Electric Studies of Excitation, Secretion & Contraction

描述(申请人提供)：松果体是大脑中的一个内分泌器官，主要由交感神经系统释放的去甲肾上腺素调节。它是我们生物钟系统的一部分，向全身广播夜间荷尔蒙--褪黑素。松果体细胞的夜间状态伴随着相对于白天状态的基因表达的广泛变化。除了哺乳动物的松果体细胞对光不敏感外，许多表达的基因都是由姐妹血统--光感受器表达的基因。我们的总体假设是松果体细胞在两种截然不同的电生理兴奋性之间切换 有助于调节褪黑激素分泌、夹带和维持昼夜节律的状态。利用生物物理技术，从离子通道补体、钙信号动力学、受体激活、第二信使水平和分泌机制等方面对比培养的大鼠松果体细胞的昼夜状态。这种状态的改变将通过将培养的原代细胞预先培养在适当的神经递质中来实现。细胞将从大鼠松果腺中分离出来，置于细胞培养中，用去甲肾上腺素处理模拟夜晚，并在显微镜下进行研究，使用全细胞巨细胞记录、钙敏感染料光度测定、神经递质的安培和高效液相分析以及使用各种活细胞指示剂作为第二信使。假设夜间松果体细胞具有与光感受器相关的电兴奋性，而白天松果体细胞被假设为相对静止。5-羟色胺是通过量子胞吐作用从松果体细胞分泌出来的，而褪黑素和N-乙酰-5-羟色胺是通过疏水扩散分泌的，这一假设也将得到验证。了解松果体的节律性分泌机制将对治疗睡眠障碍、对短时间的季节性情感反应以及因时差和轮班工作而导致的注意力丧失做出重要贡献。

DESCRIPTION (provided by applicant): The pineal gland is an endocrine organ in the brain that is primarily regulated by noradrenaline released by the sympathetic nervous system. It is the part of our circadian clock system that broadcasts the night hormone, melatonin, to all of the body. The night state of pinealocytes is accompanied by extensive changes in gene expression relative to the day state. Many expressed genes are those expressed by a sister lineage, photoreceptors, except that mammalian pinealocytes are not photosensitive. Our overall hypothesis is that pinealocytes switch between two very different electrophysiological excitability states that contribute to regulated secretion of melatonin and to entrainment and maintenance of circadian rhythms. Using biophysical techniques, the night and day states of cultured rat pinealocytes will be contrasted in terms of their ion channel complement, calcium signal dynamics, receptor activation, second messenger levels, and secretory mechanisms. The state changes will be achieved by preincubating cultured primary cells in appropriate neurotransmitters. Cells will be dissociated from rat pineal glands, placed in cell culture, treate with norepinephrine to mimic night, and studied under the microscope with techniques such as whole-cell gigaseal recording, photometry of calcium-sensitive dyes, amperometry and HPLC of neurotransmitters, and use of various live-cell indicators for second messengers. Night pinealocytes are hypothesized to have electrical excitability related to that of photoreceptors, and day pinealocytes are hypothesized to be relatively quiescent. The hypothesis that serotonin is secreted from pinealocytes by quantal exocytosis whereas melatonin and N-acetyl serotonin are secreted by hydrophobic diffusion will also be tested. Understanding the rhythmic secretory mechanisms of the pineal will make an important contribution towards treating sleep disorders, seasonal affective responses to short days, and loss of attention due to jet lag and shift work.