Elsevier

NeuroImage

Volume 87, 15 February 2014, Pages 465-475
NeuroImage

Visualization of mouse barrel cortex using ex-vivo track density imaging

https://doi.org/10.1016/j.neuroimage.2013.09.030Get rights and content

Highlights

  • Mouse barrel cortex can be visualized in high definition using stTDI.

  • stTDI produced superior barrel visualization compared to conventional MRI and FA maps.

  • stTDI is a novel imaging technique for 3D characterization of the barrel cortex.

  • stTDI can detect barrel changes resulting from infraorbital nerve cut.

Abstract

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3 mm)3 block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.

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

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