PHYSIOLOGICAL SUBSTRATES OF A CIRCADIAN OSCILLATOR

该提案代表了表征生理学特征的意图， 调节脑中主要昼夜节律振荡器的底物 大鼠视交叉上核（SCN）神经元。 这些神经元 在生成和夹带中发挥明确的关键作用 生理、代谢和行为功能的日常波动。 采用离体下丘脑脑片技术， 在完整动物中难以解决的昼夜节律机制， 但大脑切片提供了不寻常的优势。 在我们手中， 体外SCN维持放电率和肽的昼夜节律 分泌长达38小时，即使减少以去除外周 下丘脑区域通常包括在切片中。 您因前述 发现电振荡可以在体外相移， 反应曲线与完整动物相似。 因此，我们的初步 神经生理学研究表明，不仅24小时 振荡器，而且相位重置机制是SCN内生的 大脑切片中的神经元。 初步的生物化学研究表明， 特异性SCN蛋白的磷酸化状态，并表明， 磷酸化机制可以调节细胞内的昼夜振荡， 这些神经元的活动。 我们建议在体外表征SCN的相位响应性， 刺激重置电活动中的振荡， 常规神经生理学和器官培养技术。 这种精确 相位响应曲线的定义将用于生理学中， 研究表征代谢途径背后的电 振荡，特别是环核苷酸和Ca++依赖性蛋白 磷酸化以及涉及新mRNA合成的途径， 蛋白 与此同时，将进行生化研究， 确定磷蛋白及其调节剂在正常过程中的变化 昼夜节律周期和相移期间。 因为SCN集成了 昼夜节律行为和代谢通量，这项研究具有基本的相关性， 了解许多大脑和代谢功能障碍，包括某些 各种形式的精神疾病。

This proposal represents an intent to characterize the physiological substrates which modulate the primary circadian oscillator in the brain of the rat, the neurons of the suprachiasmatic nuclei (SCN). These neurons serve a well-defined and critical role in the generation and entrainment of the daily oscillations of physiological, metabolic and behavioral functions. The in vitro hypothalamic brain slice technique will be used to investigate circadian mechanisms which are difficult to address in the intact animal, but for which the brain slice offers unusual advantage. In our hands, the SCN in vitro sustains a circadian rhythm of firing rate and peptide secretion for up to 38 hr, even when reduced to remove peripheral hypothalamic regions normally included in the slice. Further, we have found that the electrical oscillations can be phase-shifted in vitro in a response curve similar to the intact animals. Thus, our preliminary neurophysiological investigations have shown that not only the 24 hr oscillator but also the phase resetting mechanism is endogenous to the SCN neurons in the brain slice. Preliminary biochemical studies demonstrate circadian changes in the phosphorylation state of specific SCN proteins and suggest that phosphorylation mechanisms may modulate circadian oscillations in the activity of these neurons. We propose to characterize the phase responsiveness of the SCN in vitro to stimuli which reset the oscillations in electrical activity using conventional neurophysiological and organ culture techniques. This precise definition of the phase-response curve will be used in physiological studies to characterize metabolic pathways underlying the electrical oscillations, particularly cyclic nucleotide- and Ca++-dependent protein phosphorylation as well as pathways involving synthesis of new mRNA and protein. Concurrently, biochemical studies will be carried out to further identify changes in phosphoproteins and their regulators during the normal circadian cycle and during phase-shifting. Because the SCN integrates most circadian behaviors and metabolic fluxes, this study has basic relevance to understanding many brain and metabolic dysfunctions, including certain forms of mental illness.