The overall picture that emerges is that the Fmr1KO mouse is hyperactive, less anxious (potentially confounded by activity), and has abnormal sensory and social responses. With the exception of the hotplate response, which was not previously reported, many of these behaviors were observed previously in Fmr1KO mice on the congenic B6 background and have been shown to be (over)corrected in Fmr1KO mice expressing transgenic FMRP [Paylor et al., 2008; Peier et al., 2000; Spencer et al., 2008]. Decreased sensitivity to the hotplate was expected based upon the self-injurious behavior of many individuals with FXS. Furthermore, it has previously been reported that Fmr1KO mice on the congenic B6 background show reduced spinal and peripheral nociceptive sensitization [Price et al., 2007]. Some of these phenotypes (anxiety-related behavior, PPI) were contrary to the expected results based upon the human phenotype. These mouse–human behavioral differences have been observed and discussed in previous studies [Paylor et al., 2008; Peier et al., 2000; Spencer et al., 2005]. For many of these phenotypes, sometimes the effect size was greater in particular backgrounds, perhaps due to different baseline responses in the F1 WT mice [see Logue et al., 1997; Owen, Logue, Rasmussen, & Wehner, 1997 for presentation of differences among F1 lines], indicating that one could select backgrounds with more robust differences to improve experimentation.
Many of these additional behavioral phenotypes in the mouse models are consistent with behaviors observed in individuals with FXS, such as ID (approximately 87%), repetitive or stereotypic behaviors (85–100%), and abnormal social behavior (61–73%) [Hagerman, 2002]. The finding of a contextual fear phenotype in the B6CD background is of particular interest given the difficulty in detecting learning and memory phenotypes in the Fmr1KO mouse model [see discussion in Spencer et al., 2006]. We previously observed a contextual fear impairment and a decreased habituation phenotype in Fmr1/Fxr2 double KO mice [Spencer et al., 2006], which suggested that Fxr2 was compensating for the loss of Fmr1 in these behavioral responses. The results of the present study suggest that the CD-1 and FVB backgrounds contribute modifier genes that over-ride compensatory mechanisms present in the B6 background. A social anxiety-like phenotype was observed previously in the congenic B6 background under special experimental conditions [Spencer et al., 2005]; however, the phenotype appears more pronounced on the B6S1 background. Differences in exploratory activity might be involved as mice on the B6S1 background are much less active than B6 mice.
Autism-Related Behaviors in Fmr1KO Mice
The core features of autism are impaired social interaction, impaired social communication, and the presence of perseverative or repetitive behaviors or restrictive interests. Considerable debate exists in defining how these human behaviors should be represented in a valid mouse model of autism. Many researchers suggest that the mouse is a good model if they exhibit only one of the core behaviors associated with autism. However, while almost all males with FXS exhibit at least one of these core symptoms of autism, only 21–50% meet the full diagnostic criteria for autism [Moss & Howlin, 2009]. Thus, the question becomes whether all valid mouse behavioral models of FXS should be considered good mouse models of autism.
Of all the core autism-related behavioral domains, social interaction has been most closely looked at in Fmr1KO mice. Previous reports have described the presence of abnormal social behavior in the Fmr1KO mice on a congenic C57BL/6 background [Mines, Yuskaitis, King, Beurel, & Jope, 2010; Mineur, Huynh, & Crusio, 2006; Spencer et al., 2005, 2008], a congenic FVB background [Liu & Smith, 2009; Moy et al., 2009], and on a B6 × FVB hybrid background [McNaughton et al., 2008]. The majority of these studies suggested elevated levels of social anxiety in the presence of unfamiliar partners [Liu & Smith, 2009; McNaughton et al., 2008; Mines et al., 2010; Moy et al., 2009; Spencer et al., 2005]. When presented with a very familiar partner, however, Fmr1KO mice respond with increased social approach, reflected mostly by increased sniffing of the partner [Spencer et al., 2005, 2008]. Importantly, this behavior was corrected by transgenic expression of FMRP in the knockout mouse, indicating that FMRP levels contribute to this social phenotype [Spencer et al., 2008]. This finding was also replicated in this study as the Fmr1KO mice on the congenic B6 background exhibited increased active social behavior in the social interaction test. Although consistent with a FXS-like phenotype [see discussion in Spencer et al., 2005], the presence of increased social approach with familiar partners is not necessarily consistent with an autism-like phenotype. The increased investigative social behavior of Fmr1KO mice on the congenic B6 and B6F backgrounds is perhaps related to their increased exploratory activity since Fmr1KO mice on these backgrounds were hyperactive in the open field. Interestingly, although B6D2-Fmr1KO mice also explored the open field more actively than their WT littermates, they showed decreased overall active social behavior with no difference in investigative social behavior.
