel stranger (stranger 2) vs. the first unfamiliar mouse (stranger 1). See text for further description. Data shown are mean + SEM for each group. n = 20 for each strain. *P < 0.05, within-group comparison between stranger 1 side and opposite side. All three strains demonstrated significant preference for spending time in the chamber containing the strange mouse.

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As shown in Fig. 3, the measure of entries into each side of the social test box did not follow the pattern seen for the measure of time spent in each chamber. Significant preferences for sociability (Fig. 3a) or preference for social novelty (Fig. 3b) were not observed for number of chamber entries in any of the inbred strains. As seen in the previous test for open-field locomotion, the DBA/2J mice appeared less active, as evidenced by significantly lower numbers of entries into each side, in comparison to the other two groups (post hoc tests following significant main effect of strain, F2,57 = 44.322, P < 0.0001).

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Figure 3. Number of entries into each side of the social test box during the procedure for(a) sociability and(b) preference for social novelty. Data shown are mean + SEM for each group. n = 20 for each strain. # P < 0.05, strain different from other two strains on comparable measures.

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Test for sociability and preference for social novelty in hybrid mice

As observed with the three inbred strains, juvenile male mice from a mixed B6/129 background had a clear preference for spending time with an unfamiliar conspecific, vs. exploring an empty chamber (Fig. 4a, left) and a significant preference for social novelty, measured by comparing time spent with the new stranger 2 as compared to time spent with the now-familiar stranger 1 (Fig. 4b, left; significant main effect of side, F1,9 = 9.645, P = 0.0126, and significant interaction between side and test condition, F1,9 = 64.427, P < 0.0001). Interestingly, in the hybrid strain, this same pattern for preference was reflected in the measure of entries, both for the sociability test (Fig. 4a, right) and the test for social novelty preference (Fig. 4b, right; significant main effect of test condition, F1,9 = 7.978, P = 0.0199, and significant interaction between side and test condition, F1,9 = 22.821, P = 0.001).

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Figure 4. Social behavior in male hybrid B6129PF2/J mice. Duration and entry measures were taken for (a) sociability, i.e. preference for a stranger mouse vs. an empty chamber, and (b) preference for a novel stranger (stranger 2) vs. the first unfamiliar mouse (stranger 1). See text for further description. Data shown are mean + SEM for each group. n = 10 mice. *P < 0.05, within-group comparison between stranger 1 side and opposite side.

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Comparison of male and female mice on sociability and preference for social novelty

Figure 5 presents the results of a comparison between duration measures in juvenile male and female C57BL/6J mice. An overall repeated measures anova indicated significant main effects of sex (F1,18 = 9.374, P = 0.0067) and a significant interaction between side and test condition (F1,18 = 108.127, P < 0.0001). Male and female mice demonstrated similar sociability, in terms of time spent in the chamber containing stranger 1 (Fig. 5a; significant main effect of side, F1,18 = 80.003, P < 0.0001), and similar preference for social novelty, in terms of time spent in the chamber containing stranger 2 (Fig. 5b; significant main effect of side, F1,18 = 63.271, P < 0.0001). Further analysis revealed that the significant effect for sex stemmed from slightly less time spent in the side compartments in the females, in comparison to the males, during the test for social novelty (Fig. 5b; significant main effect of sex, F1,18 = 9.669, P = 0.0061).

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Figure 5. Male and female C57BL/6J mice show similar sociability and preference for social novelty. Duration measures were taken for (a) sociability, i.e. preference for a stranger mouse vs. an empty chamber, and (b) preference for a novel stranger (stranger 2) vs. the first unfamiliar mouse (stranger 1). See text for further description. Data shown are mean + SEM for each group. n = 12 males and 8 females. *P < 0.05, within-group comparison between stranger 1 side and opposite side.

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There were no significant differences between the male and female mice for number of entries (Fig. 6), although a significant main effect of test condition (F1,18 = 9.722, P < 0.0059) and a significant interaction between side and test condition (F1,18 = 21.917, P < 0.0001) were found. Differences in number of entries into the two sides were not observed during the test for sociability (Fig. 6a). In contrast, both the male and female mice were found to have more entries into the chamber containing stranger 2, in comparison to the side containing stranger 1 (Fig. 6b, F1,18 = 28.972, P < 0.0001).

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Figure 6. Social behavior in male and female C57BL/6J mice. Number of entries were recorded for (a) sociability, i.e. preference for a stranger mouse vs. an empty chamber, and (b) preference for a novel stranger (stranger 2) vs. the first unfamiliar mouse (stranger 1). See text for further description. Data shown are mean + SEM for each group. n = 12 males and 8 females. *P < 0.05, within-group comparison between stranger 1 side and opposite side.

