Visualization of mouse barrel cortex using ex-vivo track density imaging
Introduction
The arrangement of whisker follicles on the rodent snout is highly conserved and can be mapped precisely onto the cytoarchitectural barrel fields located in the primary somatosensory (S1) cortex (Woolsey and Van der Loos, 1970). Whiskers actively move at high frequency and are thought to have a similar function to the human fingertips for sensing surface texture (Petersen, 2007). Functional and anatomical mapping studies have established that each whisker makes a preferential connection to a single barrel (Grinvald and Hildesheim, 2004, Simons and Woolsey, 1979, Welker, 1976). In rodents, barrels in the large barrel field, referred to as the posterior medial barrel sub-field (PMBSF), are arranged topographically in 5 rows (A–E) with 5 arcs (1–5). The cross-sectional area of PMBSF is approximately 1 mm2, which is 40% of the total barrel field area. In cross-section the principal axes of the largest barrels in the PMBSF (B1, C1) are approximately 170 and 380 μm. The stereotypical organization of the barrel field provides an important anatomical reference used to study synaptic connectivity, development, and plasticity of the sensory system (Daw et al., 2007, Petersen, 2007).
Currently, the barrel field is visualized using optical imaging methods involving immunohistological staining for choline acetyltransferase (ChAT), glucose transporter-2, the 5HTT serotonin transporter or cytochrome oxidase C (Gonzalo-Ruiz et al., 1995, Land and Simons, 1985, Voutsinos-Porche et al., 2003). While immunohistochemical methods produce clear barrel delineation, processing techniques involve sectioning or removal of the cortex from the whole brain followed by pressure flattening prior to vibratome sectioning. The loss of cortical curvature and tissue distortion not only renders three-dimensional volumetric reconstruction challenging, but also affected the accuracy measurements of individual barrel column angles (Egger et al., 2012). More recently 3D auto-fluorescence optical imaging has been used (Gleave et al., 2012). Auto-fluorescence methods are non-destructive but involve dehydration of the sample in ethanol resulting in significant artifacts from inhomogeneous tissue shrinkage.
Magnetic resonance imaging (MRI) has also been employed to visualize the barrel cortex. Using in vivo blood-oxygenation level dependent functional MRI (BOLD fMRI), the low-resolution structure of the barrel cortex has been indirectly visualized during whisker electrical stimulation through signal changes in the microvasculature surrounding each barrel column (Yu et al., 2012). Ex-vivo MRI has been used to reveal brain microstructure (Dorr et al., 2008, Ma et al., 2005, Ullmann et al., 2012); however, observation of individual barrels in the barrel cortex has not been reported.
Recently, a new super-resolution track-density imaging (TDI) technique was developed to increase the spatial resolution of reconstructed images significantly beyond the acquired MRI resolution. The technique provides very high anatomical contrast based on anatomical connectivity (Calamante et al., 2010). It entails the application of constrained spherical deconvolution (CSD) to high angular resolution diffusion imaging (HARDI) data (Tournier et al., 2007) followed by the generation of a large number of streamlines using whole brain probabilistic fiber-tracking. The intensity of TDI maps corresponds to the number of streamlines that are present within each high-resolution grid element (the voxel of the super-resolution map). To increase the contrast in ex-vivo C57BL/6J mouse brain diffusion imaging, Calamante et al. (2012a) introduced TDI maps using short tracks instead of full-length streamlines. Short tracks TDI (stTDI) maps have the advantage of reducing saturation within structures with a high-density of long tracks (such as the corpus callosum and the internal capsule). Structures with shorter streamlines within the gray matter, such as the molecular layer of the hippocampus, are more clearly visualized with stTDI (Calamante et al., 2012a). In directional encoded color stTDI (DEC stTDI), the directionality of the tracks within the grid is also color-mapped using the standard red–green–blue convention.
In this study, we compared stTDI with conventional diffusion tensor analysis and T1, T2 and T2* weighted imaging methods to visualize the stereotypical architecture of the barrel cortex. Images were compared to histological data from the same animal by registering the MRI scans with 3D reconstructions of tissue sections in which the barrel field was visualized using virus-mediated fluorescence labeling. Finally, we tested the sensitivity of stTDI to detect changes in the barrel field resulting from infraorbital nerve cut.
