Circadian clock in cell culture: I. Oscillation of melatonin release from dissociated chick pineal cells in flow-through microcarrier culture

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Abstract
The avian pineal gland contains circadian oscillators that regulate the rhythmic release of melatonin. We have developed a dissociated chick pineal cell culture system in order to begin a cellular analysis of this vertebrate circadian oscillator. Dissociated pineal cells maintained in cyclic light conditions (LD 12:12) released melatonin rhythmically. The release of melatonin was elevated during the dark and low during the light. A circadian oscillation of melatonin release persisted for at least 5 cycles in constant darkness with a period close to 24 hr; however, there was a gradual damping of the amplitude. Analysis of the rhythm revealed that the observed damping was consistent with either desynchronization of multiple oscillators within the cultures or damping of individual oscillators. The circadian oscillation of melatonin release persisted for up to 4 cycles under conditions of constant light; however, the oscillation was strongly damped and the period of the oscillation was lengthened significantly. Thus, dissociated pineal cells express a persistent circadian oscillation of melatonin release in constant darkness, and properties of this oscillation are modulated by light treatment in vitro. This flow-through cell culture method for dissociated chick pineal cells should provide a useful model for the analysis of a vertebrate circadian system at the cellular level.