Figure 5

Iron and zinc histological stains in the anterior olfactory nucleus in the Parkinson’s disease (PD) olfactory bulb. (a) Nonheme iron (brown) is present predominantly in the outer layers of the olfactory bulb and tract. Cell nuclei and neuronal bodies are counterstained with cresyl violet. (a’) Zoom of boxed region in (a) showing very little nonheme iron in anterior olfactory nucleus cells. (b) Ferrous iron (brown) is present at low levels in the olfactory bulb and tract. Cell nuclei and neuronal bodies are counterstained with cresyl violet. (b’) Zoom of boxed region in (b) showing very little ferrous iron in anterior olfactory nucleus neurons. (c) Free and loosely bound zinc (black and brown) is present throughout the olfactory bulb and tract. Cell nuclei and neuronal bodies are counterstained with cresyl violet. (c’) Zoom of boxed region in (c) showing multiple zinc granules in anterior olfactory nucleus neurons. (d) Light micrograph from the anterior olfactory nucleus showing free and loosely bound zinc (black), followed by confocal micrographs of the same cell showing that the free zinc (green, using reflected light microscopy) is located within large aggregates of phosphorylated alpha-synuclein (red). (e) Confocal micrographs showing phosphorylated alpha-synuclein (red) aggregates in cells that are positive for PGP9.5 (green), but not GFAP (purple), in the anterior olfactory nucleus. (f) Transmission electron micrograph of free zinc staining in the anterior olfactory nucleus. Cells in this region were rich in lipofuscin pigment (L) and mitochondria (M), both of which contained numerous silver granules (corresponding to free zinc). The cell nucleus (N) is also visible in this micrograph. (g) Small silver granules (open arrowheads) were common on mitochondria in cells in the anterior olfactory nucleus. (h) Lipofuscin pigment contained silver granules that were often very large (closed arrowheads). Smaller silver granules can also be seen in a mitochondrion in this image (open arrowhead). (i) Mitochondria and (j) lipofuscin did not contain silver granules under negative control conditions, where no sodium sulfide was used. Scale bars: a, b: 500 µm; a’, b’: 50 µm; c: 1 mm; c’: 40 µm; d: 10 µm; e: 20 µm; f: 2 µm; g: 500 nm; h: 1 µm; i: 250 nm; j: 1 µm.