An example of HARDI/Qball tractography demonstrates the difference in streamline profile compared to an example of standard tensor modeling (DTI). Streamlines of the optic nerves are viewed from the ventral surface of the brain and streamlines overlay FA horizontal planes at the level of the hippocampal commissure. ROIs (arrowheads) of the optic nerve were drawn as follows: left-lateral (green) and left-medial (red), and right medial (purple) and right-lateral (blue). These ROIs produced a greater delineation of ipsilateral and contralateral streamline populations following Qball modeling compared to DTI. Examples of false-positive streamlines include those indicated by arrows. The asterisk in both examples represents an example of streamlines failing to terminate and instead, crossing the caudal corpus callosum (this is dealt with elsewhere in the study). Scale bar = 1 mm.

Tractography of white matter regions of interest using Qball-FACT (A–C), CSD deterministic (D–F), CSD probabilistic (G–I) and FSL probabilistic (J–L) methods. Streamlines are three-dimensional, while FA maps are shown at two-dimensional planes: coronal, −1.70 mm Bregma; sagittal, midline; horizontal, anterior crossing at the midline. Streamlines were generated from an ROI placed at the intersection of the fimbria, stria terminalis and dorsal internal capsule (left and middle columns), or from a unilateral olfactory bulb (right column). Streamlines shown are colored according to their waypoint ROIs (Supp. Fig. 1). For the ROI of the left and middle columns a number of streamline populations were identified which accord with known anatomical tracts: hippocampal commissure (hc; yellow), stria terminalis (st; light blue), precommissural fornix (pcf; red), ventral diagonal band (of Broca) (vdb; red), dorsal tenia tecta (dtt; red), dorsal corticothalamic tracts (dct; dark blue in A, B, J and K; purple in D, E, G and H). J, K and L are maximum intensity projection (MIP) maps for the ROIs as generated in FSL. C, F, and I, Streamlines from the olfactory bulb in colour-coded schemes (L is a MIP for the olfactory bulb generated by FSL). Insert in C shows the coronal waypoint ROIs used to delineate streamline populations. The dashed line shows the point along the anterioposterior axis from which the coronal waypoint ROIs were drawn. While the anterior commissure was consistently plotted, the lateral olfactory tract (lo) was more variable depending on the method used. The lateral olfactory tract was generated by Qball/FACT but streamlines did not reach the waypoint ROI, and hence is not delineated in C. The ventricle streamlines have been eliminated from I for clarity. Additional abbreviations: aci, anterior commissure intrabulbar; cor, coronal; CPu, caudate putamen; hc, hippocampal commissure; hor, horizontal (axial); pir, piriform cortex; sag, sagittal. Scale bars = 1 mm.

Segmentation of the thalamus according to probabilistic tractography streamlines seeded in various cortical ROIs. Segmentation was made using unilateral ROIs in the somatosensory, visual, motor, auditory and the ventrolateral cortices (Supp. Fig. 1). A–C, Wholebrain CSD at 1 streamline seeded per voxel failed to map the entire thalamus, unlike 50 streamlines seeded per voxel (D–F). The same ROIs were employed for segmentation using the FSL ROI tractography approach seeded at 500 streamlines per voxel (G–I) or 5000 streamlines per voxel (J–L). Under the conditions employed, CSD probabilistic tractography produces a hard segmentation of the entire thalamic ROI, unlike FSL probabilistic tractography. Segmentation of the thalamic ROI by CSD probabilistic tractography follows the anatomical cortico-thalamic topography. The coronal plane shown correlates to Bregma - 2.06 mm, the sagittal plane corresponds to 2mm from the midline, and the horizontal plane corresponds to 2.5 mm from the interaural line from the mouse brain atlas (Franklin and Paxinos, 1997). The inserts are at 4× zoom. For similar planes, scale bars = 1 mm.

Fgf17−/− (KO) mice have frontocortical tract deficits. An ROI was drawn in the frontal cortex of one hemisphere from each wildtype (WT) and KO mouse, using the colour-FA maps (A). From this ROI, four streamline populations were delineated (B–C): those passing laterally through the rhinal and piriform cortices (noted for simplicity as lateral cortex (lc), red); those following the cerebral peduncle and pyramidal tracts, noted as the corticospinal tract (cst, light blue); those crossing the midline via the forceps minor of the corpus callosum (fmi, green) into the contralateral frontal cortex; and, those following the cingulum (cg, yellow). From this, waypoints were created (Supp. Fig. 1). D and E, Representative tractography of WT and KO brains using Qball-FACT deterministic tractography in TrackVis. Streamlines were generated from the ROI in A, then waypoint ROIs were applied. Genotype differences in streamline number for each streamline population are quantified in F. Since TrackVis generates wholebrain tractograms prior to the selection of seed ROIs, this quantification is noted as Wholebrain. * p < 0.05, compared to WT within that streamline population. G, Representative streamline populations of WT brains using CSD probabilistic tractography. Here, the parahippocampal extent of the cingulum is seen (arrowhead), as opposed to its absence in Qball-FACT deterministic tractography in D. CSD streamline number for each population was quantified by three different means: using a wholebrain (WB) tractogram (H), arbitrarily at 1000 streamlines (J), or at 50 streamlines per voxel (50 spv) (K). * p < 0.05, compared to WT within that population. The wholebrain tractogram approach was used for normalization based on the volume of the frontal cortex (I). Using the normalized streamline approach, there remains a significant difference in streamlines belonging to the lateral cortex (I), but not for the cingulum. For comparison, FSL was also used to generate streamline statistics. A representative MIP of the WT frontal cortex streamlines is shown in L, which is not markedly different from that generated by MRtrix (C). Statistical analysis reveals that once normalized, the lateral cortex streamline population is significantly different in KO mice. Histograms represent the mean ± s.e.m from three animals. Scale bar = 1 mm.