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Test for sociability in adult mice from three inbred mouse strains

Measures of duration in each side of the social test box and number of entries were taken during the five-minute habituation period, in order to demonstrate that mice did not have a bias for spending time in one particular side, and to establish baseline levels of activity when no stranger mouse was present. Overall, the mice did not show a preference for either the right or left sides of the test box during habituation (Fig. 7a; F1,27 = 0.158, P = 0.6945). The adult male mice from the A/J inbred strain spent significantly less time in either of the side chambers, in comparison to the C57BL/6J and DBA/2 animals (post hoc tests following significant main effect of strain, F2,27 = 32.234, P < 0.0001). The A/J group also made fewer entries into either side during habituation, in comparison with the other two strains, while the C57BL/6J mice had higher levels of entries than the other groups (Fig. 7b; post hoc tests following significant main effect of strain, F2,27 = 56.14, P < 0.0001).

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Figure 7. Exploration in social test box during 5-min habituation period for adult male mice from three inbred strains. Measures were taken of (a) duration in and (b) entries into the right and left sides of the social test box. All three chambers were empty during this habituation period. Data shown are mean + SEM for each group. n = 10 for each strain. # P < 0.05, strain different from other two strains on comparable measure.

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As depicted in Fig. 8a, adult male mice from the C57BL/6J and DBA/2 strains showed a preference for spending more time investigating an unfamiliar mouse, vs. time in the empty side, in the sociability test (post hoc tests following repeated measures anova, significant main effects of strain, F2,27 = 30.648, P < 0.0001; side, F1,27 = 76.646, P < 0.0001; and significant interaction between strain and side, F2,27 = 22.233, P < 0.0001). These results were analogous to the data obtained with juvenile male mice of these two inbred strains. In this set of experiments with adult males, a comparable pattern emerged for entries (Fig. 8b), where higher numbers of entries were associated with the side containing the stranger mouse in the C57BL/6J and DBA/2 strains (post hoc tests following repeated measures anova, significant main effects of strain, F2,27 = 32.245, P < 0.0001; side, F1,27 = 9.353, P = 0.005; and significant interaction between strain and side, F2,27 = 3.96, P = 0.031).

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Figure 8. Sociability in adult male mice from three inbred strains. Measures were taken of (a) duration in and (b) entries into the side of the social test box containing the unfamiliar mouse (stranger side) vs. the empty side. Data shown are mean + SEM for each group. n = 10 for each strain. *P < 0.05, within-group comparison, different from stranger side. # P < 0.05, strain different from other two strains on comparable measure. A/J was the only strain tested that failed to spend more time with a conspecific, and spent most of its time in the central chamber after the initial 5-minute habituation session.

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In contrast, adult males of the A/J strain did not spend more time in the chamber containing stranger 1 than in the empty chamber (within-group repeated measures anova, F1,9 = 0.096, P = 0.7639). As clearly indicated in Fig. 8a, the A/J mice stayed in the center region for most of the session. The low number of entries observed in the A/J mice was significantly different from both of the other inbred strains (post hoc tests following repeated measures anova, F-values above).

Sociability procedure with modified social test box

Observations of the A/J mice during the social test indicated that the animals explored the center region of the social test box, but did not readily enter the openings in the dividing walls. Therefore, these A/J mice were retested one week following the initial test, to explore the possibility that partial removal of the dividing walls would facilitate entry into the chambers adjacent to the center region. As shown in Fig. 9, the A/J group did show significant increases in time spent in the side regions (Fig. 9a, significant main effect of retest, F1,9 = 11.343, P = 0.0083) and in number of entries (Fig. 9b, significant main effect of retest, F1,9 = 13.302, P = 0.0053) when the openings between chambers were more accessible. However, A/J mice still did not show a preference for the side containing the unfamiliar mouse, in comparison to the empty side (F1,9 = 0.531, P = 0.4849).

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Figure 9. Retest, using modified social test box, in A/J adult mice. During the retest, the dividers between the sides were partially removed to allow easier access into each side. Results are for (a) duration of time in and (b) number of entries into the side of the social test box containing the unfamiliar mouse (stranger side) vs. the empty side. Data shown are mean + SEM. n = 10 mice. The retest occurred one week following test one. # P < 0.05, retest mean different from corresponding test one mean for comparable measure.

