MECHANISMS OF AN IN VITRO NEURONAL CIRCADIAN OSCILLATOR

体外神经元昼夜节律振荡器的机制

• 批准号: 3379767
• 负责人: FELIX STRUMWASSER
• 金额: $ 10.68万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3379767
• 关键词: action potentials; cell type; circadian rhythms; eye; gel electrophoresis; genetic transcription; ion transport; membrane permeability; messenger RNA; monoclonal antibody; neurons; phosphorylation; radiotracer; single cell analysis; tissue /cell culture

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.

昼夜节律(CRS)是人体生命活动的重要组成部分 时间组织的机制，在生命历史和行为中 包括人类在内的多细胞生物。基本的生物化学和 这种振荡器的细胞机制尚不清楚。的关联性 CRS对某些疾病的干扰才刚刚开始出现。 躁郁症是一种特别好的疾病候选者 它的起源可能在于两个昼夜节律振荡器的扰动 (因为)在人类中已知。锂仍然是治疗这种疾病的最有效的方法 现在已经知道，锂可以在一定剂量内延长病程。 依赖的方式，一个模型软体动物神经元CO系统。我们的研究 将使用此模型的神经元CO系统，分离出海绵状突眼。这 眼睛产生自发的复合动作电位(CAP)，这是 在体外和连续分离时，传播到视神经 夜幕降临。这些帽子的频率随着一天中的时间而变化；节奏 CAP的频率很高，在器官培养中持续1-2周。我们的 研究将集中在确定产生的生化机制 节律及其特定的细胞基础。我们的工作假设是 昼夜节律的调节是通过每日mRNA的核生产来表达的 编码调节离子通道和泵的蛋白质的转录本 膜的内表面直接，或通过中介事件， 比如蛋白质的磷酸化。我们还假设有 被编程为将一氧化碳表达为独立单位的特殊细胞。四 具体目标是：1.新经济发展模式的量化研究 合成的蛋白质(及其磷酸化状态)作为 CT.将使用放射性氨基酸前体(~3H，~35S)和2-D凝胶 分离标记的新合成的蛋白质。无机32PO4 将独立使用预标记和32P-伽马ATP后标记 实验，以评估这些蛋白质的磷酸化状态 昼夜节律。2.抗肿瘤单抗的制备 特定的细胞类型。这些单抗将被用作试剂来鉴定 蛋白质的合成在昼夜节律中发生变化的细胞类型 时尚，与时尚联系在一起。3.长期录音的发展 来自较小(即非光感受器)细胞类型的技术 培养以确定是否可以在分离的情况下观察到CoS 和孤立的神经元。4.测量可能的昼夜节律变化 通过放射性配基结合的膜通道和泵的数量或状态 实验。