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The loss of fragile X mental retardation (FMR1) gene function causes fragile X syndrome (FXS), a common mental retardation syndrome. Anxiety and abnormal social behaviors are prominent features of FXS in humans. To better understand the role of FMR1 in these behaviors, we analyzed anxiety-related and social behaviors in Fmr1 knockout (KO) mice. In the mirrored chamber test, Fmr1 KO mice showed greater aversion to the central mirrored chamber than wild-type (WT) littermates, suggesting increased anxiety-like responses to reflected images of mice. Fmr1 KO mice exhibited abnormal social interactions in a tube test of social dominance, winning fewer matches than WT littermates. In a partition test, Fmr1 KO mice had normal levels of social interest and social recognition. However, during direct interaction tests, Fmr1 KO mice showed significant increases in sniffing behaviors. We further tested the influence of environmental familiarity on the social responses of Fmr1 KO mice to unfamiliar partners. In unfamiliar partitioned cages, Fmr1 KO mice did not differ from WT mice in investigation of unfamiliar partners. However, in familiar partitioned cages, Fmr1 KO mice showed less investigation of a newly introduced partner during the first 5 min and more investigation during the last 5 min of a 20-min partition test, behaviors consistent with initial social anxiety followed by enhanced social investigation. Our findings indicate that the loss of Fmr1 gene function results in altered anxiety and social behavior in mice and demonstrate that the Fmr1 KO mouse is a relevant animal model for the abnormal social responses seen in FXS.
Fragile X syndrome (FXS) is the most common inherited cause of human mental retardation with an estimated prevalence of one in 4000 males (Turner et al. 1996). Physical features of the disorder include mild facial anomalies (long narrow face, prominent jaw and protruding ears), hyperextensible joints, flat feet and, in postpubescent males, macroorchidism. Behavioral features of the disorder include cognitive impairment, hyperactivity, attention deficits, sensory hypersensitivity and hyperarousal, social isolation and anxiety and autistic-like behaviors such as gaze avoidance, perseverative language and hand stereotypies (Hagerman 2002).
Fragile X syndrome results from the loss of expression of the fragile X mental retardation (FMR1) gene located on the X chromosome. In most cases, expansion of a CGG repeat region in the 5′ untranslated region of the FMR1 gene to over 230 copies leads to hypermethylation and subsequent transcriptional inactivation (O'Donnell & Warren 2002). FMR1 mRNA is expressed at high levels during early development and in the brain and testis in adults (Devys et al. 1993; Hinds et al. 1993). In brain, the protein product of FMR1 (FMRP) is highly expressed in the cytoplasm and dendrites of neurons (Feng et al. 1997). FMRP is an RNA-binding protein that is thought to regulate the intracellular transportation and translation of mRNAs important for synaptic function (Jin & Warren 2000). Several RNA targets related to synaptic function have been recently identified (Brown et al. 2001; Chen et al. 2003; Darnell et al. 2001; Miyashiro et al. 2003), supporting the hypothesis that FMRP plays an important role in synaptic plasticity.
Fmr1 knockout (KO) mice, which have undetectable levels of Fmr1 mRNA and FMRP (Bakker et al. 1994; but see Yan et al. 2004), exhibit several of the physical and behavioral characteristics of the human syndrome. In addition to having macroorchidism, these mice show hyperactivity and have a mild spatial learning impairment in the Morris water maze (Bakker et al. 1994). Both macroorchidism and hyperactivity were over-corrected in FMR1 yeast artificial chromosome (YAC) transgenic mice carrying the human FMR1 gene, indicating a role for FMR1 in these processes (Peier et al. 2000). Contrary to expectations based on the human phenotype, Fmr1 KO mice showed decreased anxiety-like responses in open-field and light/dark tests. Nonetheless, this phenotype was over-corrected in the FMR1 YAC transgenic mice, indicating a role for the Fmr1 gene in anxiety-related behaviors (Peier et al. 2000). The unexpected nature of the results, however, suggests the need to assess anxiety-like behavior in additional rodent tests for anxiety. In addition, it is important to note that the anxiety responses observed in FXS are predominant in social settings.
Abnormal social behaviors, especially shyness and social withdrawal, can be a prominent feature of FXS. Social anxiety is often observed in high-functioning males with only mild or moderate deficits in FMRP and is frequently the presenting problem in females with FXS (Hagerman 2002). A negative correlation between FMRP production and severity of behavioral problems such as social withdrawal or anxious/depressed behavior in girls with FXS was recently reported (Hessl et al. 2001). The association between FMRP levels and social behavior suggests that the FMR1 gene plays a role in mediating some aspects of social behavior.
Fmr1 KO and transgenic mouse models of FXS (Bakker et al. 1994; Peier et al. 2000) would be useful to determine more specifically whether the FMR1 gene plays a role in social behavior. However, despite extensive characterization of behavior in these mice, very little has been reported about their social behavior (Mineur et al. 2002). Therefore, we designed a series of experiments to further characterize the anxiety-related responses in male Fmr1 KO mice and to begin to determine whether Fmr1 KO mice display abnormal social interactions. Our findings indicate that Fmr1 KO mice display (a) a moderate increase in anxiety-related response in a mirrored chamber test and (b) abnormal social behavior that is influenced by experience.