Female CGG knock-in mice modeling the fragile X premutation are impaired on a skilled forelimb reaching task

https://doi.org/10.1016/j.nlm.2011.12.006Get rights and content

Abstract

The fragile X premutation is a tandem CGG trinucleotide repeat expansion in the fragile X mental retardation 1 (FMR1) gene between 55 and 200 repeats in length. A CGG knock-in (CGG KI) mouse has been developed that models the neuropathology and cognitive deficits reported in fragile X premutation carriers. Previous studies have demonstrated that CGG KI mice have spatiotemporal information processing deficits and impaired visuomotor function that worsen with increasing CGG repeat length. Since skilled forelimb reaching requires integration of information from the visual and motor systems, skilled reaching performance could identify potential visuomotor dysfunction in CGG KI mice. To characterize motor deficits associated with the fragile X premutation, 6 month old female CGG KI mice heterozygous for trinucleotide repeats ranging from 70–200 CGG in length were tested for their ability to learn a skilled forelimb reaching task. The results demonstrate that female CGG KI mice show deficits for learning a skilled forelimb reaching task compared to wildtype littermates, and that these deficits worsen with increasing CGG repeat lengths.

Highlights

► CGG KI mice modeling the fragile X premutation show deficits for skilled forelimb reaching. ► CGG repeat length shows a negative association with skilled reaching performance. ► CGG KI mice show visuospatial and visuomotor function impairments.

Introduction

The fragile X mental retardation 1 (FMR1) gene is polymorphic for the length of a CGG trinucleotide repeat in the 5′ untranslated region (UTR). In the general population there are fewer than 45 CGG repeats in the FMR1 gene, while in the full mutation underlying fragile X syndrome (FXS) there are greater than 200 CGG repeats and the FMR1 gene is transcriptionally silenced. In the fragile X premutation there are between 55 and 200 CGG repeats and increased transcription of FMR1 mRNA (Garcia-Arocena & Hagerman, 2010).

To investigate the pathological and behavioral consequences of the fragile X premutation, a transgenic CGG knock-in (KI) mouse was developed in which the 5′ UTR containing 8 CGG repeats in the endogenous murine Fmr1 gene was replaced, via homologous recombination, with a human Nhel–Xhol fragment containing 98 CGG repeats (Willemsen et al., 2003). Behavioral analyses of these mice have demonstrated spatiotemporal processing deficits (Hunsaker et al., 2010, Hunsaker et al., 2009) and an early motor phenotype evaluated by a skilled ladder walking task that was interpreted as impaired visuomotor processing (Hunsaker et al., 2011).

To further characterize the nature of the motor performance deficits in CGG KI mice, female CGG KI mice heterozygous for the fragile X premutation were trained on a skilled reaching task based on work by Whishaw and colleagues (Farr & Whishaw, 2002), among others (Buitrago et al., 2004, Hermer-Vazquez et al., 2007, Kolb and Gibb, 2010, Tennant and Jones, 2009). Female mice heterozygous for the fragile X premutation were chosen for this study over male mice because the frequency of fragile X premutation is higher in females than males (1:113 females vs 1:250 in males; cf., Hagerman, 2008). Additionally, there have also been emerging reports of neurocognitive abnormalities in human females carrying the fragile X premutation and heterozygous female CGG KI mice (Goodrich-Hunsaker et al., 2011a, Goodrich-Hunsaker et al., 2011b, Goodrich-Hunsaker et al., 2011c, Hunsaker et al., 2010, Hunsaker et al., 2011, Lachiewicz et al., 2006). The importance of studying female premutation carriers and CGG KI mice is that the identification of subtle phenotypes in these less affected populations may inform research into the underlying mechanisms subserving the more profound phenotypes observed in males. A skilled forelimb reaching task was chosen as it has been shown that reaching in peripersonal space (i.e., space within reach of a limb) depends upon integration of visuospatial and motor information across widespread neural circuitry involving the basal ganglia, motor and posterior parietal cortices (Beloozerova and Sirota, 2003, Beurze et al., 2010, Kolb and Gibb, 2010, Redish and Touretzky, 1994, Simon, 2008), superior colliculus, and cerebellum (MacKinnon, Gross, & Bender, 1976). Each of these structures are affected to some degree in the carriers of the fragile X premutation and the CGG KI mouse (Adams et al., 2007, Hunsaker et al., 2009, Hunsaker et al., 2010, Hunsaker et al., 2011, Keri and Benedek, 2009, Keri and Benedek, 2010, Lachiewicz et al., 2006, van Dam et al., 2005, Wenzel et al., 2010, Willemsen et al., 2003). Furthermore, previous studies with rats using single pellet reaching tasks demonstrate a relationship between impaired motor control and onset/severity of neurological disease, particularly focusing on the deleterious effects of dopamine depletion on skilled reaching performance (cf., Vergara-Aragon, Gonzalez, & Whishaw, 2003).

