MECHANISMS OF AN IN VITRO NEURONAL CIRCADIAN OSCILLATOR

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Circadian rhythms (CRs) represent one of the important integrating mechanisms, for temporal organization, in the life history and behavior of multicellular organisms including man. The fundamental biochemical and cellular mechanisms of such oscillators are not known. The relevance of disturbances in CRs to certain diseases is just beginning to emerge. Manic-depressive illness is a particularly good candidate for a disease whose origins may lie in disturbances of the two circadian oscillators (COs) known in man. Lithium remains the most effective treatment for such patients and it is now known that lithium lengthens the period, in a dose dependent manner, of a model molluscan neuronal CO system. Our research will use this model neuronal CO system, the isolated eye of Aplysia. This eye generates spontaneous compound action potentials (CAPs) which are propagated into the optic nerve, while isolated in vitro and in continuous darkness. The frequency of these CAPs varies with time of day; the rhythm of CAP frequency is robust and persists from 1-2 wks in organ culture. Our research will focus on determining the biochemical mechanisms that generate the rhythm and its cell-specific basis. Our working hypothesis is that circadian modulation is expressed through nuclear production of daily mRNA transcripts coding for proteins that modulate ion channels and pumps from the inner surface of the membrane directly, or through intermediary events, such as protein phosphorylation. We also postulate that there are specialized cells programmed to express a CO as independent units. Four specific aims are planned: 1. Quantitative studies of the pattern of newly synthesized proteins (and their phosphorylation state) as a function of CT. Radioactive amino-acid precursors (3H, 35S) and 2-D gels will be used to separate labeled newly synthesized proteins. Inorganic 32PO4 prelabeling and 32P-gamma ATP postlabeling will be used, in independent experiments, to assess the phosphorylation state of these proteins during the circadian cycle. 2. Generation of monoclonal antibodies (MAbs) to specific cell types. These MAbs will be used as reagents to identify the cell type that the proteins, whose syntheses are altered in a circadian fashion, are associated with. 3. Development of long-term recording techniques from the smaller (i.e. non-photoreceptor) cell types in primary cultures in order to determine whether COs can be observed in dissociated and isolated neurons. 4. Measurements of possible circadian changes in the numbers or state of membrane channels and pumps by radioligand binding experiments.