SIGNAL TRANSDUCTION AND GENE EXPRESSION IN LTP AND LTD

LTP 和 LTD 中的信号转导和基因表达

• 批准号: 6243157
• 负责人: HOWARD SCHULMAN
• 金额: $ 15.13万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6243157
• 关键词: biological signal transduction; cAMP response element binding protein; calcium; calmodulin dependent protein kinase; confocal scanning microscopy; gene expression; genetically modified animals; hippocampus; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rat; long term potentiation; molecular cloning; neural plasticity; neurogenetics; nitric oxide; polymerase chain reaction; protein kinase C; second messengers; single cell analysis; synapses; synaptic vesicles

The hippocampus in intact animals, in slice preparation, and as isolated neurons in culture offers an experimental opportunity to study both long- term depression (LTD) and long-term potentiation (LTD), forms of synaptic plasticity utilized in learning and memory. Ca2+ is a key signaling molecular regulating synaptic plasticity in the hippocampus and we will focus on several aspects of Ca2+ action including the activation of protein kinases/phosphatases, nitric oxide synthase, and transcription of genes that underlie changes in synaptic function during LTD and LTP. Nitric oxide (no) is a retrograde messenger that we have found to stimulate synaptic release in a Ca2+-independent manner. We have collaborated with Dr. Richard Scheller to demonstrate NO alters protein-protein interactions among the synaptic proteins VAMP, syntaxin, n-secl, and SNAP-25 that may be responsible for such release. We will define, quantitate, and identify the sites of these changes. We will determine which neurotransmitter classes are affected and whether NO alters stimulated release. We have demonstrated a mechanism by which multifunctional CaM kinase II may be switched to a Ca2+ -independent species in a stimulus frequency- dependent manner. In collaboration with Dr. Richard Tsien we will correlate activation of the kinase at various frequencies that elicit LTD or LTP in hippocampal cultures. Immunocytochemistry with phosphoselective Ab and biochemical analysis will compare antagonism between CaM kinase II and calcineurin, a Ca2+-dependent phosphatase. We will examine how Ca2+ can change the sign of the synaptic strength by favoring activation of CaM kinase II to elicit a potentiation or favoring calcineurin to elicit a depression. We will examine Ca2+ -based signaling pathways from the glutamate receptors on synaptic spines to the phosphorylation of the transcription factor CREB in the nucleus with Dr. Tsien. Transgenic animals with reporter genes driven by promoters for regulatory elements for CREB and other transcription factors will be generated to examine transcription at different stimulus frequencies. Single cell PCR will be used to correlate synaptic plasticity and induciton of genes in single cells examined morphologically under the microscopy. Finally, we have developed a method termed indexing which will be optimized to a single cell level and will allow us to compare cDNA from control, LTD or LTP neurons and thereby clone novel plasticity genes.

完整动物中的海马，切成薄片的制备和分离 文化中的神经元提供了研究长期研究的实验机会 术语抑郁（LTD）和长期增强（LTD），突触的形式 可塑性用于学习和记忆。 CA2+是关键信号 分子调节海马中的突触可塑性，我们将 专注于Ca2+动作的几个方面，包括蛋白质的激活 激酶/磷酸酶，一氧化氮合酶和基因转录 这是LTD和LTP期间突触功能的变化。 一氧化氮（NO）是我们发现刺激的逆行信使 以Ca2+非依赖性方式突触释放。 我们已经与 理查德·谢勒（Richard Scheller）博士展示没有变化的蛋白质 - 蛋白质相互作用 在突触蛋白鞋面鞋面，语法素，N-secl和SNAP-25中可能是 负责此类版本。 我们将定义，定量和确定 这些更改的站点。 我们将确定哪些神经递质类 受到影响，是否没有改变刺激的释放。 我们已经证明了一种多功能CAM激酶II的机制 在刺激频率中切换到Ca2+独立的物种 依赖方式。 与理查德·蒂恩博士合作，我们将 在引起Ltd的各种频率下激活激活 或海马培养物中的LTP。 磷酸化的免疫细胞化学 AB和生化分析将比较CAM激酶II之间的拮抗作用 和钙调神经酶，一种Ca2+依赖性磷酸酶。 我们将研究CA2+如何 可以通过赞成激活CAM来改变突触强度的迹象 激酶II引起增强或赞成钙调神经酶引起 沮丧。 我们将检查来自谷氨酸受体的基于CA2+的信号通路 在转录因子CREB的磷酸化的突触刺上 在Tsien博士的核中。 带有记者基因的转基因动物 由促进者的CREB和其他调节元素的驱动器驱动 将生成转录因子以检查在 不同的刺激频率。 单细胞PCR将用于关联 所检查的单细胞中基因的突触可塑性和诱导 在显微镜下形态学。 最后，我们开发了一种方法 称为索引，将优化为单个单元格，将 允许我们比较来自Control，Ltd或LTP神经元的cDNA，从而克隆 新型可塑性基因。