Bidirectional coupling of neuronal Ca²⁺ and nitric oxide signals visualized by a dual biosensor

Nitric oxide (NO) and calcium (Ca²⁺) are tightly interconnected mediators of neuronal signaling, yet their dynamic relationship in primary neurons has remained widely unexplored. Here we report a bicistronic dual biosensor that combines jGCaMP8s for high-sensitivity Ca²⁺ imaging with the orange fluorescent NO reporter O-geNOps, delivered by adeno-associated virus (AAV) to primary hippocampal neurons. We establish imaging conditions optimized for physiological temperature and oxygen, which critically shape NO bioavailability. Under these conditions, spontaneous Ca²⁺ spikes and network activity are largely insufficient to elicit detectable NO production, whereas robust Ca²⁺ elevation by high extracellular K⁺ (50 mM) or glutamate (30 μM) evokes robust NO signals that surpass responses to exogenous NO donors. Conversely, both endogenous and exogenously applied NO dampen spontaneous Ca²⁺ spikes, revealing feedback control of neuronal excitability in the hippocampus. Together, these findings demonstrate bidirectional functional coupling between Ca²⁺ and NO signals in primary hippocampal neurons and introduce an informative approach for dual-color visualization of both messengers under near-physiological conditions.