Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the ZnT3 gene

Abstract

The mammalian protein ZnT3 resides on synaptic vesicle membranes of zinc-containing neurons, suggesting its possible role in vesicular zinc transport. We show here that histochemically reactive zinc, corresponding to the zinc found within synaptic vesicles, was undetectable in the brains of mice with targeted disruption of the ZnT3 gene. Total zinc levels in the hippocampus and cortex of these mice were reduced by about 20%. The ultrastructure of mossy fiber boutons, which normally store the highest levels of vesicular zinc, was unaffected. Mice with one normal ZnT3 allele had reduced levels of ZnT3 protein on synaptic vesicle membranes and had intermediate amounts of vesicular zinc. These results demonstrate that ZnT3 is required for transport of zinc into synaptic vesicles and suggest that vesicular zinc concentration is determined by the abundance of ZnT3.

Figure 1

Targeted disruption of the mouse ZnT3 gene. (A) Diagram of the ZnT3 wild-type allele, targeting vector, and predicted mutant allele. The targeting strategy placed nlacZ into the ZnT3 locus, using neo^r for positive selection and herpes simplex virus thymidine kinase (TK) genes for negative selection. The eight numbered boxes represent exons. Homologous recombination in the regions indicated with an X should result in a mutant allele as shown at the bottom. The black bar represents the 380-bp TaqI/EcoRI probe used for Southern hybridization in B. A, AflII; N, NarI; Nh, NheI; No, NotI; S, StuI. (B) Southern blot of NheI-digested genomic DNA from ZnT3^+/+, ZnT3^+/−, and ZnT3^−/− mice. The probe detects a 7.9-kb mutant and 3.2-kb wild-type fragment. (C) Western blot of brain homogenates from 10-wk-old ZnT3^+/+, ZnT3^+/−, and ZnT3^−/− mice. Equal amounts of total protein were loaded into each well. ZnT3 protein, which migrates as a 39-kD band, was undetectable in the brains of the mutants and reduced in the heterozygotes. The upper, nonspecific band controls for loading differences.

Figure 2

ZnT3 is expressed in the central nervous system. (A) ZnT3 mRNA levels in developing mouse brain, assessed by solution hybridization. Data are presented as mean ± SEM. (B–F) Expression of nlacZ (under the control of the ZnT3 promoter) in the central nervous system of ZnT3^+/− mice, as detected by 5-bromo-4-chloro-3-indolyl-β d-galactoside (X-gal) staining (white nuclei, indicated by black arrows). nlacZ is expressed in s granulosum of the dentate gyrus (B), the pyriform cortex (C), and the cochlear nucleus (D). nlacZ expression was not completely penetrant, as evident by the absence of staining (white arrows) in many areas where ZnT3 is normally expressed. (E and F) nlacZ is expressed in laminae I, II, III, and IV of the spinal cord dorsal horn. Sections were photographed under dark-field illumination. [Bars = 500 μm (B and D); 200 μm (C and E); 100 μm (F).]

Figure 3

Total zinc is reduced in the hippocampus and cortex of ZnT3^−/− mice. Selected regions of the brain were dissected, and elemental analysis was performed for 22 elements, including zinc (see Materials and Methods). Total zinc was reduced by about 20% in the hippocampus and cortex of ZnT3^−/− mice, and ZnT3^+/− mice had a 10% reduction in total zinc in these same regions. Data are presented as mean ± SEM (n = 4).

