Intracellular calcium responses of circadian pacemaker neurons measured with fura-2

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni2+. To test directly if an influx of Ca2+ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K+. Calcium levels in the BRNs remained elevated during treatments with 50 mM K+ lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca2+ (approx. 1.7 x 10(-7) M) during depolarization blocked the rise in intracellular Ca2+, verifying that a Ca2+ influx is required. The sustained Ca2+ elevation during depolarization was also prevented with 50 mM Ni2+, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni2+, which does not block phase shifts. The initial rise in [Ca2+]i in response to 50 mM K+ was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca2+ signal.