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Repeated testing for sociability

To assess replicability of the procedure when the same mouse was tested twice, a subset of the C57BL/6J and DBA/2 mice was given a retest, using procedures identical to their first test. As shown in Fig. 10, a preference for spending more time with the unfamiliar mouse, in comparison to exploring an empty chamber, was again evident in both strains when retested 11–12 days following the first test (Fig. 10a; significant main effect of side, F1,9 = 285.705, P < 0.001; and significant interaction between side and retest, F1,9 = 7.16, P = 0.0254). Further analysis revealed that the DBA/2 mice did not show any differences in duration between the first and second tests, while the C57BL/6J mice had lower times in the stranger side during the retest (post hoc test following within-group comparison, significant main effect of side, F1,4 = 72.372, P = 0.001; and significant interaction between side and retest, F1,4 = 35.876, P = 0.0039).

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Figure 10. Repeat of sociability test in C57BL/6J and DBA/2 adult mice. Results are for (a) duration of time in and (b) number of entries into the side of the social test box containing the unfamiliar mouse (stranger side) vs. the empty side. Data shown are mean + SEM. The retest was conducted 11–12 days following the first test. n = 5 C57BL/6J mice and 6 DBA/2 mice. * P < 0.05, within-group comparison between stranger side and empty side. # P < 0.05, mean for DBA/2 different from corresponding C57BL/6J mean for comparable measure.

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Differences between the two strains emerged for the measure of entries. DBA/2 mice tended to have lower numbers of entries than the C57BL/6J mice (Fig. 10b; significant main effect of strain, F1,9 = 16.156, P = 0.003; retest, F1,9 = 36.215, P = 0.0002; and side, F1,9 = 8.723, P = 0.0161). DBA/2 mice did not show significant differences between number of entries recorded for the first test and the second test. C57BL/6J mice showed higher numbers of entries into the empty side of the social test box during the retest, in comparison to the first test (post hoc tests following within-group comparison, significant main effect of retest, F1,4 = 103.143, P = 0.0005; and significant interaction bet- ween side and retest, F1,4 = 8.963, P = 0.0402).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Deficits in social interaction and communication are primary diagnostic indicators of autism (e.g. American Psychiatric Association 1994; Lord et al. 2000b). From an early age, autistic children show low levels of eye contact and affective responses, less orientation toward others and language deficits, with a concomitant paucity of speech and social communication (for review, see Tager-Flusberg et al. 2001). Given the primacy of social impairment in autism, we considered this symptom to be fundamental to a mouse model of autism (Insel 2001). Therefore, the present methods were designed to quantitate levels of sociability, i.e. tendency to initiate social contact, and preference for social novelty, i.e. tendency to initiate social contacts with a new individual as compared to someone familiar from past experience, using a simple scoring approach for laboratory mice.

In the present studies, sociability was defined as the tendency to approach and remain proximal to an unfamiliar conspecific, vs. avoidance of the stranger mouse by remaining in the center chamber or exploring an equally novel chamber devoid of another mouse. Overall, four of the five inbred and hybrid strains tested spent more time in the side of the social test box containing the unfamiliar stranger, vs. time in the empty side. Juvenile male mice from three different inbred strains (C57BL/6J, DBA/2J and FVB/NJ) and one hybrid cross (B6129PF2/J), as well as older adult male mice (C57BL/6J and DBA/2), all showed significant levels of sociability, as evidenced by longer durations for time in the stranger side. Similar levels of sociability were clearly evident in a group of young female C57BL/6J mice.

When a novel unfamiliar conspecific was placed in the formerly empty side, a reversal of chamber preference was noted in all groups of juvenile mice from the three inbred strains and the hybrid cross. The higher duration of time spent with the novel stranger (stranger 2) was interpreted as a demonstration that the mice preferred to approach a new stranger, rather than restrict contact to a more familiar individual. It was also an indication of the ability of the mice to discriminate between the two strangers, and to recognize the one that had not been encountered before. Previous work in social recognition has shown that mice show both habituation of olfactory responses directed toward an unfamiliar conspecific, and dishabituation, with a reinstatement of social sniffing, when a novel stimulus animal is presented (Bluthe et al. 1993; Dluzen & Kreutzberg 1993; Ferguson et al. 2000; Kogan et al. 2000; Winslow & Camacho 1995). The choice procedure used in the present study allows the investigator to determine whether the test mice prefer proximity to a novel stranger mouse, vs. proximity to a previously encountered mouse, which may be more relevant to the core symptoms of autism than simple recognition tests.

