NeuroImage 53 (2010) 1023–1029 Contents lists available at ScienceDirect NeuroImage journal homepage: www.elsevier.com/locate/ynimg Anatomical phenotyping in a mouse model of fragile X syndrome with magnetic resonance imaging Jacob Ellegood a,⁎, Laura K. Pacey b, David R. Hampson b, Jason P. Lerch a, R. Mark Henkelman a a Mouse Imaging Centre, The Hospital for Sick Children, Toronto, Ontario, Canada b Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada article info abstract Article history: Fragile X Syndrome (FXS) is the most common single gene cause of inherited mental impairment, and Received 2 September 2009 cognitive deficits can range from simple learning disabilities to mental retardation. Human FXS is caused by a Revised 11 March 2010 loss of the Fragile X Mental Retardation Protein (FMRP). The fragile X knockout (FX KO) mouse also shows a Accepted 12 March 2010 loss of FMRP, as well as many of the physical and behavioural characteristics of human FXS. This work aims Available online 19 March 2010 to characterize the anatomical changes between the FX KO and a corresponding wild type mouse. Significant volume decreases were found in two regions within the deep cerebellar nuclei, namely the nucleus Keywords: fragile X syndrome interpositus and the fastigial nucleus, which may be caused by a loss of neurons as indicated by histological magnetic resonance imaging analysis. Well-known links between these nuclei and previously established behavioural and physical deep cerebellar nuclei characteristics of FXS are discussed. The loss of FMRP has a significant effect on these two nuclei, and future immunohistochemistry studies of FXS should evaluate the biochemical, physiological, and behavioral consequences of alterations in these key nuclei. © 2010 Elsevier Inc. All rights reserved. Introduction opment and/or maturation in the absence of FMRP (Grossman et al., 2006; Irwin et al., 2000; Koekkoek et al., 2005). The first brain The most common inherited form of mental retardation, Fragile X abnormality reported in a Magnetic Resonance Imaging (MRI) study Syndrome (FXS), affects approximately 1 in 4000 males and 1 in 8000 of human FXS carriers was underdevelopment of the cerebellar females (Crawford et al., 2001; Visootsak et al., 2005). FXS results vermis (the tissue connecting the right and left hemisphere of the from a trinucleotide repeat expansion mutation in the X-linked Fragile cerebellum) (Reiss et al., 1988). Abnormalities in the development of X Mental Retardation 1 (FMR1) gene. Methylation of the FMR1 the cerebellar vermis have been mentioned in a large number of promoter causes gene silencing and loss of expression of the protein studies, and are thought to be directly related to some of the product, fragile X mental retardation protein (FMRP). Individuals with behavioural deficits in fragile X syndrome, since the vermis has FXS exhibit a range of symptoms, including mild to moderate mental connections to the amygdala and hippocampus (Hessl et al., 2004). retardation, anxiety, hyperactivity, autistic-like behaviours and Other MRI studies have shown abnormalities in the fourth ventricle seizures (O'Donnell and Warren, 2002). Common physical character- (Mostofsky et al., 1998; Reiss et al., 1991), the hippocampus (Reiss et istics include a long narrow face, large protruding ears and enlarged al., 1994), and the amygdala (Reiss et al., 1995). Diffusion Tensor testicles (Visootsak et al., 2005). Imaging (DTI) has also found lower Fractional Anisotropy (FA) values The FMR1 knockout mouse (FX KO), first created in 1994 (Dutch- in fronto-striatal pathways and in parietal sensory motor tracts Belgium Fragile X Consortium, 1994), is the most common animal (Barnea-Goraly et al., 2003) indicating a loss of order/structure in model used to study FXS. The FX KO does not express FMRP and those pathways. displays many characteristics associated with FXS in humans ApreviousMRIstudyontheFXKOmousedidnotfind any including learning deficits, hyperactivity, seizures and enlarged volume changes in the brain (Kooy et al., 1999); however, the testicles (Musumeci et al., 2000; Oostra and Hoogeveen, 1997; analysis was performed on very large brain structures and the Pacey et al., 2009). Both humans and mice also exhibit an abundance resolution was quite low. The purpose of this paper was to of long, thin immature dendritic spines in the cerebral cortex, determineifthereareanydetectable anatomical and/or white hippocampus and cerebellum, suggesting abnormal synaptic devel- matter structural changes in the FX KO mouse model when comparedtothewildtype(WT)miceusinghigh-resolutionMRI and DTI. Furthermore, we assess changes to the craniofacial ⁎ Corresponding author. Mouse Imaging Centre (MICe), Hospital for Sick Children, structure with high-resolution micro-CT in order to look for skeletal Toronto Centre for Phenogenomics, 25 Orde Street, Toronto, Ontario, Canada M5T 3H7. Fax: +1 647 837 5832. changes due to the known facial dismorphism associated with E-mail address: [email protected] (J. Ellegood). human