In the present study, CGG KI mice were trained to extend their forelimb through an opening to grasp and retrieve a sucrose pellet. We demonstrated that heterozygous female CGG KI mice took between 1 and 2 days longer to reach asymptotic performance on the skilled forelimb reaching task and that the CGG KI mice failed to reach the same asymptotic level of performance as wildtype littermates (i.e., never reached with the same level of success). Furthermore, a CGG repeat length dosage effect was evident within the CGG KI mice: such that mice with longer CGG repeat lengths (136–200) had a lower percentage of successful reaches than CGG KI mice with more intermediate length CGG repeats (70–116), and took longer to acquire the task. Furthermore, Within the CGG KI mice there was a negative association between increasing CGG repeats and performance on the skilled reaching task.

Section snippets

Animals

Twelve female CGG KI mice heterozygous for the fragile X premutation at 6 months of age and 6 female wildtype mice of the same age were used as subjects for this task. All wildtype mice were littermates with CGG KI mice included in the study. All CGG KI mice were bred onto a congenic C57BL/6 J background over greater than 12 generations from founder mice on a mixed FVB/N x C57BL/6 J background (Willemsen et al., 2003). Mice were housed in same sex, mixed genotype groups with three or four mice per

Pretraining

Mice were food deprived to 90–95% free feeding weight and given access to 20 mg banana flavored sucrose pellets in their home cage to habituate to the food reward for 2 days. Throughout experimentation mice were provided sufficient food to maintain 95% free feeding weight 30 min after experimentation each day.

Training

On days 3–5, mice were placed in the apparatus with sucrose pellets on the floor and in the open window within reach of the mouse’s tongue for 30 min and allowed to consume sucrose pellets. On

Results

For all mice, data were grouped by CGG repeat length (wildtype, Low CGG, High CGG) and analyzed across days of training using repeated measures ANCOVA with percent successful reaches as the dependent variable, CGG repeat group as the grouping factor, and day of training as a repeated within-subjects factor with total number of attempted reaches during each session as a covariate. All p values have been FDR adjusted per Benjamini et al. (2001). There was a main effect of CGG repeat length group (

Discussion

The current experimental results provide evidence for impaired reaching abilities in CGG KI mice that are modulated by CGG repeat length in female CGG KI mice modeling the fragile X premutation. These data model subclinical motor features present in female carriers of the fragile X premutation as young as 36 years of age that show no features of FXTAS (Narcisa et al., 2011). These female premutation carriers demonstrated impaired finger tapping used as a measure of manual coordination in both

Acknowledgments

The authors wish to thank Lara E. Cardy, Gian G. Greenberg, Lindsey C. Curley, and Ramona E. von Leden for assistance with preliminary versions of this experiment, and Binh T. Ta for assistance with mouse genotyping.

Funding: This work was supported by National Institute of Health (NIH) Grants, NINDS RL1 NS062411 and TL1 DA024854. This work was also made possible by a Roadmap Initiative Grant (UL1 DE019583) from the National Institute of Dental and Craniofacial Research (NIDCR) in support of the

References (52)

  • S. Keri et al.

    Visual pathway deficit in female fragile X premutation carriers: A potential endophenotype

    Brain and Cognition

    (2009)
  • S. Keri et al.

    The perception of biological and mechanical motion in female fragile X premutation carriers

    Brain and Cognition

    (2010)
  • B. Kolb et al.

    Tactile stimulation after frontal or parietal cortical injury in infant rats facilitates functional recovery and produces synaptic changes in adjacent cortex

    Behavioural Brain Research

    (2010)
  • K.G. Melvin et al.

