Variability of brain anatomy for three common mouse strains
Introduction
Unlike outbred strains, inbred strains are strains with little or no genetic variation between individuals due to inbreeding over multiple generations. Inbred strains are popular in research because they exist in many genetically modified versions that address specific cellular and molecular processes. Neuroanatomical studies have shown that within-strain variance is smaller than between strain variance in inbred strains. More specifically, MRI-base segmentation of the brain of eleven inbred mouse strains showed that volume variation between strains was significantly larger than within strains for 23 out of 33 regions (Badea et al., 2009). This suggests that different strains form relatively well-separated tight clusters of anatomical variation.
Inbred strains have been described as more stable, uniform, and genetically better defined than outbred strains. It could thus be argued that the use of genetically identical mice should lead to more consistent results, thus requiring fewer individuals to demonstrate the significance of a specific effect However, in the literature there is still no clear consensus whether outbred strains are indeed more variable. Some studies have argued that inbred strains are more variable or just as variable as outbred strains in terms of body size and physiological characteristics (Biggers, J.D., et al., 1958, McLaren, A. and Michie, D., 1956). Some studies suggest that outbred strains are more variable (Jay, G.E., 1955, Stehelin, D., et al., 1976). The argument for larger variance in outbred strains usually involves the hypothesis that phenotypic variance is composed of genetic and environmental components (Chia et al., 2005). Following this logic, inbred strains should vary less due to the absence of genetic variation. Due to their genetic heterogeneity, however, outbred strains do allow one to draw conclusions that are less likely to reflect strain-specific idiosyncrasies (Miller et al., 1999). Therefore, outbred strains might constitute a better model of humans and their natural genetic variability. In addition, outbred mice are more efficient breeders and have large litter sizes (Aldinger et al., 2009). In light of these advantages of outbred mice, it is curious that so few studies have specifically investigated whether they do indeed come at the price of increased phenotypic variability (Vaickus et al., 2010). Prior studies suggest that certain anatomical features, such as mandible shape, are more variable (Stehelin et al., 1976). For neuroscientific studies it is of particular interest whether strain differences in anatomical variability are related to variability in brain function and behaviour.
Chen et al. (2006) report a measure of morphological variability averaged across the whole brain using ex-vivo MRI of two inbred (CD-1, 129S1) and one outbred strain (C57BL/6). Thus it is not possible to attribute increased variance to specific brain regions. Badea et al. (2009) report MRI-based volumes of individual anatomical regions, but only within inbred strains. Quantitative trait locus (QTL) mapping studies probe the link between phenotype and genotypic data (Kearsey, 1998). Sizes of the neocortex (Gaglani et al., 2009) and the barrel cortex (Jan et al., 2008) measured on histological sections have been found to be associated with QTLs on specific chromosomes across large numbers of inbred mouse strains. These studies suggest that particular genes might determine the size and shape of specific brain regions (Hager et al., 2012). This hypothesis raises the question whether the increased genetic heterogeneity of outbred mice is associated with increased variance in the size and shape of neuroanatomy.
Here we use ex vivo magnetic resonance imaging (MRI) to test specifically for variability in intact brain anatomy using two inbred strains (C57BL/6, 129S6) and one outbred strain (CD-1). Mouse models in conjunction with advanced imaging methods have proven to be a valuable tool to investigate how genetic and environmental factors affect brain structure (Lerch et al., 2011a). Anatomical MR imaging followed by automatic registration-based volume estimation is sensitive enough to even detect the subtle morphological changes associated with skill training (Lerch, J.P., et al., 2011b, Scholz, J., et al., 2014) and environmental enrichment (Scholz et al., 2015). However, the effect sizes are small and close to minimal detection thresholds. Future studies could thus benefit from a better characterization of the variability associated with a specific genetic background. A procedural question is how to best characterize this variability. To answer these questions we demonstrate that neuroanatomical shape variability can be quantified using surface shape models in conjunction with principal component analysis (PCA). Specifically, we test whether variability is localized to functionally relevant anatomical regions and whether this variability is strain-dependent. Second, we test whether variability is increased in the outbred strain. Third, we test whether anatomical variability is a distinguishing feature between the three strains.
Briefly, we base our estimates of anatomical variability on principal components derived from applying PCA to surface shape models of the whole brain and specific anatomical structures, including the hippocampus and striatum. We use the Generalised Procrustes Analysis (GPA) framework which brings the surface point clouds into alignment and optionally adjusts for differences in scale. Variants of PCA have been successfully used to characterize phenotypic variation in the mouse cranium (Hallgrímsson, B., et al., 2009, Hallgrímsson, B., et al., 2007) and morphological asymmetry in the primate and human brain (Gómez-Robles et al., 2013). Here we show that GPA can reveal meaningful components of neuroanatomical variation in the mouse brain, some of which are associated with strain differences.
Section snippets
Mice
Three mouse strains were used: 129S6/SvEvTac/J (N = 25), C57BL6J/Tcp (N = 25), and Crl:CD1(ICR)/Tcp (N = 27). Strains are referred to as 129S6, C57BL/6, and CD-1 for brevity throughout the manuscript. All mice were male and 8 (± 0.17) weeks old. Brains were perfusion-fixed and subsequently immersion fixed for 24 h (Cahill et al., 2012).
MR image acquisition
Brains were scanned within their skulls to minimize perturbations (Badea et al., 2007). A custom-built 16-coil solenoid array was used to image 16 samples concurrently (
Whole brain surface
The GPA of the whole brain surface resulted in a number of PCs of quickly decreasing importance (N = 77, Fig. 1). While the first PC explains about 64% of total variance, the following PCs explain only 13, 4, 4, and 2%. Seven components are sufficient to explain 90% of total brain surface shape variance.
We used Levene's test on the first seven PC scores to test for differences in variance between strains. The strains differed only in the variance associated with motor cortex (M1) size, i.e. PC2
Discussion
Here we present the first systematic evaluation of neuroanatomical shape variability across three common laboratory mouse strains including both inbred and outbred strains. Among the components that explained 90% of total variance we found at least three that seemed to have some degree of anatomical or even functional relevance as suggested by their spatial specificity and symmetry. Further, we were able to quantify inter-strain variance as well as strain-independent variance, with the former
Limitations
Our aim was the investigation of the anatomical variability across several common laboratory mouse strains. We chose PCA-based shape analysis as a method that is well established in related anatomical studies of faces (Claes et al., 2014) and skeletal features (Hallgrímsson, B., et al., 2007, Hallgrímsson, B., et al., 2009, Cates, J., et al., 2008). We could have varied our methods in several ways to conduct a comparable analysis. First, PCA can be applied to voxel-wise data as well to reduce
Conclusions
In summary, using two inbred strains and one outbred strain we were able to quantify strain-dependent neuroanatomical shape variance. Our analysis suggests that outbred strains might not come at the price of larger variability. Neuroanatomical studies could thus benefit from their increased disease resistance, high fecundity, and low neonatal mortality without sacrificing sensitivity and power of the analysis. Future studies will have to investigate how the variability of molecular, cellular
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