The isolated eyes of several marine mollusks express circadian rhythms in spontaneous optic nerve impulse frequency. In Bulla gouldiana, one of the most intensively studied preparations, the ocular rhythm is generated among approximately 100 neurons at the retinal base (basal retinal neurons). These neurons are electrically coupled to one another and fire in synchrony. Recently completed experiments with basal retinal neurons plated at low density in dispersed cell culture reveal circadian rhythms in membrane conductance. Therefore, circadian rhythm generation appears to be a property of individual neurons and does not require a tissue level of organization. The overall goal of the proposed research is to obtain an understanding of the cellular mechanisms underlying circadian rhythm generation, entrainment and expression. Experiments are directed towards seven specific aims: 1) The single neuron circadian oscillator model will be rigorously tested by determining whether completely isolated neurons remain rhythmic. The single cell model will then be developed by comparing the circadian properties of isolated neurons with the intact retina. 2) A similar analysis will be conducted for the eye of Aplysia. 3) The role of a transmembrane calcium signal for pacemaker entrainment will be explored in Bulla and in Aplysia. The temporal profile of the calcium signal and the type(s) of calcium channels involved will be identified. 4) The specific K+ conductances generating the circadian rhythm in membrane potential will be identified and the importance of phosphorylation in modulation of K+ conductances investigated. 5) The importance of changes in membrane potential and the underlying conductances in circadian rhythm generation will be evaluated. 6) The role of efferent FMRFamide projections in the modulation of pacemaker properties will be explored. 7) The mechanisms responsible for induction of """"""""phase-jumps"""""""" between the mutually coupled ocular pacemaker will be identified. The research program will employ extracellular, intracellular sharp electrode, whole-cell and patch-clamp electrophysiological recording procedures and digital imaging with fluorescent calcium probes. The development of a single neuron model for circadian oscillator research provides a unique opportunity to study the cellular basis of biological timing. The generation, synchronization and modulation of circadian rhythms represent fundamental questions in neurobiological research.
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