Fig. 1

Axonal spheroids are formed in the hippocampo-septal tracts of 5xFAD; Thy1-YFP transgenic mice. A Whole-brain imaging revealed numerous axonal spheroids in the hippocampo-septal tracts of 5xFAD; Thy1-YFP transgenic mice. Scale bars = 3 mm for the dorsal view, 300 μm for the lateral view. B Sagittal sections and higher magnification images of the septum revealed that the transgenic mice had insufficient axonal fasciculation and a distinct morphology with a series of bulbous YFP-positive projections with enlarged ends. Scale bars = 400 μm for the left, and 200 μm for the right. C Schematic drawing for the active clarity method in conjunction with immunostaining and representative coronal sections showing aggregation of YFP-positive axons in the septum of 5xFAD; Thy1-YFP mice. MBP (red) was used to detect the axons in the sections. Scale bars = 200 μm. All schematic drawings were created with Biorender.com. D The presence and location of axonal spheroids in relation to amyloid plaques in the transgenic mice were studied using the ACT combined with immunostaining for Aβ (Yellow arrow: the septum, scale bar: 3 mm). Higher magnification images show that the axonal spheroids contained vesicular features (bright YFP signals in the spheroid) and were found at the end of MBP-stained axons around Aβ plaques. Whole brain imaging of Thy1-YFP WT mouse brain and higher magnification Thy1-YFP expression in the septum did not reveal any spheroids. Scale bars = 2 mm (whole brain), 10 μm (middle), 100 μm (rightmost). E A schematic drawing depicts the injection of AAV into the somatosensory cortex. A low magnification image under the drawing shows a tiled coronal image of AAV expression. The numbered magnified images are presented on the right panels with dendrite (1), callosal axons crossing the corpus callosum (2), corticothalamic axonal ends (3), and callosal axons reaching the contralateral cortex (4). AAV-hSyn-EGFP virus was injected into the somatosensory cortex of 6-month-old (1–4) and 8-month-old (1*–4*) 5xFAD mouse brains to determine the selectivity of neuronal compartments for spheroid development in the presence of Aβ plaques. The spheroid-like or bead-like big bulbous formations were not found in the somatic or dendritic compartments (1, 1*) or in a callosal bundle of nerve fibers in the corpus callosum (2, 2*). However, the spheroids near Aβ plaques were typically observed at the axon terminals of the ipsilateral corticothalamic route (3, 3*) and the callosal axons reaching the contralateral cortex (4, 4*; asterisk (*). AAV-hSyn-EGFP virus was injected into the somatosensory cortex of 8-month-old C57BL/6 mice (5) and the typical axon terminals of the ipsilateral corticothalamic route (5*) was presented (n = 4). Scale bars = 100 μm except for the leftmost image, scale bar = 500 μm and 4* = 20 μm