Abstract

Hydrogen sulfide ($\text{H}_2\text{S}$) is increasingly recognized as a crucial gasotransmitter that modulates numerous physiological processes in the central nervous system, including neuroprotection against oxidative stress and regulation of synaptic function. Real-time monitoring of endogenous $\text{H}_2\text{S}$ dynamics in living cells, particularly in sensitive primary neuronal cultures, is essential for understanding its precise role in cell biology and pathology. This study investigates the feasibility of using a novel Acedan-based fluorescent probe to monitor $\text{H}_2\text{S}$ fluctuations in primary neuronal cultures. The probe's performance was first characterized in vitro, demonstrating high selectivity and sensitivity towards $\text{H}_2\text{S}$ over other biologically relevant thiols and reactive oxygen species. The mechanism of action involves a $\text{H}_2\text{S}$-triggered reaction that leads to a significant turn-on fluorescence signal, ideal for live-cell imaging. Application of the probe to primary hippocampal and cortical neuronal cultures successfully visualized both basal and stimulated $\text{H}_2\text{S}$ production. Specifically, the probe was able to detect the rapid increase in $\text{H}_2\text{S}$ concentration following the administration of $\text{H}_2\text{S}$-generating enzymes' substrates. These findings confirm the probe's utility as a valuable tool for studying $\text{H}_2\text{S}$ signaling pathways, offering a non-invasive method to link $\text{H}_2\text{S}$ dynamics to neuronal function and dysfunction in a cell biology context.