The second core feature of autism is impaired social communication. In mice, social communication has routinely been assessed by measuring USV, especially isolation-induced vocalization in neonatal mice [Branchi et al., 2001; Scattoni et al., 2009]. To our knowledge, the experiment evaluating pup USVs in the B6D2-Fmr1 mice is the first published study of USVs in any Fmr1 mouse model. As a first attempt to assess social communication in these mice, this experiment was performed only in the background (B6D2) that was most promising as a potential model for autistic-like traits. Many mouse models of autism-related neurodevelopmental disorders have been analyzed in this manner and alterations in the total number of “calls” at particular ages or the developmental trajectory have been observed in the majority of these models [reviewed in Moy & Nadler, 2008]. It is logical that most investigators would have predicted a priori that pups would vocalize less in response to isolation since humans with autism spectrum disorders (ASD) usually are less communicative [Crawley, 2004]. However, while some of the genetic mouse models associated with autism, such as neuroligin-3 [Radyushkin et al., 2009] and neuroligin-4 [Jamain et al., 2008] do show decreased USVs, other genetic models such as the Mecp2 null Rett syndrome mouse [Picker, Yang, Ricceri, & Berger-Sweeney, 2006] and the 15q11–13 duplication mouse [Nakatani et al., 2009] exhibited increased calling during the neonatal period. The inconsistency of these results together with inconsistent reports regarding the amount of crying in human infants who are later diagnosed with autism, has led some to question whether isolation-induced vocalizations in rodent pups are relevant to social communication in autism.
Infant cries are the earliest form of social communication, intended to elicit caregiver attention. Similar to human infants, the calls of neonatal mice elicit retrieval behavior in the parents [Ehret, 2005], thus there is face validity for studying this behavior as a model for human infant social communication. The natural process of call followed by response can be altered by abnormal calling by the infant or by abnormal response of the parent. Furthermore, maternal response can feedback to modulate infant calling; in mice, there is a correlation between maternal responsiveness and the number of isolation-induced pup USVs such that pups that receive less maternal care call more than pups that receive more maternal care [D'Amato, Scalera, Sarli, & Moles, 2005]. In the case of the B6D2-Fmr1KO mice, there was an increased number of calls as compared to their WT littermates, a finding which is similar to other genetic mouse models of autism mentioned above but counter to what may have been expected a priori for an autism-related phenotype. We suggest that the increased calling in the B6D2-Fmr1KO neonatal mice and possibly other mouse models of autism could be a reflection of less maternal care, which could reflect the quality of the pups' communication. The quality of human infant cries could be an important early characteristic of autism. Analysis of retrospective videotapes of infants with autism has revealed that the acoustic properties of their cries are different from the cries of typically developing infants and their cries are considered more aversive and distressing to adult caregivers [Esposito & Venuti, 2010]. Thus, further studies in the B6D2-Fmr1KO mouse model will be important to analyze the detailed characteristics of the vocalization patterns, such as performed for BTBR mice [Scattoni, Gandhy, Ricceri, & Crawley, 2008], and also to assess whether there are any differences in the maternal responses to Fmr1KO compared to WT offspring. Future studies of vocalizations during adult social interaction will also be informative, since communication difficulties persist throughout life for individuals with autism.