The social preference pattern observed for the duration measures was not evident for the number of entries into each side for the initial set of juvenile male mice (C57BL/6J, DBA/2J and FVB/NJ). In fact, within each strain, the number of entries into either side of the social test box was almost equal for both the sociability and social novelty tests. However, differences between the three inbred strains were found, with the DBA/2J mice having significantly lower entries into each side, in comparison to the C57BL/6J and FVB/NJ mice. While the DBA/2J group also had significantly lower levels of locomotor activity in the open field test, the significantly higher locomotor scores observed in the FVB/NJ strain, in comparison to the other two groups, were not reflected in an overall higher number of entries. Therefore, for the juvenile male mice, number of entries did not reflect the trends for social preference seen in the duration measures, and only partially correlated with their locomotor activity independently quantitated in the open field.

In contrast, the hybrid B6129PF2/J mice, when tested for sociability, had more entries into the side of the social test box containing the unfamiliar stranger, in comparison to the empty side. There was a reversal of this pattern during the social novelty test, when a higher number of entries were made into the side with the second unfamiliar stranger (formerly the empty side). The pattern for the entries measured in the hybrid mice reflected the social preference pattern observed for the duration measure. In the second group of juvenile C57BL/6J mice, both males and females made equal numbers of entries into each side of the social test box during the assay for sociability, but showed an increased number of entries into the side of the social box containing the second, ‘novel’ unfamiliar stranger during the test for social novelty. Finally, the adult C57BL/6J and DBA/2 mice had higher numbers of entries into the side of the chamber containing the unfamiliar conspecific in the sociability test. Therefore, number of entries appears to vary in terms of its correlation to other parameters. Future studies with these and other inbred strains will be designed to understand the usefulness of the number of entries parameter as a measure of sociability and preference for social novelty.

Based on the present results, time spent in each chamber appears to be the most useful parameter. However, it will be important to differentiate time spent in the chamber from true social investigation of the stranger target mouse in its wire cage. One approach to confirming the social nature of the chamber exploration is to score sniffs directed towards the stranger. Number of sniffs of the wire cage containing the stranger vs. number of sniffs of an empty wire cage were compared in a companion study with an automated version of the present social test chambers (Nadler et al. 2004). Higher numbers of sniffs were directed toward stranger 1 than towards the empty wire cage, and towards the novel stranger 2 vs. the now-familiar stranger 1, in C57BL/6J, DBA/2J and FVB/NJ male mice (Nadler et al. 2004).

A particularly interesting finding from the present strain distribution analysis is the low level of sociability in the A/J inbred strain. Adult A/J males failed to show a significant preference for the side containing stranger 1, as compared to the empty side. The A/J mice spent significantly more time in the center chamber, indicating a strong preference for the start box that was not seen in the other strains. These findings may indicate a specific social deficit in the A/J strain. Alternatively, these data may reflect the low levels of open field exploratory behaviors and high levels of anxiety-like behaviors in the light/dark transitions test reported for A/J mice in other studies (Bouwknecht & Paylor 2002; Mathis et al. 1994, 1995; Thifault et al. 2002; Wahlsten et al. 2003) and reflected in the low number of entries in the present study. Arguing against an artifactual interpretation that hypolocomotion caused the social deficit in the A/J group are the data of Fig. 8. Repeated testing of A/J in the modified apparatus increased the number of entries, but did not yield significant sociability scores. In addition, low locomotor scores in DBA/2J and high locomotor scores in FVB/NJ appeared to be unrelated to amount of time spent in the chamber with stranger 1 and stranger 2. It will be important to address the potential confounding variable of locomotor activity in future investigations.

The present procedures for the sociability and social novelty tests are designed to be simple, quantitative and easily mastered by investigators. Methods are based on procedures examining pair-bonding in voles (Dean, S.M. & Vandenbergh, J.G. as above), as well as many other elegant tests for social behavior in rodents (Bluthe et al. 1993; Carter et al. 1995; Dluzen & Kreutzberg 1993; Gheusi et al. 1994; Insel and Young 2001; Kogan et al. 2000; Winslow & Camacho 1995; Winslow 2003). In most cases, these procedures have involved placing two animals together and recording behavioral responses, or using a stimulus animal that is tethered or otherwise confined to one side of a multichambered testing apparatus. Observations of mice in these settings have shown that exposure to an unfamiliar stranger typically evokes a period of intense social sniffing, which may be followed by escalating aggressive responses or sexual behavior, dependent upon such factors as age and sex of the unfamiliar conspecific (Bluthe et al. 1993; Kogan et al. 2000; Winslow & Camacho 1995). Use of the three-chambered apparatus for demonstrating social preference (Insel 2001) was adapted by Brodkin et al. (2004) to investigate social approach and avoidance in female mice. The center chamber was smaller than the side chambers, and Plexiglas cylinders with small air holes were used to contain the stranger. Juvenile Balb/cJ females demonstrated social avoidance, rather than social approach, in this study (Brodkin et al. 2004). The present protocol used a small wire cage to enclose the stimulus mouse, which allowed visual, auditory and olfactory contact between the mice, but prevented the occurrence of aggressive and other responses. This method also simplified the quantification of data. As noted by Winslow (2003), the analysis of the full behavioral series that can occur in social tests demands familiarity with the mouse ethogram, extensive training and the ability to discern the sometimes rapid and fleeting responses that mice can emit. In our experience to date with the present procedures, human observers attain a high level of proficiency after only one or two practice sessions, with high rates (greater than 95%) of interrater reliability between trained observers.

