1. Background Fragile X Syndrome is an inherited form of mental retardation that affects 1 in 4000 human males. One-third of those with Fragile X also have autism, often leading to severe social deficits. Mice produce ultrasonic vocalizations (USVs) that range from 30 to 110 kHz in frequency (Figure 1). These USVs are often associated with social activity, including mating. Prior studies have suggested that higher frequency vocalizations (towards 70kHz) show positive affect while lower pitch vocalizations (towards 20 kHz) and audible squeaks show negative affect. Prior social test results for Fragile X (FX) knockout type 1 (ko1) mice have shown reduced social activity in some tests (e.g. with ovariectomized females; Mineur et. al., 2005), but in other studies FX ko1 male mice were at least as likely to socially interact as wild-type (wt) mice (Spencer et. al, 2005; Spencer et. al., 2008). Since both mental retardation and autism are known to affect human speech, an examination was made here of USV production in mice with alterations in Fragile X (Fmr1) gene expression. Mouse models of this disease have been created by making an insertional mutation in the Fmr1 gene’s exon 5 (“ko1”) or by knocking out the Fmr1 promoter (“ko2”; Figure 2). This leads to little (ko1) or no (ko2) Fragile X Mental Retardation Protein (FMRP) being produced. The present experiments also studied the effects of one and two copies of a human FMR1 transgene expressed in place of, or in addition to, mouse Fmr1. We previously showed that human FMR1 transgenes were expressed at the same protein and RNA levels as in the mouse, and except for developing sperm, in the same tissue patterns. RNA expression of Fmr1, Foxp2 (a speech- related gene in humans), and many other genes were further assessed here using QPCR and microarrays (Figure 3). Characterization of Social Behaviors Reveals a Novel Ultrasonic Vocalization Deficit in Fragile X Syndrome Mouse Models of Mental Retardation Ben R. Bauchwitz and Robert P. Bauchwitz, Amerandus Research, BNY Mellon Center, 1735 Market St., Suite 3750, Philadelphia, PA 19103 USA Figure 10. Interest in novel mice as assessed by face to cage contacts Social novelty behavior was analyzed in adult FVB/NJ mice with the genotypes indicated (wt, Fragile X ko1, or Fragile X ko2). The number of investigations of cages showed that all mice seemed to prefer an occupied cage to an empty cage; however, this effect was only statistically significant in ko1 and ko2 mice. Wt mice, on the other hand, showed a significant preference for a novel mouse during the second period of the test, while the ko1 and ko2 mice approached both of the cages equally. F(11, 76) = 2.787, p =.0043 (**). Significant differences between pairwise comparisons as assessed by Tukey-Kramer post hoc analysis were as indicated in the chart. Figure 11. Social Transmission of Food Preference (STFP)-like oral-oral contacts between male mice does not show a Fragile X Social Deficit Adult male FVB/NJ mice with the FX genotypes indicated (wt, ko1, or ko2) were observed as they interacted socially with male mice in cages in the open field. STFP-like contacts produced by these mice were considered to be evidence of social interaction. Wt mice produced equal contacts with a cage containing a novel mouse as they did with a cage containing a mouse who was not novel. Ko1 and ko2 mice appeared to produce more contacts with a novel mouse, although this effect was not statistically significant. F(8, 57) = 3.471, p =.0025. Dunn’s multiple comparison tests indicated that significant differences were associated with the ko1 and ko2 L/O conditions, i.e. that the Fragile X mice initially showed an elevated preference for the occupied cage, but then habituated to both cages. Power tests indicated that more mice would have to be tested to assess statistical differences for L and R conditions based on the means and standard deviations. Duration of USV was studied in adult male FVB mice with a silent ko2 female in the home cage test. Ko1 USV duration was significantly lower than wt (p =.0011, **)and transgenic “Tg+” mice (p <.001, ***). Ko2 USV duration was also significantly lower than wt (p =.0128, *) and Tg+ (p =.0010, ***). For the effects of Tg dose, see Figure 12. Figure 2. Fmr1 mutations Figure 4. USV recording equipment and pup test platform (Left) Pettersson D240x ultrasonic microphone and time expansion digitizer on an elevated platform used to record pup