Circadian clock in cell culture: II. In vitro photic entrainment of melatonin oscillation from dissociated chick pineal cells

The avian pineal gland contains circadian oscillators that regulate the rhythmic synthesis of melatonin. We have developed a flow-through cell culture system in order to begin to study the cellular and molecular basis of this vertebrate circadian oscillator. Pineal cell cultures express a circadian oscillation of melatonin release for at least 5 cycles in constant darkness with a period close to 24 hr. In all circadian systems, light regulates the rhythm by the process of entrainment that involves control of the phase and period of the circadian oscillator. In chick pineal cell cultures we have investigated the entraining effects of light in 2 ways: by shifting the light-dark cycle in vitro and by measuring the phase-shifting effects of single light pulses. A 6 hr advance or delay of a LD 12:12 light-dark cycle produced a corresponding shift in the melatonin rhythm. The phase shifts of the rhythms persisted after transfer to constant darkness, showing that the underlying circadian oscillator was entrained. Photic entrainment of the oscillator was further characterized by measuring the phase-shifting effects of single 6 hr light pulses. Single pulses of light shifted the phase of the circadian oscillator in a phase-dependent manner. Light pulses beginning early in the subjective night delayed the phase of the oscillation 8 hr relative to dark controls. Conversely, light pulses beginning late in the subjective night advanced the phase of the oscillation nearly 8 hr. Thus, photoreceptors within the cell cultures can mediate entrainment of the pineal oscillators. Phase-dependent phase shifts to light pulses (as described in a 'phase-response curve') are a fundamental characteristic of all circadian systems. The expression of this circadian property in the pineal cell cultures should permit a mechanistic analysis of the photic pathway.