Mapping somatosensory connectivity in adult mice using diffusion MRI tractography and super-resolution track density imaging
Introduction
The rodent “whisker pathway” is an important model system, used to explore the architecture and function of local and long-range circuits in the developing, diseased and injured brain. Rodents use whisking to probe textures and surfaces of their local environment and a stereotypical link from individual facial whisker to Layer IV cortical barrel comprises the somatosensory pathway, for reviews see Fox (2008) and Petersen (2007). The topographic map of projections, arranged in rows and arcs, is preserved in the brainstem barrelettes (Belford and Killackey, 1979, Erzurumlu and Killackey, 1982, Killackey and Leshin, 1975, Ma and Woolsey, 1984), the thalamic barreloids (Van Der Loos, 1976), and cortical barrels (Woolsey and Van der Loos, 1970). This study describes the first mesoscopic (20 μm) mapping of trigemino-thalamo-cortical structures and connectivity in mice achieved by diffusion MRI micro-imaging methods obtained using ultra-high field MRI.
The first sensory relay location of whisker primary afferents is the brainstem barrelettes (Ma, 1991). Second order neurons then project to the ventral posteromedial (VPM) thalamic nucleus via the lemniscal pathway (Bates and Killackey, 1985). Projections from the VPM barreloids then cluster as dense arborisations terminating in Layer IV primary somatosensory cortex that appear as “barrel” shaped structures (Woolsey and Van der Loos, 1970). The structural composition and connectivity of the “whisker pathways” have been described using 2D histologic and functional activation methods. Network tracking techniques include lesion studies (Killackey and Fleming, 1985, Killackey and Leshin, 1975), carbocyanine dyes (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; DiI, DiA) (Kivrak and Erzurumlu, 2013, Seehaus et al., 2013) and myelin staining (Barrera et al., 2012); these techniques are also complemented by Lenti and Adeno-associated viral vector expression of fluorescent proteins (Aronoff et al., 2010, Dittgen et al., 2004, Wimmer et al., 2010). A range of macroscopic to microscopic functional connectivity mapping techniques include functional MRI (Kim et al., 2012, Yang et al., 1996), and 2-photon imaging of electrical activity using voltage-sensitive dyes (Petersen et al., 2003a, Petersen et al., 2003b) and channel-rhodopsins to map neuronal connectivity have also been conducted (Paz et al., 2011, Petreanu et al., 2007).
Spatial preservation of whole brain networks and global architecture using 3D imaging methods delivers considerable advantage over 2D histological techniques and partial mapping of cortical microcircuits. Diffusion MRI is an important mapping tool for tissue structure and connectivity, including information about fiber orientation and integrity and has been used to visualize tissue microstructure in humans and animal models (Behrens et al., 2003, Calamante et al., 2010, Calamante et al., 2012a, Jiang and Johnson, 2011, Kim et al., 2012, Moldrich et al., 2010).
More recently, the technique of super-resolution track density imaging (TDI) has been introduced as a means to achieve detailed visualization of structures at spatial resolutions beyond that of the acquired voxel resolution (Calamante et al., 2010, Calamante et al., 2011). In particular, when combined with ultra-high field MRI, this technique provides very high-resolution images with rich anatomical contrast in the mouse brain (Calamante et al., 2012b).
The TDI method was recently shown to allow a clear delineation of the barrel cortex (Kurniawan et al., 2014), leading to a much improved visualization of this discriminate structure than could be achieved with either conventional high resolution relaxation-weighted MRI or diffusion tensor imaging. In this study we sought to build on this finding, to afford 3D topographical mapping of the three major relay locations of the trigemino-thalamo-cortical pathway, and the connectivity between these locations. We report that virus-mediated fluorescent labeling of the thalamo-cortical afferents from the VPM to Layer IV cortex is recapitulated using targeted diffusion MRI fiber tracking of thalamo-cortical connectivity. We present compelling evidence that the super-resolution TDI method provides a unique platform for “virtual slicing”, and informs the 3D structural topography of the trigemino-thalamo-cortical pathway in mice.
Section snippets
Tissue — diffusion weighted imaging
Adult male C57Bl/6 mice (n = 3) were used for diffusion MRI tractography analysis. Animals were housed at the Florey Institute of Neuroscience and Mental Health animal facility under a 12-hour light/dark cycle with food and water ad libitum. All procedures involving mice were approved by the local Animal Ethics Committee and were conducted in accordance with the current National Health and Medical Research Council of Australia Code of Practice for the Care and Use of Animals for Scientific
Barrel cortex topography identified using stTDI
High-resolution diffusion MRI data was obtained with a signal-to-noise ratio of the b = 0 s/mm2 ~ 70, measured using the dual acquisition, subtraction method described by Firbank et al. (1999). The posterior medial barrel sub-field was clearly identified in Layer IV somatosensory cortex using stTDI; these results were consistent with previous findings (Kurniawan et al., 2014) (Figs. 1C & D).
Thalamo-cortical tracking using TDI recapitulates projections labeled using virus mediated fluorescent protein expression
We then examined the projection pattern of long-range connectivity for the thalamo-cortical projections,
Discussion
This study presents data supporting the complementary role of the super-resolution TDI approach compared to histology methods for mapping brain structures and connectivity of the somatosensory pathway in mice. Our principal finding is the pattern of somatosensory connectivity revealed by diffusion MRI that is consistent with the stereotypical pattern of thalamo-cortical projections, visualized using a virus encoded expression system and, in a separate set of experiments, DiI tracing of the
Conclusion
In summary we have shown in regions of complex anatomy, MRI diffusion micro-imaging informed at an unprecedented level, both structure and connectivity patterns at a high concordance with histologic methods. In addition, targeted tracking TDI results presented in this study suggest a key role to reveal long-range connectivity by providing spatial preservation of whole brain networks at a mesoscopic level.
The following are the supplementary data related to this article.
Acknowledgments
This work was supported by the Victorian Government through the Operational Infrastructure Scheme. We thank the Queensland NMR Network (QNN) and the National Imaging Facility (NIF) for instrument access and technical support. This work was funded by the Australian Research Council (ARC DP120104112; FT110100726; CE140100007), the National Health and Medical Research Council (NHMRC Program Grant 628952) and fellowship (1005050).
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- 1
Current address: Department of Biomedical Engineering, Division of Imaging Sciences & Biomedical Engineering, King's College London, London, UK.
- 2
These authors contributed equally.