vocalizations. (Right) A 14 day old pup on the recording platform. (The pup is just before the developmental stage when its eyes will open.) Figure 5. Open field with SocT cages (Left) Overview of the social test (SocT) arena. (Right) An oral-oral Social Transmission of Food Preference-like (STFP-like) contact made through the cage mesh. A SSESSING SOCIAL V OCALIZATION BEHAVIOR HYB (C57 x FVB) wt mice vocalized for significantly shorter periods of time than FVB wt mice (p <.001, **) C57 wt mice almost never vocalized under the conditions of these home cage tests; this was a very significant effect: C57 wt vs. HYB wt (p <.001, ***), and C57 wt vs FVB wt (p <.001, ***). This suggests that C57 inbred mice have a dose-dependent USV stimulatory allele. Although HYB Tg+ mice showed elevated average USV duration, this result was not statistically significant. Figure 8. Male vs. male (open field) USV duration Discussion: Social Tests Wt mice tended to show fewer cage investigations than FX ko1 and ko2 mice in all categories except for when a novel mouse was present in the right cage, where they were approximately comparable to ko1 and ko2. The elevated contacts in ko1 and ko2 may be a function of altered motor behavior (Figures 6 and 7), or may indicate a lack of social inhibition (but see below). Wt mice investigated the second (novel) mouse significantly more than the original mouse, which is considered to be a normal behavior for mice. The ko1 and ko2 did not show a clear preference for the second mouse. However, when STFP-like oral-oral social contacts were examined, it was found that wt mice were not socially interacting more with the novel mouse during the second period. This suggests an interest in novelty but not necessarily social novelty. Consequently, STFP- like contacts may be a better measure of social interaction than simple investigation. Ko1 and ko2 mice both appeared very social in terms of STFP-like contacts. Furthermore, they had a larger reduction in their social contacts than did the wt mice, indicating that they were able to inhibit their behaviors at least as well as wt mice. Actual STFP was not impaired in ko2 mice (Rhian Gunn and Richard Brown, personal communications). Therefore, while FX mice showed strong deficits in terms of their ability to produce USVs, they were not obviously less social than wt mice. Consequently, although FX mice may have vocal and motor deficits consistent with Fragile X Syndrome in people, it is not clear that their responses to social novelty are consistent with the social withdrawal characteristic of autism. Figure 7. Total track length Figure 6. Center square entries Discussion: Ultrasonic Vocalization USVs were recorded and analyzed as a measure of social interaction. It was found that adult, but not postnatal day 10-14, Fragile X ko mice displayed a pronounced deficit in total duration of USVs compared to wt mice (Figures 8 and 9), even though other measures of social interaction did not show a difference between the two (Figures 10 and 11). FX ko mice who had at least one copy of a human FMR1 transgene were as vocal as wt mice, confirming the significance of Fmr1 on vocalization (Figure 9). Additional copies of human FMR1 or mouse Fmr1 did not further increase the amount of USV, suggesting a ceiling effect (Figure 12). It was also discovered that there was an additional genetic effect on USV duration that depended on the inbred strain background of the mice (Figure 13). Prior studies had shown that seizures in FX mice were much lower in those with a C57 parent compared to those with an FVB/NJ (FVB) parent (Yan et. al., 2005). Furthermore, such seizures could be suppressed by glycogen synthase kinase 3 (GSK3) inhibitors such as lithium. In this study it was found that lithium present in the diet increased USV of FX mice dramatically (Figure 14). None of the FX mice who underwent USV testing were found to have any reduction in mobility in a tail suspension test relative to wt mice, before or after lithium treatment, which suggests that lithium was not affecting mood. Instead, these data indicate that Fmr1 and lithium are acting in a common vocalization pathway, possibly involving GSK3 (Figure 15). Pairs of adult mice were allowed to freely roam in an open field. USV during social interactions were recorded. FEMALE mice are specified as such. Mice were also recorded while alone in the field. Male urine was present in the field to induce female vocalizations. (No