SIGNAL TRANSDUCTION AND GENE EXPRESSION IN LTP AND LTD

完整动物的海马、切片制备和分离 培养的神经元提供了一个实验性的机会， 时程抑制（LTD）和长时程增强（LTD），突触形式 学习和记忆中的可塑性。 Ca 2+是一个关键的信号 分子调节海马突触可塑性，我们将 重点介绍了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， 突触释放以Ca 2+非依赖性方式。 我们与 博士Richard舍勒证明NO改变蛋白质-蛋白质相互作用 在突触蛋白VAMP、突触融合蛋白、n-secl和SNAP-25中， 负责这种释放。 我们将定义，量化，并确定 这些变化的地点。 我们将确定哪类神经递质 以及NO是否改变刺激释放。 我们已经证明了多功能钙调蛋白激酶II可能 在刺激频率下转换为Ca 2+非依赖性物质- 依赖的方式。 与钱存训博士合作，我们将 在不同频率下激活激酶， 或海马培养物中的LTP。 磷酸选择性免疫细胞化学 抗体和生化分析将比较CaM激酶II之间的拮抗作用 和钙调神经磷酸酶，一种钙依赖性磷酸酶。 我们将研究Ca 2 + 可以通过促进钙调素的激活来改变突触强度的符号 激酶II引起增强作用或有利于钙调磷酸酶引起增强作用。 萧条 我们将研究谷氨酸受体的基于Ca 2+的信号通路， 对转录因子CREB磷酸化的影响 和钱博士在一起 具有报告基因的转基因动物 由CREB和其他调节元件的启动子驱动 将产生转录因子以检查转录， 不同的刺激频率 单细胞PCR将用于关联 突触可塑性和基因在单个细胞中的诱导 在显微镜下的形态学。 最后，我们开发了一种方法 称为索引，其将被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

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.