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Free, ionic zinc (Zn2+) modulates neurotransmitter dynamics in the brain. However, the sub-s effects of transient concentration changes of Zn2+ on neurotransmitter release and uptake are not well understood. To address this lack of knowledge, we have combined the photolysis of the novel caged Zn2+ compound [Zn(DPAdeCageOMe)]+ with fast scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes in live, whole brain preparations from zebrafish (Danio rerio). After treating the brain with [Zn(DPAdeCageOMe)]+, Zn2+ was released by application of light that was gated through a computer-controlled shutter synchronized with the FSCV measurements and delivered through a 1 mm fiber optic cable. We systematically optimized the photocage concentration and light application parameters, including the total duration and light-to-electrical stimulation delay time. While sub-s Zn2+ application with this method inhibited DA reuptake, assessed by the first-order rate constant (k) and half-life (t1/2), it had no effect on the electrically stimulated DA overflow ([DA]STIM). Increasing the photocage concentration and light duration progressively inhibited uptake, with maximal effects occurring at 100 μM and 800 ms, respectively. Furthermore, uptake was inhibited 200 ms after Zn2+ photorelease, but no measurable effect occurred after 800 ms. We expect that application of this method to the zebrafish whole brain and other preparations will help expand the current knowledge of how Zn2+ affects neurotransmitter release/uptake in select neurological disease states.
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Spatiotemporal Imaging of Zinc Ions in Zebrafish Live Brain Tissue Enabled by Fluorescent Bionanoprobes
The zebrafish is a powerful model organism to study the mechanisms governing transition metal ions within whole brain tissue. Zinc is one of the most abundant metal ions in the brain, playing a critical pathophysiological role in neurodegenerative diseases. The homeostasis of free, ionic zinc (Zn2+) is a key intersection point in many of these diseases, including Alzheimer’s disease and Parkinson’s disease. A Zn2+ imbalance can eventuate several disturbances that may lead to the development of neurodegenerative changes. Therefore, compact, reliable approaches that allow the optical detection of Zn2+ across the whole brain would contribute to our current understanding of the mechanisms that underlie neurological disease pathology. We developed an engineered fluorescence protein-based nanoprobe that can spatially and temporally resolve Zn2+ in living zebrafish brain tissue. The self-assembled engineered fluorescence protein on gold nanoparticles was shown to be confined to defined locations within the brain tissue, enabling site specific studies, compared to fluorescent protein-based molecular tools, which diffuse throughout the brain tissue. Two-photon excitation microscopy confirmed the physical and photometrical stability of these nanoprobes in living zebrafish (Danio rerio) brain tissue, while the addition of Zn2+ quenched the nanoprobe fluorescence. Combining orthogonal sensing methods with our engineered nanoprobes will enable the study of imbalances in homeostatic Zn2+ regulation. The proposed bionanoprobe system offers a versatile platform to couple metal ion specific linkers and contribute to the understanding of neurological diseases.
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- Award ID(s):
- 2108448
- PAR ID:
- 10426761
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 28
- Issue:
- 5
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 2260
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules28052260
