Elsevier

Neurobiology of Disease

Volume 36, Issue 2, November 2009, Pages 361-373
Neurobiology of Disease

Fmr1 knockout mice show reduced anxiety and alterations in neurogenesis that are specific to the ventral dentate gyrus

https://doi.org/10.1016/j.nbd.2009.08.001Get rights and content

Abstract

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the selective loss of the expression of the Fmr1 gene. Key symptoms in FXS include intellectual impairment and abnormal anxiety-related behaviors. Fmr1 knockout (KO) mice exhibited reduced anxiety on two behavioral tests as well as a blunted corticosterone response to acute stress. Spatial learning and memory was not impaired when tested with both the classic Morris water and Plus-shaped mazes. Adult hippocampal neurogenesis has been associated with spatial learning and memory and emotions such as anxiety and depression. The process of neurogenesis appears abnormal in young adult Fmr1 KO mice, with significantly fewer bromodeoxyuridine-positive cells surviving for at least 4 weeks in the ventral subregion of the dentate gyrus (DG), a hippocampal subregion more closely associated with emotion than the dorsal DG. Within this smaller pool of surviving cells, we observed a concomitant increase in the proportion of surviving cells that acquire a neuronal phenotype. We did not observe a clear difference in cell proliferation using both endogenous and exogenous markers. This work indicates that loss of Fmr1 expression can alter anxiety-related behaviors in mice as well as produce region-specific alterations in hippocampal adult neurogenesis.

Introduction

Fragile X syndrome (FXS) is the most common form of inherited mental retardation (Bagni and Greenough, 2005) affecting an estimated 1 in 4000 males and 1 in 8000 females (Turner et al., 1996). The loss of transcription of the Fmr1 gene on the fragile tip of the X chromosome leads to loss of production of the fragile X mental retardation protein (FMRP) (Fu, 1991, Verkerk, 1991). Normally, FMRP is highly expressed in principal cells in the brain, particularly in hippocampal neurons. It remains unclear if the loss of expression of this gene in the hippocampus results in the same behavioral abnormalities that are characteristic of FXS in humans (i.e., intellectual impairment and emotional dysfunction).

The cognitive and behavioral symptoms of FXS in humans include reduced IQ, increased anxiety, attention deficit/hyperactivity and autistic behaviors (Jacquemont et al., 2007). Curiously, mice lacking the Fmr1 gene show decreased anxiety (Mineur, 2002, Peier, 2000, Qin, 2002, Qin, 2005, Restivo, 2005, Yan, 2005) and show no robust deficits in learning when tested on the Morris water maze (MWM) 1994, D'Hooge, 1997, Gantois, 2001, Paradee, 1999, Peier, 2000, Qin, 2002. Based on these behavioral observations, we hypothesize that the neural circuitry associated with anxiety is altered by a loss of Fmr1 expression.

The ventral hippocampus is a brain region strongly linked to anxiety-related behaviors (Bannerman, 2004, Bannerman, 1999, Degroot and Treit, 2004, Engin and Treit, 2007), while the dorsal hippocampus appears to be subservient to spatial learning and memory (Engin and Treit, 2007, Maurer, 2005, Moser, 1993). There are notable differences in the afferent and efferent projections to other limbic structures along the longitudinal axis of the hippocampus that appear to support this hypothesis (Bannerman, 1999, Dolorfo and Amaral, 1998, Pikkarainen, 1999, Pitkanen, 2000, van Groen, 2003). In addition, the different subfields of the hippocampus (i.e., the dentate gyrus (DG), CA3 and CA1) have been associated with different aspects of spatial learning behavior. For example, lesions of the CA1 of the dorsal hippocampus can disrupt spatial memory (Duva et al., 1997), while the selective deletion of the NMDA receptor subunit NR1 in the DG impairs pattern separation in a contextual fear-conditioning task without impairing performance in the classic MWM (Goodrich-Hunsaker, 2008, Hunsaker, 2008, McHugh, 2007).

A relatively unique phenomenon observed across the longitudinal axis of the hippocampus, specifically within the DG, is adult neurogenesis: the production of new neurons into adulthood (for a review, see Christie and Cameron, 2006, Gage, 2002). Prevailing theories regarding the function of adult neurogenesis typically involve learning and memory (van Praag et al., 1999), but there has been recent speculation that adult neurogenesis may also play important roles in emotional disorders such as depression and anxiety (Sahay, 2007, Sahay and Hen, 2007, Santarelli, 2003, Wang, 2008). In addition, several laboratories have also suggested that the dorsal and ventral aspects of the hippocampus may play different roles in spatial and emotional learning (Quinn, 2005, Rogers and Kesner, 2006, Snyder, 2009). Furthermore, neurogenesis in these subregions may also be specifically involved in different forms of learning (Snyder et al., 2009). In the current study, we examined whether there were region-specific abnormalities in adult neurogenesis in the dorsal and ventral DG, and whether these abnormalities were correlated with altered performance in spatial memory-related and anxiety-related behavioral tasks in Fmr1 KO mice.

Section snippets

Animals

Sixty-nine C57BL/6 male Fmr1 knockout (KO) (n = 37) and Fmr1 wild-type (WT) (n = 32) littermate mice were generated by breeding WT male C57BL/6 (obtained from Jackson Laboratories) with female C57BL/6 mice heterozygous for the Fmr1 gene (Fmr1 KO mice were produced from founders originally provided by Dr. Mark Bear, MIT). The C57BL/6 background strain was employed because they perform well on spatial learning and memory tasks and show substantial neurogenesis (Holmes, 2002, van Praag, 1999). All

Anxiety in Fmr1 KO mice

Several specific behaviors in the open field were assessed, including the time spent in the center of the arena, the rate of movement and the number of defecations. In comparison to WT littermates, Fmr1 KO mice traveled significantly more in the center of the open field (WT: 10.13 ± 0.77%; Fmr1 KO: 13.65 ± 1.14%; t(13) = 2.43, p = 0.03; Fig. 2A), indicating they were less anxious about being exposed to the open environment. Similarly, the inverse of this measure, percentage of time spent in the

Discussion

We have observed selective abnormalities in a unique form of plasticity, neurogenesis, in young adult Fmr1 KO mice, in an animal model for FXS. The results are intriguing considering that neurogenesis has been associated with both learning and anxiety in rodents (Sahay, 2007, Sahay and Hen, 2007, Santarelli, 2003, Wang, 2008), domains clearly impaired in patients with FXS (Jacquemont et al., 2007). Our results show that Fmr1 KO mice (1) exhibit decreased anxiety on two separate behavioral

Acknowledgments

The authors thank Dr. M Bear for generously providing founder Fmr1 KO mice for our colony, Drs. YT Wang and B Chow for technical assistance, Lynn Huang and Lindsay Evangelista for assistance with genotyping and A Titterness for critical reading of earlier versions of the manuscript. This research was supported by grants from SRCFC, FXRFC, NSERC and CIHR to BRC. BDE holds a UBC MD-PhD Program/CIHR/VCHRI Award. JGM held a post-doctoral fellowship from the Foundation for Science and Technology

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