SIGNAL TRANSDUCTION AND GENE EXPRESSION IN LTP AND LTD

完整动物的海马体，在切片制备中，以及在分离的情况下 培养中的神经元提供了一个实验机会来研究长时间的 突触形式的时程抑制(LTD)和长时程增强(LTD) 用于学习和记忆的可塑性。CA2是一个关键的信号转导 调节海马区突触可塑性的分子，我们将 关注钙离子作用的几个方面，包括蛋白质的激活 激酶/磷酸酶、一氧化氮合酶与基因转录 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，我们已经发现它能刺激 以非钙依赖的方式释放突触。我们已经与 理查德·谢勒博士将证明不会改变蛋白质之间的相互作用 在突触蛋白VAMP、Synaxin、n-secl和SNAP-25中，可能是 对这种释放负有责任。我们将定义、量化和确定 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，以及是否没有变化刺激释放。 我们已经证明了多功能CaM激酶II的一种机制 在刺激频率下转换成不依赖于钙离子的物种- 依赖的态度。通过与钱存训博士的合作，我们将 在不同的频率上相互关联的激活的激酶导致了LTD 或在海马区培养的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的拮抗作用 还有钙调神经磷酸酶，一种依赖于钙离子的磷酸酶。我们将研究钙离子是如何 能通过促进CaM的激活改变突触强度的符号 激酶II诱导增强或有利于钙调神经磷酸酶诱导 抑郁症。 我们将研究谷氨酸受体中基于钙的信号通路。 关于转录因子CREB磷酸化的突触棘突 和钱博士一起在原子核里。携带报告基因的转基因动物 由CREB和其他监管要素的推动者推动 转录因子将被生成以检查转录在 不同的刺激频率。单细胞聚合酶链式反应将被用于关联 单细胞突触可塑性和基因诱导的研究 显微镜下的形态。最后，我们开发了一种方法 称为索引，该索引将优化到单个单元级，并将 使我们能够比较对照、LTD或LTP神经元的cDNA，从而克隆 新的可塑性基因。

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.