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S U M M A R Y The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2-to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. (J Histochem Cytochem 56:3-6, 2008) K E Y W O R D S mossy fibers ZNT3 glutamate zinc release hilus X-ray fluorescence knockout MORE THAN A DECADE AGO, Dr. Richard Palmiter discovered and cloned a gene (Slc30a3) the protein from which (ZNT3) he showed to be selectively located on the vesicles of zinc-secreting neurons, such as those comprising the hippocampal mossy-fiber pathway (Palmiter et al. 1996;Wenzel et al. 1997). Mice congenitally lacking the ZNT3 protein (ZNT3 knockout mice) were developed by Cole et al. (1999), and these mice proved to have no histochemically detectable zinc whatsoever in their mossy-fiber pathway, or in other zinc-secreting pathways (Cole et al. 1999); implying that the ZNT3 protein serves as a zinc transporter, responsible for the loading of zinc into those vesicles. The experiments performed by both Palmiter et al. (1996) and Cole et al. (1999) used immunocytochemistry to show that ZNT3 in the hippocampus is localized to the CA4/hilar region of the dentate gyrus, the stratum lucida of CA3, and the pyramidal cells of CA3 and CA1. Cole et al. (1999) also performed an elemental analysis of digested brain regions, demonstrating that total hippocampal zinc was reduced by 20%. The present work was undertaken to confirm that knocking out ZNT3 results in the loss of elemental zinc from the normally zincenriched regions of the hippocampus by using a novel non-destructive technique, microprobe synchrotron X-ray fluorescence (mSXRF).The specific question under examination in the present work was, Does the ZNT3 protein control the amount of zinc stored in vesicles of zinc-secreting neurons, or does ZNT3 merely control the amount of zinc that is detectable by histochemical methods, i.e., control the speciation, or distribution between bound and free (rapidly exchangeable) zinc in the vesicle? The answer we have obtained by quantitative imaging of total elemental zinc, using mSXRF, in the hippocampal regions studied by Cole et al. (1999), of ZNT3 knockout mice and wild-type mice is unambiguous: mice lacking the ZNT3 protein have no detectable enrichment of elemental zinc in their vesicles. Moreover,...

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There is considerable evidence suggesting that metals play a central role in the pathogenesis of Alzheimer's disease. Reports suggest that elevated dietary metals may both precipitate and potentiate an Alzheimer's disease phenotype. Despite this, there remain few studies that have examined the behavioral consequences of elevated dietary metals in wild type and Alzheimer's disease animals. To further investigate this in the current study, two separate transgenic models of AD (Tg2576 and TgCRND8), together with wild type littermates were administered 10 ppm (0.153 mM) Zn. Tg2576 animals were maintained on a zinc-enriched diet both pre- and postnatally until 11 months of age, while TgCRND8 animals were treated for five months following weaning. Behavioral testing, consisting of "Atlantis" and "moving" platform versions of the Morris water maze, were conducted at the end of the study, and tissues were collected for immunohistochemical analysis of amyloid-beta burden. Our data demonstrate that the provision of a zinc-enriched diet potentiated Alzheimer-like spatial memory impairments in the transgenic animals and was associated with reduced hippocampal amyloid-beta plaque deposits. Zinc-related behavioral deficits were also demonstrated in wild type mice, which were sometimes as great as those present in the transgenic animals. However, zinc-related cognitive impairments in transgenic mice were greater than the summation of zinc effects in the wild type mice and the transgene effects.