Gap junction-mediated spontaneous Ca2+ waves in differentiated cholinergic SN56 cells

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca2+ waves. These waves were unaffected by tetrodotoxin (1 μM), but blocked by removal of extracellular Ca2+, or addition of non-specific Ca2+ channel inhibitors (Cd2+ (0.1 mM) or Ni2+ (1 mM)). Combined application of antagonists of NMDA receptors (AP5; 100 μM), AMPA/kainate receptors (NBQX; 20 μM), nicotinic AChR receptors (hexamethonium; 100 μM) or inotropic purinoceptors (brilliant blue; 100 nM) was also without effect. However, Ca2+ waves were fully prevented by carbenoxolone (200 μM), halothane (3 mM) or niflumic acid (100 μM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd2+, Ni2+ or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca2+ waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.