Figure 4

Timm stain is undetectable in the brains of ZnT3^−/− mice. Comparison of Timm stain between brains of ZnT3^+/+ (A, E, and H), ZnT-3^+/− (B, F, and I), and ZnT-3^−/− mice (C, G, and J). (A–C) Coronal sections through the midbrain. Timm stain in the hippocampus (H), piriform cortex (P), neocortex (N), and amygdala (A) was conspicuous in the ZnT3^+/+ brain (A), reduced in the ZnT3^+/− brain (B), and undetectable in the brains of ZnT3^−/− mice (C). (D) Higher magnification of the choroid plexus from the lateral ventricle (indicated area in C). Timm stain was unperturbed in the ZnT3^−/− choroid plexus. (E–G) Higher magnification of Timm-stained hippocampi from ZnT3^+/+ (E), ZnT3^+/− (F), and ZnT3^−/− (G) mice. Timm stain was reduced (ZnT3^+/−) or absent (ZnT3^−/−) in the hilus (h), s lucidum (luc) of CA3, and s oriens (or) and s radiatum (rad) of CA1 and CA3. (H–J) Electron micrographs of Timm-stained MFBs in s lucidum of CA3, taken from a ZnT3^+/+ (H), a ZnT3^+/− (I), and a ZnT3^−/− (J) mouse. Timm-positive vesicles were abundant in ZnT3^+/+ MFBs, whereas fewer vesicles were Timm-positive in ZnT3^+/− MFBs, and no Timm-positive vesicles were present in ZnT3^−/− MFBs. Arrowheads represent synaptic contacts made with a dendrite (D) and dendritic spines (S).

Figure 5

ZnT3 immunocytochemistry in the ZnT3^+/+ (A and B), ZnT3^+/− (C and D), and ZnT3^−/− (E and F) hippocampus. Compared with the ZnT3^+/+ hippocampus (A), ZnT3 immunoreactivity was reduced in many areas of the ZnT3^+/− hippocampus (C) and was undetectable in the ZnT3^−/− hippocampus (E). (B, D, and F) Electron micrographs of MFBs in s lucidum (boxed area in A, C, and E). ZnT3 immunoreactivity was conspicuous on synaptic vesicle membranes of ZnT3^+/+ MFBs (B), reduced on vesicle membranes of ZnT3^+/− MFBs (D), and undetectable on vesicle membranes of ZnT3^−/− MFBs (F). The number of immunoreactive vesicles in the ZnT3^+/− and ZnT3^+/+ MFBs was approximately the same. Arrowheads point to synaptic contacts made with dendritic spines (S). H, hippocampus; DG, dentate gyrus; h, hilus; luc, s lucidum; or, s oriens; rad, s radiatum; ml, molecular layer.

Figure 6

TSQ fluorescence in the hippocampus. (A) Computer-generated images of TSQ fluorescence in the hippocampi of ZnT3^+/+ (Left), ZnT3^+/− (Center), and ZnT3^−/− (Right) mice. The bright fluorescence in the hilus (Hi), s oriens and s lucidum of CA3, and s oriens and s radiatum of CA1 was reduced in the ZnT3^+/− hippocampus and undetectable in the ZnT3^−/− hippocampus. TSQ staining was also reduced in the neocortex (N). TSQ fluorescence in the hippocampus of the mutants was less than the autofluorescence of the overlying corpus callosum (cc). (B) Quantification of TSQ fluorescence in regions within the hippocampus by computer-assisted laser cytometry. Data are expressed as mean ± SEM (n = 5). (C) TSQ-stained section of a single seminiferous tubule from the testis of a ZnT3^−/− mouse. Spermatids poised at the lumen of the tubule fluoresced with TSQ, similar to wild-type spermatids (not shown). (D) TSQ-stained pancreas from a ZnT3^−/− mouse, illustrating a single Islet of Langerhans, composed of beta cells that have an abundance of histochemically reactive zinc packaged in secretory granules. [Bars = 50 μm (C); 100 μm (D).]

Figure 7

MFBs in the ZnT3^−/− hippocampus show normal ultrastructure. Electron micrographs of MFBs in s lucidum of CA3, taken from a ZnT3^+/+ (A) and a ZnT3^−/− (B) mouse. ZnT3^−/− MFBs were normal with respect to their size, the approximate number of small, clear synaptic vesicles contained within them, the numbers and types of synaptic contacts made with dendritic spines, and the presence of mitochondria. Note the asymmetric synaptic contacts (arrowheads) on dendritic spines (S) and a pyramidal cell dendrite (D).