PHYSIOLOGICAL SUBSTRATES OF CIRCADIAN OSCILLATOR

下丘脑的视交叉上核（SCN）是内源性的 振荡器，服务于一个明确的，关键的作用， 产生和夹带的日常（昼夜）振荡， 哺乳动物的生理、代谢和行为。 我们广泛的研究 目的是了解分子，细胞和 SCN保持24小时时间的神经生理机制。 研究的模型动物是大鼠。 SCN起搏器存活 在下丘脑脑切片中是完整的， 旨在剖析细胞机制的实验。 我们 方法学方法将脑切片培养与 神经生理学技术，测量昼夜节律， 整体神经元活动和全细胞记录在 切片以及生化分析， 核苷酸水平、酶活性和磷酸化的蛋白质， 和免疫细胞化学/原位杂交技术， 识别和定位感兴趣分子。 目前的建议自然发展从我们的发现， SCN节律可以在脑切片中重置， cAMP-、cGMP-或百日咳毒素敏感途径。 此外，即使 在恒定的切片室条件下， 底物发生变化，因此节律被cAMP重置， 主观白天、主观夜间cGMP和褪黑素 在日/夜过渡期间。 我们的具体目标包括：A） 更全面地探讨cAMP/蛋白激酶A在SCN中的作用 功能（通过检查活动，浓度和 蛋白激酶A（PKA）的磷酸化状态，定位 cAMP和PKA作用的位点，以及 cAMP刺激中的转录/翻译）; B）更充分地 探讨cGMP/蛋白激酶G在夜间的作用（通过测定 蛋白质活性、浓度和磷酸化状态 激酶G（PKG），定位cGMP和PKG效应; C）检查 环核苷酸磷酸二酯酶的调节（通过测定 cAMP和cGMP磷酸二酯酶水平及其调节 D）了解其他第二类活动的关系 信使/激酶系统产生昼夜节律时间（通过探索 蛋白激酶在计时中的相互作用）。 因为 SCN整合了大多数昼夜行为和代谢变化， 这项研究对了解许多大脑具有基本意义， 代谢功能障碍，包括睡眠障碍和某些形式的 精神疾病

The suprachiasmatic nuclei (SCN) of the hypothalamus are endogenous oscillators that serve a well-defined, critical role in the generation and entrainment of the daily (circadian) oscillations of physiology, metabolism and behavior of mammals. Our broad research objective is to understand the molecular, cellular and neurophysiological mechanisms by which the SCN keep 24-hr time. The model species studied is the rat. The SCN pacemaker survives intact in the hypothalamic brain slice where it is accessible to experiments aimed at dissecting cellular mechanisms. Our methodological approach combines brain slice culture with neurophysiological techniques that measure the circadian rhythm of the ensemble neuronal activity and whole cell recordings in the slice as well as biochemical analyses that measure cyclic nucleotide levels, enzyme activities and proteins phosphorylated, and immunocytochemical/in situ hybridization techniques that identify and localize molecules of interest. The present proposal develops naturally from our finding that the SCN rhythm can be reset in the brain slice by treatments affecting cAMP-,cGMP- or pertussis toxin-sensitive pathways. Further, even under the constant conditions in the slice chamber, the pacemaker substrates are changing so that the rhythm is reset by cAMP in subjective day, by cGMP during subjective night and by melatonin during the day/night transition. Our specific aims include: A) to more fully explore the role of cAMP/protein kinase A in SCN function (by examining the activity, concentration and phosphorylation state of protein kinase A (PKA), localizing the sites of cAMP and PKA effects, and the involvement of transcription/translation in cAMP stimulation); B) to more fully explore the role of cGMP/protein kinase G at night (by determining the activity, concentration and phosphorylation state of protein kinase G (PKG), localizing cGMP and PKG effect; C) to examine the regulation of cyclic nucleotide phosphodiesterase (by determining the level and regulation of cAMP and cGMP phosphodiesterase activities) and D) to understand the relationship of other second messenger/kinase systems in generating circadian time (by exploring the interactive role of protein kinases in time-keeping). Because the SCN integrates most circadian behaviors and metabolic changes, this study has basic relevance of understanding many brain and metabolic dysfunctions, including sleep disorders and certain forms of mental illness.