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: 6111547
• 负责人: HOWARD SCHULMAN
• 金额: $ 15.44万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6111547
• 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.

完整动物的海马体、切片制备和分离的海马体 培养中的神经元提供了研究长期神经元的实验机会 长时程抑制（LTD）和长时程增强（LTD），突触的形式 用于学习和记忆的可塑性。 Ca2+ 是关键信号传导 调节海马突触可塑性的分子，我们将 关注 Ca2+ 作用的几个方面，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期间突触功能变化的基础。 一氧化氮（NO）是一种逆行信使，我们发现它可以刺激 突触以不依赖 Ca2+ 的方式释放。 我们合作过 Richard Scheller 博士证明 NO 会改变蛋白质-蛋白质相互作用 突触蛋白 VAMP、突触蛋白、n-secl 和 SNAP-25 可能是 负责此类发布。 我们将定义、量化和识别 这些变化的地点。 我们将确定哪些神经递质类别 是否受到影响以及 NO 是否会改变刺激释放。 我们已经证明了多功能 CaM 激酶 II 可能的机制 在刺激频率下切换到 Ca2+ 独立物种 依赖方式。 与钱博士合作，我们将 将引起 LTD 的各种频率下的激酶激活相关联 或海马培养物中的 LTP。 磷酸选择性免疫细胞化学 Ab和生化分析将比较CaM激酶II之间的拮抗作用 和钙调神经磷酸酶，一种 Ca2+ 依赖性磷酸酶。 我们将研究 Ca2+ 如何 可以通过促进 CaM 的激活来改变突触强度的符号 激酶 II 引起增强或有利于钙调神经磷酸酶引起 沮丧。 我们将检查来自谷氨酸受体的基于 Ca2+ 的信号传导途径 突触棘对转录因子 CREB ​​磷酸化的影响 与钱博士一起在核心。 带有报告基因的转基因动物 由 CREB ​​和其他监管要素的发起人推动 将生成转录因子来检查转录 不同的刺激频率。 单细胞 PCR 将用于关联 检查单细胞中的突触可塑性和基因诱导 在显微镜下观察形态。 最后我们开发了一个方法 称为索引，它将优化到单个单元格级别，并且将 允许我们比较来自对照、LTD 或 LTP 神经元的 cDNA，从而克隆 新的可塑性基因。