FUNCTIONAL IMAGING OF PIEZO1 IN HIPPOCAMPAL NEURONS REVEALS THE CHANNEL ROLE IN ISCHAEMIC NEUROPATHOLOGY

Piezo1 is a mechanically gated ion channel that converts mechanical stimuli, including tissue stiffness, fluid sheer stress and pressure, into rapid intracellular signalling responses. While Piezo1 was extensively studied across various organs, its expression, cell-type distribution, and functional role in the brain remain unclear. Emerging evidence implicates Piezo1 in neurodevelopment and neuropathological conditions such as ischaemic stroke, traumatic brain injury, and tumours; however, direct functional evidence in central neurons is absent. Here, we performed two-photon imaging of immunostained hippocampal tissue to demonstrate comparable membrane localisation of Piezo1 in CA1 and CA3 pyramidal neurons. To directly assess Piezo1 function, we employed GenEPi, a genetically encoded, Piezo1-specific fluorescent sensor previously validated in cardiomyocytes and in vivo in zebrafish. We developed a protocol using lipofectamine-based gene transfer to achieve robust neuronal GenEPi expression in organotypic hippocampal slices within 1–3 weeks post-transduction. Two-photon imaging confirmed reliable sensor performance in the organised brain tissue: mechanical stimulation of GenEPi-expressing cells elicited transient fluorescence responses, and pharmacological activation with the selective Piezo1 agonist Yoda-1 evoked robust signals, validating functional Piezo1 in neurons. To examine the Piezo1 contribution in neuropathology, we modelled ischaemic injury ex-vivo using oxygen-glucose deprivation (OGD). OGD significantly increased Piezo1 immunosignal in pyramidal neurons. Real-time monitoring of GenEPi dynamics revealed Piezo1-mediated Ca2+ influx upon OGD that persisted or further increased during reperfusion, consistent with intracellular Ca2+ overload. Overall, our findings provide evidence of functional Piezo1 in brain neurons and establish GenEPi as a valuable tool for studying Piezo1-dependent mechanisms in health and disease.