Diffusion tensor magnetic resonance imaging and tract-tracing analysis of Probst bundle structure in Netrin1- and DCC-deficient mice

Abstract

In many cases of callosal dysgenesis in both human patients and mouse models, misguided fibers from the cortex form abnormal bilateral, barrel-shaped structures known as Probst bundles. Because little is known about how axons are arranged within these anomalous fiber bundles, understanding this arrangement may provide structural and molecular insights into how axons behave when they are misguided in vivo. Previous studies described these bundles as longitudinal swirls of axons that fail to cross the midline (Ozaki et al., 1987). However, recent studies on human acallosal patients using diffusion tensor magnetic resonance imaging (DTMRI) technology suggest that axons project in an anteroposterior direction within the Probst bundle (Lee et al., 2004; Tovar-Moll et al., 2007). This led us to ask the question, is DTMRI an accurate method for analyzing axonal tracts in regions of high axon overlap and disorganization, or is our current perception of axon arrangement within these bundles inaccurate? Using DTMRI, immunohistochemistry, and carbocyanine dye tract-tracing studies, we analyzed the Probst bundles in both Netrin1 and deleted in colorectal cancer (DCC) mutant mice. Our findings indicate that DTMRI can accurately demonstrate fiber tract orientation and morphology where axons are in ordered arrays such as in the dorsal part of the bundle. In ventral areas, where the axons are disorganized, no coordinated diffusion is apparent via DTMRI. In these regions, a higher-resolution approach such as tract tracing is required. We conclude that in DCC and Netrin1 mutant mice, guidance mechanisms remain in the dorsal part of the tract but are lost ventrally.

Figure 1.

Probst bundles in E18 DCC and Netrin1 mutant brains. Immunostaining of coronal and horizontal sections from E18 Netrin1 and DCC mutant brains and wild-type controls with GAP43 antibody, an axon marker, and visualization by a nickel–DAB chromagen are shown. A, D, Control sections show normal formation of the corpus callosum in the coronal (A) or horizontal (D) view. B, C, E, F, In contrast, Probst bundles can be clearly observed in sections from either Netrin1-deficient (B, E) or DCC-deficient (C, F) brains (boxes in A–F indicate the areas shown in higher power in A′–F′). After immunohistochemistry, where the entire Probst bundle is stained, swirling fibers can be observed within the bundle, especially in the coronal view (B′, C′, arrows) and in the ventral region of the bundle. Scale bar: (in F′) A–F, 1 mm; A′–F′, 250 μm.

Figure 2.

DTMRI and tract-tracing analysis of Probst bundles in DCC knock-outs. Using both DTMRI scanning and carbocyanine dye tract tracing, the fiber structure of Probst bundles was analyzed in E18 DCC knock-out (DCC KO) brains (C–F, I–L) and compared with wild-type controls (A, B, G, H). Colored arrows in A indicate fiber direction in DTMRI color maps, with the rostrocaudal direction in red, the mediolateral direction in green, and the dorsoventral direction in blue. A, B, G, H, In control embryos at E18, the corpus callosum (CC; in green) can be visualized in the horizontal view (A, G) and can be recapitulated via dye tract tracing (B, H). C–F, I, In DCC knock-out brains, Probst bundles (C, I, outlined by white dashed lines) form instead. Under DTMRI in the horizontal view, the dorsal part of the bundle is pseudo-colored red (C), indicating fibers projecting in a rostrocaudal direction, and a large fiber bundle in the rostrocaudal axis can also be seen by DiI tract tracing (E, F, arrows; E is a higher-power view of the boxed area in D). I–L, In contrast, the ventral region of the bundle is black by DTMRI, indicating fibers that do not project in any specific direction (I), and under DiI tract tracing, Probst fibers indeed appear disorganized (K, L, arrows; K is a higher-power view of the boxed area in J). Scale bar: (in L) A, C, D, G, I, J, 1 mm; B, H, 500 μm; E, F, K, L, 250 μm.

Figure 3.

DTMRI and tract-tracing analysis of Probst bundles in Netrin1 mutants. Using both DTMRI scanning and carbocyanine dye tract tracing, the fiber structure of Probst bundles were analyzed in E18 Netrin1 mutant brains (B–D, F–H; magnified in F′–H′) and compared with wild-type controls (A, E, E′). Similar to the DCC knock-outs, Probst bundles are pseudo-colored red in the dorsal part of the brain (B, C), again indicating rostrocaudal projecting axons, the presence of which is verified by DiI tract tracing (F′, G′, arrow). In contrast, DiI-labeled fibers in the ventral region of the Probst bundle project in a disorganized manner (H′, arrow). Scale bar: (in H′) A–D, 1 mm; E–H, 600 μm; E′–H′, 300 μm.

Figure 4.

DTI-tractography in DCC and Netrin1 mutant brains. Trajectories of fibers entering and traveling within the corpus callosum or Probst bundle (in white) and the hippocampal commissure (in yellow). A–C, ROIs used for DTI-tractography. D, G, In wild-type controls, cortical fibers cross the midline to form the corpus callosum (D), whereas fibers emerging from the dentate gyrus (G, arrow) cross to form the hippocampal commissure. E, F, In DCC and Netrin1 mutant brains, hippocampal fibers from the fimbria either terminate at the midline or project ipsilaterally into the fornix (F, arrow), whereas cortical fibers form bilateral Probst bundles (E, F). The Probst bundle comprises of axons originating from throughout the cortex (E, arrows). H, I, Within the PB, fibers project in a rostrocaudal direction (white tract) and remain separate from the hippocampal fibers (yellow tract). I is a higher-power view of F. CC, Corpus callosum; HC, hippocampal commissure; PB, Probst bundle; Fim, fimbria. Orientation of the brain is as follows: R, right; L, left; A, anterior; D, dorsal. Scale bar: (in I) A–C, 1 mm; D–G, 1.5 mm; H, I, 750 μm.