FXS. 1053-8119/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2010.03.038 1024 J. Ellegood et al. / NeuroImage 53 (2010) 1023–1029 Materials and methods create a population atlas representing the average anatomy or skeletal structure of the study sample. All registrations are performed using Specimen preparation the mni_autoreg tools (Collins et al., 1994). The result of the registration is to have all scans deformed into exact alignment with Fourteen, fixed C57BL6 mouse brains left in the skull (7 FX KO mice each other in an unbiased fashion. This allows for the analysis of the and 7 WT mice) were examined at age P30 (postnatal day 30). P30 deformations needed to take each individual mouse's anatomy into was chosen because this is the age where the FX KO mice were the this final atlas space, the goal being to model how the deformation most susceptible to the audiogenic seizures associated with the fields relate to genotype (Lerch et al., 2008; Nieman et al., 2006). The mouse model (Musumeci et al., 2000; Pacey et al., 2009). Initially the determinants of the deformation fields were then calculated as mice were anesthetized with ketamine/xylazine and intracardially measures of volume at each voxel. Significant volume and shape perfused with 30 mL of 0.1 M PBS containing 10 U/mL heparin changes can then be calculated by warping a pre-existing classified (Sigma) and 2 mM ProHance (a Gadolinium contrast agent) followed MRI atlas onto the population atlas (Dorr et al., 2008), which allows by 30 mL of ice cold 4% paraformaldehyde (PFA) containing 2 mM the volume of 62 segmented structures encompassing cortical lobes, ProHance (Spring et al., 2007). Perfusions were performed with a large white matter structures (i.e. corpus callosum), ventricles, Pharmacia minipump at a rate of approximately 100 mL/h. After cerebellum, brain stem, and olfactory bulbs (Dorr et al., 2008)tobe perfusion, mice were decapitated and the skin, lower jaw, ears, and assessed in all 14 brains. Since the mice at P30 are still developing, the the cartilaginous nose tip were removed. The brain and remaining 62 volumes were calculated as a percentage of total brain volume; skull structures were incubated in 4% PFA+2 mM ProHance over- furthermore, the voxel-wise analyses were also corrected for total night at 4 °C then transferred to 0.1 M PBS containing 2 mM ProHance brain volume. For the DTI data set, the transformations applied to the and 0.02% sodium azide for at least 7 days prior to MRI scanning. b=0 images during the registration process were applied to the calculated FA map for each brain, from which average FA maps were Magnetic resonance imaging calculated for both the FX KO and the WT mouse. The intensities (i.e. the FA values) of the FA maps were then compared. FA differences A 7.0 Tesla MRI scanner (Varian Inc., Palo Alto, CA) with a 6-cm inner were assessed on a voxel by voxel basis constrained by the white bore diameter insert gradient (max gradient 100 G/cm) was used for all matter structures in the classified atlas (Dorr et al., 2008), and by MRI scans. Three custom built solenoid coils were used to image three measuring the mean FA values of the same white matter regions brains in parallel (Bock et al., 2005). Parameters used for anatomical MRI assessed in the anatomical MRI measurements. Multiple scans were optimized for high efficiency and gray/white matter comparisons were controlled for using the False Discovery Rate contrast: T2-weighted, 3D fast spin echo, with a TR of 325 ms, and TEs (FDR) (Genovese et al., 2002). of 10 ms per echo for 6 echoes, with the centre of k-space being acquired on the 4th echo, four averages, field-of-view of 14×14×25 mm3 and Immunohistochemistry matrix size of 432×432×780 giving an image with 0.032 mm isotropic voxels. Total imaging time for this MRI sequence is ∼12 h (Henkelman The 14 fixed mouse brains were removed from the skull and et al., 2006). paraffin embedded. 32 consecutive coronal slices (thickness 5 µm) DTI scanning was performed with a 3D diffusion-weighted fast were taken from each of the 14 mice through the cerebellum which spin echo sequence. Parameters used in the DTI sequence: echo train included contributions from the dentate nucleus, fastigial nucleus, length of 6, TR of 325 ms, first TE 30 ms and a TE of 6 ms for the and nucleus interpositus. The vestibulocerebellar nucleus was not remaining 5 echoes, ten averages, field-of-view of 14×14×25 mm3 quantified. 8 slices each were stained with the following four and a matrix size of 120×120×214 yielding an image with 0.117 mm antibodies: rabbit anti-Glial Fibrillary Acidic Protein (GFAP, 1:100, isotropic voxels. One b=0 image (with minimal diffusion weighting) SIGNET), mouse anti-Neuronal Nuclei (NeuN, 1:100, Chemicon), and 6 high b-value images (b=1956 s/mm2) were acquired in six rabbit anti-Vesicular GABA Transporter (VGAT, 1:1000, Synaptic different directions [(1, 1, 0),(1, 0, 1),(0, 1, 1),(−1, 1, 0),(−1, 0, 1),(0, Systems), and mouse anti-Vesicular Glutamate Transporter 1 1, −1)] of (Gx, Gy, Gz).