MECHANISMS OF AN IN VITRO NEURONAL CIRCADIAN OSCILLATOR

昼夜节律（CRs）代表重要的整合之一， 机制，时间组织，在生活史和行为的 包括人类在内的多细胞生物。 这种振荡器的细胞机制是未知的。 的相关性 CRs对某些疾病的干扰才刚刚开始出现。 躁狂抑郁症是一种特别好的疾病候选者， 其起源可能在于两个昼夜节律振荡器的干扰 (COs)锂仍然是治疗这种疾病最有效的方法。 病人，现在知道锂延长时间，在剂量 依赖的方式，模型软体动物神经元CO系统。 我们的研究 将使用这个模型神经元CO系统，孤立的眼睛的Aaplasia。 这 眼睛产生自发的复合动作电位（CAP）， 增殖到视神经，而在体外和连续分离， 山中贼易 这些CAP的频率随一天中的时间而变化;节律 CAP频率的降低是稳健的，并且在器官培养中持续1 - 2周。 我们 研究将集中在确定产生的生物化学机制， 节奏及其细胞特异性基础。 我们的假设是 昼夜节律调节通过细胞核产生每日mRNA来表达 转录本编码调节离子通道和泵的蛋白质， 膜的内表面直接，或通过中间事件， 例如蛋白质磷酸化。 我们还假设， 被编程为将CO表达为独立单位的专门细胞。 四 具体目标是：1。新的模式的定量研究 合成的蛋白质（及其磷酸化状态）， CT. 将使用放射性氨基酸前体（3H，35S）和2-D凝胶 来分离标记的新合成蛋白质。 无机32PO4 将使用预标记和32 P-γ ATP后标记， 实验，以评估这些蛋白质的磷酸化状态， 生理周期 2.产生针对以下的单克隆抗体（MAb）： 特定细胞类型。 这些单克隆抗体将被用作试剂，以鉴定 细胞类型的蛋白质，其合成在昼夜节律改变， 时尚，与之相关。 3.长期记录的发展 技术从较小的（即非感光细胞）细胞类型在初级 培养以确定是否可以在解离的细胞中观察到CO 和孤立的神经元。 4.测量可能的昼夜节律变化， 通过放射性配体结合的膜通道和泵的数量或状态 实验

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.