Quantitative Control of Zn²⁺ Photorelease: A Step toward Decoding Mechanisms of Subsecond Metal Signaling in the...

Free ionic zinc (Zn²⁺) exerts control over many aspects of neuronal function after its exocytotic release from vesicles by associating with conserved binding sites on regulatory proteins, including the dopamine transporter. While the use of photoreactive cages to investigate the influence of subsecond Zn²⁺ transients on neurotransmitter release and uptake is useful, a quantitative understanding of Zn²⁺'s regulatory effects requires knowing how much Zn²⁺ has been released. Here, we have addressed this issue by directly measuring 4-methoxy-3-nitrobenzaldehyde (3-NPA), a photoreaction byproduct from [Zn(DPAeCageOMe)]⁺, as a reporter to quantify Zn²⁺ photorelease. We optimized a sawhorse waveform, which eliminated fouling and provided a linear oxidation current response as concentration was increased. We then demonstrated the utility of our method by measuring released zinc ions evoked by light exposure in whole brains harvested from adult zebrafish ex vivo. Our findings revealed that zinc, which was released at concentrations of about 1 to 4 μM in response to light application durations of 300 to 900 ms, significantly increased dopamine overflow and inhibited dopamine uptake. The profound effects of low concentrations of Zn²⁺ on release and uptake raise the possibility that Zn²⁺, which is released from terminals at concentrations of 10 to 20 μM, acts as a volume neuromodulator, with targets beyond respective postsynaptic terminals. Given that Zn²⁺ dysregulation is a well-established phenomenon in Parkinson's and Alzheimer's diseases, application of our newly developed method may provide insight into these disorders.