Perseveration, resistance to change, or stereotypic/ repetitive behavior is another core feature of autism. Children with autism often show both repetitive motor mannerisms and cognitive rigidity [Carcani-Rathwell, Rabe-Hasketh, & Santosh, 2006]. In mice, these behaviors have been modeled in two different ways. One approach has been to look at reversal learning paradigms such as Morris water maze or appetitive t-maze [Crawley, 2007; Moy et al., 2007, 2008a, b]. In early reports about Fmr1KO mice on a mixed genetic background, impairments in reversal learning were noted in the Morris water maze assay [Bakker et al., 1994; D'Hooge et al., 1997; Kooy et al., 1996]. Some investigators suggested that this is an example of resistance to change that supports the Fmr1KO mouse model as a model for autism [Bernardet & Crusio, 2006; Moy, Nadler, Magnuson, & Crawley, 2006]. However, it is difficult in the Morris water maze to ensure that the mice are at equivalent levels in the initial test before initiating the reversal trials. This is especially a concern when there are reports of learning tasks in which Fmr1KO mice performed better than their WT littermates [Fisch, Hao, Bakker, & Oostra, 1999; Frankland et al., 2004]. Mice that are overtrained relative to their controls would be expected to reverse more slowly. Furthermore, swim latencies were used as the primary evidence for impaired reversal, and swim latencies can be influenced by many factors that are not specific to learning. For example, Fmr1KO mice have been reported to have mild motor learning impairments [Peier et al., 2000] and in one water maze experiment it was noted that the Fmr1KO mice did not swim as fast in later trials [D'Hooge et al., 1997]. Thus, previous reports of impaired reversal learning in the Morris water maze are not particularly convincing with respect to a perseverative behavioral phenotype in Fmr1KO mice. We suggest that clearer conclusions would be obtained by using assays such as a t-maze task that has defined indicators of learning such as trials to criterion [Crawley, 2007; Moy et al., 2007, 2008a, b]. One assay we recommend is a simple perseveration assay in a water t-maze in which mice are trained to go either left or right to an escape platform. This task takes advantage of a behavior in which mice naturally perseverate. For each individual mouse, after achieving nine out of ten correct trials for 2 consecutive days, the platform is moved to the opposite location and the number of trials to reach a criterion of nine out of ten correct trials in the new location is determined. We have performed this experiment with Fmr1KO mice on the congenic B6 background and did not find evidence of perseveration. No difference was observed between Fmr1KO and WT littermates in the number of trials to criterion for the initial task to find the escape platform in one arm nor for the reversal task in which the escape platform was moved to the other arm nor for a second reversal trial [Spencer, unpublished results]. We believe that behavioral tasks such as this may be more appropriate to test for perseverative or inflexible learning behavior in mice.
Another approach to determining if mice are resistant to change is to look directly at the reaction of mice when confronted with a reversal learning task. Moon et al.  looked at the behaviors of Fmr1KO mice on a B6 × FVB hybrid background as they were confronted with changing task demands during olfactory discrimination learning. Although there was no difference in measures of learning during acquisition and reversal, Fmr1KO mice showed increased activity and wall-climbing when contingencies were changed in the reversal learning task and also in trials that followed an error. In another study using the five-choice visual attention task, these authors found that Fmr1KO mice were more impulsive (making more premature responses), had difficulty maintaining attention, and exhibited increased arousal (more wall-climbing) after task changes or errors [Moon et al., 2006]. These studies offer compelling evidence that Fmr1KO mice exhibit heightened emotional responses when confronted with changes during learning paradigms.
Repetitive or stereotypic behaviors in mice can also be studied by observing the mice in their home-cage or in novel environments. Thus, it is a challenge to detect repetitive behavior in otherwise normal-looking mice. Indeed, to our knowledge, there have been no published reports of repetitive behavior in Fmr1KO mice until this study. Our laboratory recently reported that the marble bury assay reflects repetitive digging behavior [Thomas et al., 2009]. Although marble bury was originally identified as a test for anxiety-like responses [Broekkamp, Rijk, Joly-Gelouin, & Lloyd, 1986; Njung'e & Handley, 1991], this activity did not correlate with anxiety-related measures in the open field test or with light–dark, but instead with digging behavior and the stereotypy measure in the open field test [Thomas et al., 2009]. Thus in the present study, we included both stereotypy measures from the open field test and the marble bury test in our comprehensive test battery. Holeboard exploration was recently recommended as a test for perserverative or repetitive behavior [Moy et al., 2008a, b], and we have observed that Fmr1KO mice on the congenic B6 background show more repetitive behavior than WT littermates in this assay [Spencer, unpublished results]. Thus, we think the holeboard assay will be useful in further screening and characterizing repetitive behavior in Fmr1KO mice.