Nicotinic Acetylcholine Receptor‐Mediated Metabotropic Signalling in Human Microglia

Nicotinic acetylcholine receptors (nAChRs) are well-recognized as ionotropic ligand-gated ion channels in the central and peripheral nervous systems. However, their role in non-neuronal cells such as microglia is less well understood due to challenges in detecting ion channel activity in the plasma membrane of immune cells, which hampers functional characterization. This study investigated nAChR-mediated intracellular signaling pathways in human microglia, exploring possible mechanisms underlying cholinergic modulation of neuroinflammation. We verified transcript expression of nAChR subunits α7, α9, and α10 in human C06 microglia and demonstrated that acetylcholine (ACh) triggers intracellular signaling consistent with nAChR-mediated metabotropic responses, concurrent with pharmacological ablation of muscarinic activity. In the absence of extracellular Ca²⁺, ACh evoked transient elevations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in functionally enriched microglia. These responses were sensitive to U73122 and 2-APB, indicating the mobilization of internal Ca²⁺ stores via the phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP₃) pathways, respectively. In C06 microglia, extracellular Ca²⁺ is crucial for replenishing Ca²⁺ stores. Once replenished, repeated ACh exposure enhanced both the incidence and amplitude of microglial [Ca²⁺]_i responses, indicating agonist-induced sensitization. These findings uncover previously unrecognized pathways for nAChR signaling in human microglia, potentially opening new therapeutic avenues for suppressing inflammation.