The long-term objectives of this project are to understand the cellular and molecular mechanisms by which steroid and related hormones regulate the development of neuronal circuits. These hormones mediate profound structural and functional changes in the developing central nervous system that are crucial for the maturation of normal behavior. In the proposed study the mechanisms and consequences of hormone action will be investigated in detail by exploiting the advantages of the relatively simple insect nervous system. During insect metamorphosis, identified motor neurons are modified structurally and functionally to accommodate changes in behavior, and several lines of evidence implicate the steroid 20-hydroxyecdysone (20-HE) as a controlling factor. To investigate how ecdysteroids mediate these developmental changes identified motor neurons will be placed in primary cell culture after the incorporation of non-toxic fluorescent labels, which will facilitate cell identification. The ability of identified motor neurons to retain their cell-specific and stage- specific properties in culture will be investigated using a morphometric system to analyze process outgrowth. Intracellular recordings and whole- cell voltage clamp techniques will be used to describe the ligand-gated and voltage-gated currents in neurons isolated from animals of different stages of life. The developmental effects of 20-HE alone, or in combination with other insect hormones. The effects of these hormones on process outgrowth and biophysical properties will be compared for different neurons, and for the same neuron isolated from animals at different stages of development. Finally, the molecular mechanisms of steroid action will be investigated through the addition of protein and mRNA synthesis inhibitors to the culture medium for short periods of time following ecdysteroid exposure. Information obtained by use of this simple system will provide insights into the mechanisms by which steroid hormones and other developmental signals operate in more complex systems.
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