Spatial relationship between synapse loss and beta-amyloid deposition in Tg2576 mice

Abstract

Although there is evidence that beta-amyloid impairs synaptic function, the relationship between beta-amyloid and synapse loss is not well understood. In this study we assessed synapse density within the hippocampus and the entorhinal cortex of Tg2576 mice at 6-18 months of age using stereological methods at both the light and electron microscope levels. Under light microscopy we failed to find overall decreases in the density of synaptophysin-positive boutons in any brain areas selected, but bouton density was significantly decreased within 200 mum of compact beta-amyloid plaques in the outer molecular layer of the dentate gyrus and Layers II and III of the entorhinal cortex at 15-18 months of age in Tg 2576 mice. Under electron microscopy, we found overall decreases in synapse density in the outer molecular layer of the dentate gyrus at both 6-9 and 15-18 months of age, and in Layers II and III of the entorhinal cortex at 15-18 months of age in Tg 2576 mice. However, we did not find overall changes in synapse density in the stratum radiatum of the CA1 subfield. Furthermore, in the two former brain areas we found a correlation between lower synapse density and greater proximity to beta-amyloid plaques. These results provide the first quantitative morphological evidence at the ultrastructure level of a spatial relationship between beta-amyloid plaques and synapse loss within the hippocampus and the entorhinal cortex of Tg2576 mice.

Figure 1

Sampling method for electron microscopy. Figure 1 represents an ultra-thin section mounted on a 400 mesh grad. The red circle in the center represents an amyloid plaque. From the edge of the plaque, one photograph per grid (i.e., 1, 2, 3….) was taken, randomly choosing one grid from 4 different directions. The black circle line indicates proximity to the nearest plaque. The distance between grid bars is 62 μm.

Figure 2

Panel A: Semi-thin sections of the hippocampus used to identify hippocampal subregions for ultra-thin section identification. Compact β-amyloid plaques (P) in semi-thin sections are distinguishable with Nissl staining. SO: stratum oriens; SP: stratum pyramidale; SR: stratum rediatum; LM: stratum lacunosum-moleculare; ML: molecular layer of dentate gyrus; DG: dentate granule cell layer. Panel B: Electron micrographs used for synapse counting. Images were scanned into a computer dataset and stereological methods were used for synapse counting. The red dots indicate counted synapses.

Figure 3

Amyloid and synaptophysin double-labeling in entorhinal cortex of Tg2576 mice at 18 months. Panel A: β-Amyloid plaque labeled with Cy3. Panel B: Synaptophysin labeled with alex 488. Panel C: Intense synaptophysin immunostaining on the periphery of the plaque (arrow). Bar in C also for A,B = 50 μm.

Figure 4

Electron microscope photographs of the entorhinal cortex of Tg2576 mice at 18 months of age. Panel A: Compact β-amyloid plaque and dystrophic neuritis. Panel B: Neuropil at 62 μm from a β-amyloid plaque, synapse density is significantly decreased in association with degenerated neurites. Panel C: Neuropil at 124 μm from the edge of a β-amyloid plaque, showing decreased synape density number as compared to an area distant from the plaque (>300 μm, Panel D). The red dots indicate counted synapses. P: the core of β-amyloid plaque; DN: dystrophic neuritis. Bar in D also for A,B,C = 1.5 μm.

Figure 5

Synapse density in proximity to compact β–amyloid plaques in the hippocampus and the entorhinal cortex in Tg+ mice at 15–18 months of age (see text for statistics). Tg+P: In this group, sampling regions were located from 62 to 248 μm from plaques (P) in Tg+ mice; Tg+NP: In this group, sampling regions were located in areas with no plaques (NP) in Tg+ mice; Tg-: In this group, sampling regions were located in analogous areas as those sampled in Tg+P and Tg+NP mice.