The Hunt for Mechanisms of Circadian Timing in the Eye of Aplysia

The goals of our research are to understand how circadian oscillations in the eye of Aplysia califomica are generated and how entraining agents regulate these oscillations. These goals require identification of the molecular components of the oscillator and entrainment pathways as well as elucidation of the biochemical processes by which these components interact with one another. Our experimental strategy entails tracing environmental information along an entrainment pathway until the last component of the pathway is reached. The isolated eye of Aplysia exhibits a circadian rhythm of optic nerve impulses. This rhythm is regulated by at least two entrainment pathways. A photic pathway entrains the rhythm to light-dark cycles and an efferent serotonergic pathway relays neural information from the CNS to the oscillator. Phase shifting by light appears to involve an increase in the levels of cGMP, depolarization, and protein synthesis. Phase shifting by serotonin appears to involve an increase in the levels of cAMP, hyperpolarization, and protein synthesis. The involvement of protein synthesis in the entrainment pathways, together with the findings that brief treatments of inhibitors of protein synthesis phase shift the rhythm and that continuous treatments of these inhibitors alter the period of the rhythm, indicates that translation is part of the oscillator mechanism. Recent evidence indicates that transcription may also be part of the oscillator mechanism. Brief treatments with DRB, a reversible transcription inhibitor, phase shift the rhythm while continuous treatments with DRB lengthen the period of the rhythm. A comparison of the effects of transcription and translation inhibitors on the rhythm indicates that transcription and translation are closely coupled in the eye circadian system. To know the precise role of transcription and translation in the circadian system, it is necessary to identify and then study specific proteins and mRNAs important for circadian timing. To identify putative oscillator proteins (POPs), we have hunted for proteins whose synthesis or phosphorylation was altered by the entraining agents light and 5-HT and by other agents that perturb the circadian rhythm. By exposing eyes to labeled amino acids in the presence of phase-shifting treatments and then using two-dimensional gel electrophoresis to separate proteins, we found eight proteins that may be considered POPs. To elucidate the cellular function of POPs, we have begun to obtain their amino acid sequences. A 40,000, pi 5.6 protein (POP-1) was identified as a member of the lipocortin family of proteins. Lipocortins are Ca2+-phospholipid binding proteins whose functions include inhibition of PLA2. This finding indicates that the arachidonic acid metabolic pathway may play a role in the Aplysia eye circadian system. Preliminary results with inhibitors of the arachidonic acid metabolic pathway indicate that this indeed might be the case. We are currently characterizing other POPs. Our results have begun to yield a skeleton of the eye circadian system (chronoskeleton) and perhaps a glimpse into the biochemical structure of the pacemaker itself. Certainly, the skeletal description obtained thus far provides a framework upon which to launch further studies into the molecular structure of the pacemaker.