After being habituated with two objects for two consecutive days in the arena, Fmr1 KO mice failed to recognize the novel object introduced by replacing one of the familiar objects on the third day of the test.

In contrast, WT and dKO mice were able to distinguish the new object with high fidelity when tested under same conditions. Interestingly, S6K1 KO mice were impaired in their ability to discriminate between familiar and novel objects. These findings indicate that removal of S6K1 in Fmr1 KO mice can restore appropriate novel object recognition memory. We also examined whether the four genotypes showed differences in their ability to learn and recall a task and NVP-BGJ398 price GSI-IX datasheet their flexibility in modifying their responses when the task was changed using a water-based Y-maze test. All four genotypes responded to training and learned the test with comparable efficiency on the first day (Figure 6D). Memory

recall for the arm location of the hidden platform also was robust on the day after training for all four genotypes, indicating that there were no deficits in long-term memory (Figure 6D). When the platform location was reversed, Fmr1 KO mice displayed impairments in reversal learning, requiring an additional 15 trials to meet criterion as compared to WT, S6K1 KO, and dKO mice ( Figures 6D and 6E). These findings suggest that upregulation of S6K1 signaling plays a key role in behavioral inflexibility in FXS model mice. Fmr1 KO mice also have been reported to show increased ambulatory behavior in the open field test ( Spencer et al., 2005), which we reproduced in our behavioral cohort ( Figures S6A and S6B). In contrast, S6K1 KO littermates exhibited significantly decreased exploration and preferred the peripheral

areas versus the center of the arena. Interestingly, dKO mice displayed open field exploration indistinguishable and from the Fmr1 KO mice, indicating that the deletion of S6K1 did not correct increased ambulatory behavior in FXS model mice. In addition, marble-burying behavior, a phenotype that has been used to model obsessive-compulsive behavior in mice, was enhanced in Fmr1 KO mice compared to WT littermates. We found that Fmr1 KO, S6K1 KO, and dKO mice buried a higher number of marbles compared to WT littermates ( Figure S6C). These findings indicate that the enhanced repetitive behavior of Fmr1 KO mice is not rescued by the abrogation of S6K1. FXS patients display neuroendocrine dysfunction that is reflected in a generalized increase in total body weight, macrocephaly, and enhanced stature (Penagarikano et al., 2007). Another frequently associated feature is macro-orchidism (enlarged testicles), first observed in male patients immediately after attaining puberty (Hagerman et al., 1983).