Several additional behavioral assays were performed in the initial set of juvenile male mice (C57BL/6J, DBA/2J and FVB/NJ). These assays were designed as controls for sensory and motor abilities, etc., to ensure that the test mice were physically able to perform the procedures necessary for the social tasks described above. Further, we included home cage observational periods to identify other behavioral parameters that could be useful in a mouse model of autism. Nesting and sleep patterns, and any incidents of stereotypy, hyperactivity and seizures were noted, relevant to common symptoms of autism. Observational findings in some of these behavioral domains have been reported in some targeted gene mutations, e.g. deficits in social interaction within the home cage have been reported for mice deficient in the dishevelled-1 gene (Lijam et al. 1997) or the NMDA NR1 subunit (Mohn et al. 1999). In the present study, the juvenile male mice made nests and huddled together while sleeping. General health, neurological reflexes, activity levels, motor functions and sensory abilities were normal in most regards for the strains tested. All of the inbred strains were able to locate buried food, supporting an interpretation that none were anosmic, which could have severely impacted social behaviors by blocking detection of olfactory cues emitted by the stranger mice. The FVB/NJ mice demonstrated a significantly shorter latency for locating the buried food, but as they did not have markedly higher levels of sociability or preference for social novelty, it seems likely that the shorter latency reflected higher levels of exploratory activity in these mice, as shown in Table 1.

These control parameters are not only important for the interpretation of the results from the social tests, but suggest additional components for putative mouse models of autism. For example, some autistic patients demonstrate aberrant motor responses, including repeated hand flapping, jumping and other repetitive and stereotyped behaviors (Bodfish et al. 2000; Tager-Flusberg et al. 2001). Altered sensory responses have been reported, including both hypo- and hyper-reactivity to environmental stimuli (Baranek 2002). Children with fragile X syndrome often show autistic behavior (Hagerman et al. 1986), and the mouse model of fragile X syndrome has been characterized by changes in activity (Bakker et al. 1994; Peier et al. 2000) and reactivity to acoustic stimuli (Chen & Toth 2001; Nielson et al. 2002). Further work has suggested that the changes found in the mouse models of fragile X are dependent upon the particular background strain for the mutation (Dobkin et al. 2000; unpublished observations from our laboratory).

Development of a new animal model of a human disease is a long and iterative process. The present mouse tasks were designed to maximize face validity to some of the core symptoms of autism. Issues of construct validity and predictive value remain to be investigated. For example, the low sociability scores of the A/J strain require further consideration in terms of sex differences, age of onset and lifetime expression, previous life experiences and potentially confounding artifacts. Following detection of a social deficit in a line of mice, the model can be expanded to encompass a wider set of parameters, including other complex and sensitive measures of social behaviors, quantitated by videotracking software. More in-depth analyses of other behaviors potentially relevant to autism will be useful, including measures of motor stereotypies, sleep dysfunction, sensory hypersensitivity and anxiety-like behavior. Potential applications of this social task include the testing of hypotheses about the etiology of autism, investigating transgenic and knockout mice with mutations in candidate genes for autism, screening large numbers of offspring obtained from chemical mutagenesis for mutations relevant to autism and comparing strains of mice with varying levels of social skills using DNA microarray and proteonomics analyses. Most importantly, the utility of these social behavior tasks in mice will require comprehensive testing to determine their predictive value for evaluating potential therapeutic approaches to treat or prevent the social deficits in autistic patients.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

We wish to thank Dr S. Miles Dean and Dr John G. Vandenbergh of the North Carolina State University Department of Zoology for assisting in the development of the social behavior assays. Supported by the National Institute of Mental Health Intramural Research Program and grants from the National Institutes of Health (MRDDRC P30 HD03110 and STAART U54 MH66418).