Section snippets
Sample preparation
Twelve-week old adult C57BL/6J mice were anesthetized and perfused with 4% paraformaldehyde containing 0.5 mM Magnevist®. The brains were removed from the skull and placed in PBS containing 0.5 mM Magnevist® for 4 days. The samples were imaged using perfluoroether Fomblin Y06/06 solution medium (Solvay Solexis, Italy). All mice were housed and handled in accordance with the Queensland Animal Care and Protection Act 2001 and the current NHMRC Australian Code of Practice for the Care and Use of
Lack of visualization of the barrel structures using 3D GE, SE and FSE images
3D GE images of the samples prepared using Magnevist® concentrations at 0.5 mM (Figs. 1A, B) and 2.5 mM (Figs. 1C, D) and incubation times of 1 week (Figs. 1A, C) and 1 month (Figs. 1B, D) could not produce consistent enhanced visualization of the cortical barrel structures. These structures were also invisible in individual brains used to generate the Australian Mouse Brain Mapping Consortium (AMBMC) adult C57BL/6J mouse brain model (Ullmann et al., 2012). Although signal to noise and contrast
Discussion
In this study, we found that visualization of barrels in the ex-vivo mouse brain is greatly enhanced with super-resolution stTDI compared to conventional FA maps, GE, b0 and ADC maps. Visualization was superior on 10 μm stTDI maps from 48 μm HARDI data compared to 10 μm stTDI maps reconstructed from 100 μm HARDI data. The barrels appeared smaller and the septa wider in the lower resolution HARDI data, indicating that the stTDI method is affected by the quality and the resolution of raw HARDI data.
Conclusion
This study is the first to show a robust visualization of the barrel fields in the mouse brain using MRI. 3D structure of the barrel fields can be clearly and reliably visualized using the super-resolution stTDI technique. These maps produced superior contrast compared to DTI parametric images and conventional MRI and provide a significant advance in our ability to map structural and functional changes in the somatosensory barrel cortex caused by genetic or acquired lesions.
The following are
Acknowledgments
We are grateful to the National Health and Medical Research Council (NHMRC) of Australia and the Australian Research Council (ARC) for their support. The Florey Institute of Neuroscience and Mental Health is supported by Victorian State Government infrastructure funds. We thank the Queensland State Government support for the operation of the 16.4 T through the Queensland NMR Network. JUY was supported by the Chilean National Scholarship. We thank Prof. LJ Richards for critical reading of the
References (39)
- et al.
Preparation of fixed mouse brains for MRI
Neuroimage
(2012) - et al.
Track-density imaging (TDI): super-resolution white matter imaging using whole-brain track-density mapping
Neuroimage
(2010) - et al.
Track density imaging (TDI): validation of super resolution property
Neuroimage
(2011) - et al.
Super-resolution track-density imaging studies of mouse brain: comparison to histology
Neuroimage
(2012) - et al.
A generalised framework for super-resolution track-weighted imaging
Neuroimage
(2012) - et al.
Structural correlates of active-staining following magnetic resonance microscopy in the mouse brain
Neuroimage
(2011) - et al.
The effects of brain tissue decomposition on diffusion tensor imaging and tractography
Neuroimage
(2007) - et al.
High resolution three-dimensional brain atlas using an average magnetic resonance image of 40 adult C57Bl/6J mice
Neuroimage
(2008) - et al.
Microscopic diffusion tensor imaging of the mouse brain
Neuroimage
(2010) - et al.
Microscopic diffusion tensor atlas of the mouse brain
Neuroimage
(2011)
Waxholm space: an image-based reference for coordinating mouse brain research
Neuroimage
Vibrissaeless mutant rats with a modular representation of innervated sinus hair follicles in the cerebral cortex
Exp. Neurol.
Vesicular glutamate transporters VGLUT1 and VGLUT2 in the developing mouse barrel cortex
Int. J. Dev. Neurosci.
A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy
Neuroscience
Comparative mouse brain tractography of diffusion magnetic resonance imaging
Neuroimage
The functional organization of the barrel cortex
Neuron
Functional organization in mouse barrel cortex
Brain Res.
Direct estimation of the fiber orientation density function from diffusion-weighted MRI data using spherical deconvolution
Neuroimage
Robust determination of the fibre orientation distribution in diffusion MRI: non-negativity constrained super-resolved spherical deconvolution
Neuroimage
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