Sexual dimorphism revealed in the structure of the mouse brain using three-dimensional magnetic resonance imaging

Abstract

A large variety of sexual dimorphisms have been described in the brains of many vertebrate species, including humans. Naturally occurring sexual dimorphism has been implicated in the risk, progression and recovery from numerous neurological disorders, including head injury, multiple sclerosis and stroke. Genetically altered mice are a key tool in the study of structure–function relationships in the mammalian central nervous system and serve as models for human neuropsychiatric and neurological disorders. However, there are a limited number of quantitative three-dimensional analyses of the adult mouse brain structures. In order to address limitations in our knowledge of anatomical differences, a comprehensive study was undertaken using full 3D magnetic resonance imaging (MRI) to examine sexual dimorphisms in the C57BL/6J whole mouse brain. An expected difference in overall brain size between the sexes was found, where male brains were 2.5% larger in volume than female brains. Beyond the overall brain size differences in the sexes, the following significantly different regions were found: males were larger in the thalamus, primary motor cortex and posterior hippocampus, while females were larger in posterior hypothalamic area, entorhinal cortex and anterior hippocampus. Using high-definition 3D MRI on a normal inbred mouse strain, we have mapped in detail many sex-associated statistically significant differences in brain structures.

Introduction

Numerous sexually dimorphic characteristics have been identified both in the human and animal brain (Ho et al., 1980, Witelson and Kigar, 1988, Witelson, 1989, Witelson, 1991, Kimura, 1992, Cowell et al., 1994, Kulynych et al., 1994, Schlaepfer et al., 1995, Murphy et al., 1996, Luders et al., 2004). Indeed, sexual differences in the nervous system have been described at virtually every anatomical level including molecular, cellular and neural system levels (reviewed in Cooke et al., 1998). Neuroimaging and morphometric studies of human subjects have shown sexual dimorphisms in brain regions such as the amygdala (Goldstein et al., 2001) and hypothalamus (Swaab and Fliers, 1985) along with differences in neuron numbers across the entire cortex, although these results lack consistency (Witelson et al., 1995, Harasty et al., 1997, Pakkenberg and Gundersen, 1997). Along with structural distinctions, sexual differences have also been reported in performance on cognitive tasks and brain physiology (Kimura, 1992). It has also been shown that naturally occurring sexual dimorphism has implications in the risk, development and recovery from numerous neurological disorders. These include head injury, multiple sclerosis, stroke and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease (reviewed in Shulman and Bhat, 2006, Webber et al., 2005).

The incidence and progression of some psychiatric disorders have also been shown to exhibit gender differences. For example, males are known to suffer from schizophrenia more than two and half times more often than females (Castle and Murray, 1991). Males are also prone to a more severe form of the disorder, experience an earlier onset and exhibit more structural brain abnormalities. Relapses are more severe, and their response to neuroleptic medication is less favorable (Castle and Murray, 1991). Work to identify the neuroanatomical regions of significance in patients diagnosed with schizophrenia compared to healthy counterparts has shown that differences seem most common in the frontal lobes, hippocampus and temporal lobes (Green, 2001, Honea et al., 2005). These differences are heavily linked to the neurocognitive deficits which often occur with schizophrenia, particularly in areas of memory, attention, problem solving and social cognition. Neurodevelopmental disorders, such as autism which has no X- or Y-linked pattern of inheritance, are also more prevalent in males than in females (reviewed in Rutter et al., 2003). Therefore, research into sexual dimorphism has become mandatory in the understanding of a host of brain disorders with sex differences in their incidence and nature.

The use of inbred and genetically altered mice has become a leading approach in the study of structure–function relationships in the central nervous system. Furthermore, these tools have served as models for many human neuropsychiatric and neurological disorders (reviewed in Crawley and Paylor, 1997, Nieman et al., 2006). Furthermore, analysis into the tissue-specific expression and regulation of genes in mice has shown that hundreds of genes are sexually dimorphic in the brain (Yang et al., 2006). However, unlike in human studies, there are a limited number of quantitative 3D analyses of adult mouse brain structures (Fransen et al., 1998, Airey et al., 2001, Zygourakis and Rosen, 2003). Until now, there has not been completed a high-definition and comprehensive study that utilized full 3D analysis and a large number of individuals to examine in detail sexual differences over the whole mouse brain. The purpose of this work was to utilize 3D fixed magnetic resonance imaging (MRI) of male and female brains to study such differences.

Magnetic resonance (MR) images are digital and therefore quantitative data can be readily extracted from inbred mouse strains (Chen et al., 2006). In comparison to live specimen imaging, the imaging of fixed brains can produce images with improved resolution as fine as 32 μm (this study; Natt et al., 2002, Nieman et al., 2005) or even 21 μm (Badea et al., 2006). This technique was chosen over traditional histological methods as the ideal tool for visualizing sexual dimorphism in mouse brains for a number of other reasons as well. Firstly, it minimizes distortion artifacts produced by extreme treatment of brain tissue during fixation, embedding, sectioning and mounting and also allows for greater accuracy in determining tissue differences. Secondly, the gross anatomy of the specimens is conserved due to the maintenance of brains in a natural conformation in the skull during imaging. Finally, this technique also allows for whole brain coverage and fully 3D analysis, characteristics not shared in other more frequently used visualization techniques.

In view of the fact that structural differences have been observed in many vertebrate brains, we expect to observe mouse sexual dimorphisms that coincide with those observed in other mammal groups. For example, the density of neurons in the dentate gyrus is significantly larger in male than in female rats, as is both the volume of the CA1 region and the number of pyramidal cells (Madeira and Lieberman, 1995). Therefore, there is an expectation that the mouse brain will show sexual differentiation within this region with the male presenting with a larger hippocampal structure. Previous reports also indicate that the corpus callosum, the fiber tract that connects the two cerebral hemispheres in some vertebrates, exhibits sexual dimorphism. In one report, male mice of approximately 16 weeks of age were shown to possess a greater total corpus callosum area compared with females of the same age when differences in brain weight were taken into account (Berrebi et al., 1988).

Clearly there is a need to identify the dimorphisms between normal (non-diseased) male and female mouse brains before differences in diseased counterparts can be fully interpreted. The results of this work provide a comprehensive map of sexual differences in the mouse brain that can be readily compared to findings gathered in human studies. These comparisons will offer details and possibly an explanation about diseases and/or disorders that appear to be sex biased. This work corroborates previous work and additionally reports many novel findings, enabling a better overall understanding of sexual dimorphism in the mouse brain and augmenting previous mouse brain atlas studies (Ma et al., 2005).