    Pallidal deep brain stimulation and L-dopa do not improve qualitative aspects of skilled reaching in Parkinson’s disease

    Behavioural Brain Research

    (2005)
  • P.K. Mutha et al.

    Coordination deficits in ideomotor apraxia during visually targeted reaching reflect impaired visuomotor transformations

    Neuropsychologia

    (2010)
  • J.M. Sprague et al.

    The role of the superior colliculus in visually guided behavior

    Experimental Neurology

    (1965)
  • K.A. Tennant et al.

    Sensorimotor behavioral effects of endothelin-1 induced small cortical infarcts in C57BL/6 mice

    Journal of Neuroscience Methods

    (2009)
  • D. van Dam et al.

    Cognitive decline, neuromotor and behavioural disturbances in a mouse model for fragile-X-associated tremor/ataxia syndrome (FXTAS)

    Behavioural Brain Research

    (2005)
  • H. Wang et al.

    FMRP acts as a key messenger for dopamine modulation in the forebrain

    Neuron

    (2008)
  • H.J. Wenzel et al.

    Ubiquitin-positive intranuclear inclusions in neuronal and glial cells in a mouse model of the fragile X premutation

    Brain Research

    (2010)
  • I.Q. Whishaw et al.

    Neurotoxic lesions of the caudate-putamen on a reaching for food task in the rat: Acute sensorimotor neglect and chronic qualitative motor impairment follow lateral lesions and improved success follows medial lesions

    Neuroscience

    (2007)
  • J.S. Adams et al.

    Volumetric brain changes in females with fragile X-associated tremor/ataxia syndrome (FXTAS)

    Neurology

    (2007)
  • P. Barneoud et al.

    Evaluation of simple and complex sensorimotor behaviours in rats with a partial lesion of the dopaminergic nigrostriatal system

    European Journal of Neuroscience

    (2000)
  • P.M. Bays et al.

    Integration of goal- and stimulus-created visual signals revealed by damage to human parietal cortex

    Journal of Neuroscience

    (2010)
  • I.N. Beloozerova et al.

    Integration of motor and visual information in the parietal area 5 during locomotion

    Journal of Neurophysiology

    (2003)
  • R.F. Berman et al.

    Mouse models of fragile X-associated tremor ataxia

    Journal of Investigative Medicine

    (2009)
  • Cited by (27)

    • Gene × environment interactions in autism spectrum disorders

      2023, Current Topics in Developmental Biology
    • Mouse Models of the Fragile X Tremor/Ataxia Syndrome (FXTAS) and the Fragile X Premutation

      2015, Movement Disorders: Genetics and Models: Second Edition
    • Effects of acute sleep deprivation on motor and reversal learning in mice

      2014, Neurobiology of Learning and Memory
      Citation Excerpt :

      Prior work assessing the inactivity of C57BL/6 mice for >40 s showed 92% agreement with sleep/wake states evaluated by polysomnography (Pack et al., 2007). We designed a skilled reaching chamber for mice as previously described (Diep et al., 2012; Farr & Whishaw, 2002). The chamber is a rectangular box constructed of clear acrylic measuring 23 cm long × 6.25 cm wide × 25 cm tall.

    • Reduced activity-dependent protein levels in a mouse model of the fragile X premutation

      2014, Neurobiology of Learning and Memory
      Citation Excerpt :

      As it has been shown that there is an increase in dendritic complexity in the motor cortex of mice after training on a skilled forelimb reaching task (Greenough, Larson, & Withers, 1985; Xu et al., 2009), it is possible that elevations in Fmrp levels in response to neuronal activity result in, or are the result of increased synaptic density in the cortex. The present experiment was designed to examine Fmrp levels in response to training on a skilled forelimb reaching task in mice, based on work by Whishaw and colleagues (Diep et al., 2012; Farr & Whishaw, 2002) to determine whether there is an activity-dependent increase in Fmrp levels in the motor cortex of mice. As skilled reaching is the product of several discrete movements, neuronal plasticity may link areas responsible for learning the fluid motion necessary for successful reaching (Whishaw, Whishaw, & Gorny, 2008).

    View all citing articles on Scopus
    View full text