Electrophysiological analysis of purinergic signaling of microglia as an approach to studying the immune mechanisms of Alzheimer's disease

Abstract

Neuroinflammation mediated by chronic microglial activation is considered a key factor in the pathogenesis of Alzheimer's disease (AD). Purinergic signaling, particularly P2X4 and P2X7 receptors activated by extracellular ATP, plays an important role in modulating microglial function. Understanding the ionic mechanisms triggered by these receptors is essential for identifying therapeutic targets and requires high-precision electrophysiological methods. The aim of this study was to investigate ATP-induced ionic mechanisms in BV-2 microglial cells to better understand the role of purinergic signaling in neuroinflammation and neurodegenerative diseases, including AD. The study was performed on the immortalized BV-2 mouse microglia line and HEK293 cells, used to optimize recording parameters. Electrophysiological measurements were performed in the whole-cell configuration at a holding potential of –60 mV. Purinergic receptors were activated by local application of ATP (1 mM). Conditions for stable gigaohm seal formation and transition to the whole-cell configuration were optimized for cells with amoeboid morphology. ATP application induced inward currents in all BV-2 cells studied (n=10), with a mean amplitude of 188 ± 33 pA. Responses showed slow development kinetics and no rapid desensitization during agonist exposure. Unlike primary human cultures, BV-2 cells did not exhibit the classic biphasic kinetics associated with full opening of the P2X7 receptor pore. This may indicate predominance of the P2X4 subtype or specific regulation of purinergic receptors in immortalized cell lines. The results confirm the effectiveness of patch-clamp for assessing microglial functional state and support BV-2 cells as a model for primary screening of purinergic signaling modulators.