Spontaneous, synchronous zinc spikes oscillate with neural excitability and calcium spikes in primary hippocampal neuron culture.

Zinc has been suggested to act as an intracellular signaling molecule due to its regulatory effects on numerous protein targets including enzymes, transcription factors, ion channels, neurotrophic factors, and postsynaptic scaffolding proteins. However, intracellular zinc concentration is tightly maintained at steady levels under natural physiological conditions. Dynamic changes in intracellular zinc concentration have only been detected in certain types of cells that are exposed to pathologic stimuli or upon receptor ligand binding. Unlike calcium, the ubiquitous signaling metal ion that can oscillate periodically and spontaneously in various cells, spontaneous zinc oscillations have never been reported. In this work, we made the novel observation that the developing neurons generated spontaneous and synchronous zinc spikes in primary hippocampal cultures using a fluorescent zinc sensor, FluoZin-3. Blocking of glutamate receptor-dependent calcium influx depleted the zinc spikes, suggesting that these zinc spikes were driven by the glutamate-mediated spontaneous neural excitability and calcium spikes that have been characterized in early developing neurons. Simultaneous imaging of calcium or pH together with zinc, we uncovered that a downward pH spike was evoked with each zinc spike and this transient cellular acidification occurred downstream of calcium spikes but upstream of zinc spikes. Our results suggest that spontaneous, synchronous zinc spikes were generated through calcium influx-induced cellular acidification, which liberates zinc from intracellular zinc binding ligands. Given that changes in zinc concentration can modulate activities of proteins essential for synapse maturation and neuronal differentiation, these zinc spikes might act as important signaling roles in neuronal development.

Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn2+ signals.

Although numerous fluorescent Zn2+ sensors have been reported, it is unclear whether and how Zn2+ can be released from the intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn2+. Here, we create a genetically encoded sensor, GZnP3, which demonstrates unprecedented sensitivity for Zn2+ at sub-nanomolar concentrations. Using GZnP3 as well as GZnP3-derived vesicular targeted probes, we provide the first direct evidence that Zn2+ can be released from endolysosomal vesicles to the cytosol in primary hippocampal neurons through the TRPML1 channel. Such TRPML1-mediated Zn2+ signals are distinct from Ca2+ in that they are selectively present in neurons, sustain longer, and are significantly higher in neurites as compared to the soma. Together, our work not only creates highly sensitive probes for investigating sub-nanomolar Zn2+ dynamics, but also reveals new pools of Zn2+ signals that can play critical roles in neuronal function.