The Role of Extracellular Calcium in Generating and in Phase-Shifting the Bulla Ocular Circadian Rhythm

Since extracellular calcium is known to be involved in the entrainment of the circadian pacemaker in the retina of Bulla gouldiana, we have assessed the requirement for extracellular calcium in the generation of the circadian rhythm. To enable us to assay the state of the pacemaker during low-calcium treatment, which often obscures rhythmicity, long-duration pulses of low-calcium artificial seawater (no added calcium, 10 mM EGTA, calculated calcium concentration = 4.5 x 10^-10 M) were applied, and the phase of the subsequent rhythm was measured. Pulse treatments started at zeitgeber time (ZT) 6, and durations ranged from 4 to 72 hr. Although no phase shifts followed pulses ending before the next projected dawn (ZT 24), phase delays of up to 4 hr followed pulses ending after projected dawn, and delays of up to 8 hr followed pulses spanning two dawns. Some activity records exhibited unequivocal circadian rhythmicity during the long low-calcium treatments, with phases and periods similar to untreated control eye records; this finding suggests that the phase delays observed following long low-calcium pulses are attributable to the pulsatile nature of the treatment. These data suggest that extracellular calcium is not an essential requirement for the pacemaker in generating the circadian rhythm.