ko2 FEMALE vocalizations when alone in the field have yet been observed.) Figure 1. Adult male and female ultrasonic vocalizations Figure 13. Strain effects on USV duration Figure 9. Male vs. female (home cage) USV durations Figure 14. FVB ko2 mice on a lithium diet for two weeks showed significantly elevated levels of ultrasonic vocalization (p =.0157, *). HYB ko2 mice almost doubled their average USV duration after a lithium diet, but the difference was not statistically significant. There was no notable change in the duration of HYB wt mice fed lithium, suggesting that lithium and USV strain alleles were not acting in the same pathway. Figure 15. The presence of one or two copies of FMR1 transgene, and zero or one functional copy of mouse Fmr1, raised USV to a maximum “ceiling” (i.e. seemingly dose-independent). A lithium diet also substantially raised the USV levels in FX mice, consistent with earlier studies showing the effect of Fmr1 through GSK3. In addition, USV were affected by the strain background. The strain effect was not substantially influenced by lithium. 2 nd Pathway (G2) Figure 3. RNA expression RNA and cDNA were prepared from whole brain. cDNA was used to assess Fmr1 and FoxP2 levels by quantitative PCR (QPCR) using TaqMan probes. Fmr1 was present at 161% of wt levels in Tg mice, 25% in ko1 mice, and 0% in ko2 mice. These were very large and reproducible differences in Fmr1 expression. Although Foxp2 was reduced by about 10% on average in ko1 and ko2 mice, and it was slightly elevated in the presence of the FMR1 transgene, it was not elevated in lithium treated, highly vocal ko2 mice; the differences among the various genotypes were not significant. Quantitative real-time PCR RNA expression levels Figure 12. USV duration with 1x vs 2-3x Fmr1 or FMR1 Methods Social interaction, ultrasonic vocalization, and motor behavior were assessed with an ultrasonic microphone and digitizer (Figure 4) in a 72 cm by 72 cm square enclosure (open field). Motor behavior was recorded for 5 minutes when male mice were 30 days old (Figures 6 and 7). Two cages were added to the open field enclosure (Figure 5, left) to measure social behavior of 2 month old male mice (primarily their preference for novelty; it was found that USV production was consistent up to 10 months of age). For the first 5 minute period, one cage had a mouse inside, while the other remained empty. The number of times the free mouse investigated (put its nose in close proximity to) each cage was recorded as well as the number of times a pair of mice performed a Social Transmission of Food Preference- like (oral-oral) contact (see Figure 5, right). After five minutes a mouse would be added to the second cage (leaving the first mouse in its cage) and the process would be repeated. In some experiments, the open field social test was followed by a tail suspension test, after which the mice were placed on a lithium diet for 10 days (as per Min et. al., 2009). Following lithium treatment, the open field social test and tail suspensions were repeated. Most of the above social tests involved only males, but females were also tested in the open field, after males, to elicit USV upon contact with male urine. It was found that ko2 females only very rarely made USVs; consequently, a “home cage” USV test was performed in which ko2 females were placed into the home cages of individual adult males of varying genotypes. Fmr1/FMR1 Dose Effect: Both one copy of human FMR1 and one copy of the endogenous mouse Fmr1 had a similar significant effect on USV duration in the FVB background; therefore, these data were combined (“1x”). No significant difference was seen based on the number of active Fmr1+FMR1 gene copies. Given the standard deviation of the data and the number of mice tested, 80% power (to avoid a false negative) would have required a mean USV duration difference of 33.79 sec. If additional FMRP has a biologic effect, it is not very substantial. Motor Behavior: Average CSE counts were consistent between mice of various genotypes (Figure 2), although a few Fragile X (FX) knockout 2 (ko2) mice tended to move in a tight circular motion, producing very high CSE counts (Figure 6). Ko1 mice showed significantly elevated average track length when compared with ko2 and wt mice (Figure 7). It was elevated track lengths (hyperactivity-like behavior) rather than extreme circling among ko1 mice which accounted for occasional elevated ko1 CSE counts