CRCNS: Quantitative description of initial biochemical steps in LTP

LTP由Ca ^* 通过NMDAR进入触发;随后Ca ^* 激活钙调蛋白（CaM）， 然后激活CaMKII。尽管广泛的研究证明了CaMKII在LTP中的关键作用， 记忆，其在活细胞中的激活机制尚不清楚。拟议工作的目标是 从数量上详细了解这些机制。这需要测量生化事件的方法 在接近光学分辨率极限的单个脊柱中， 这些反应。由于实验和计算方法以前不可用， 将是第一次尝试解释活细胞中酶的测量激活。 目标1.将测量系统的关键定量性质，包括游离CaMKI 1， CaM、Ng和CaMKII。这将使用校准的光学方法完成。 目标2.为了模拟钙调素的激活，需要关于棘突中钙离子浓度的时空梯度的信息。 2-谷氨酸的光子释放将用于以受控的方式激活NMDAR;产生的Ca ^* 将测量大量棘细胞质中的升高。 目标3.使用荧光寿命方法（FLIM）），在单个细胞中CaMKIl激活的时间过程 将测量脊柱。然后将使用计算机模拟来预测Ca ^* 的海拔高度，如 在目的2中确定的，导致CaMKII激活。为此需要在目标1中确定的参数 计算.将该预测的CaMKII激活与测量的激活进行比较。 目标4。神经颗粒蛋白（Ng）是一种丰富的突触后蛋白，可与钙调素结合，可能在 控制可用于激活CaMKII的CaM。将研究Ng敲除的影响。 相关性（参见说明）： 这项拟议中的研究与成瘾有关，成瘾涉及突触强度的持续变化。的 CaMKII在突触强度的持续性中的作用最近已经被证明;特别是生物化学 CaMKII的攻击逆转了突触强度。因此，有可能攻击的代理人 CaMKII可用于逆转成瘾基础的突触变化。

LTP is triggered by Ca^* entry through the NMDAR; subsequently Ca^* activates calmodulin (CaM), which then activates CaMKIl. Despite extensive studies demonstrating the pivotal role of CaMKIl in LTP and memory, the mechanisms of its activation in living cells is not known. The goal ofthe proposed work is to understand these mechanisms in quantitative detail. This requires methods to measure biochemical events in single spines near the limit of optical resolution and a sophisticated modeling framework for simulating these reactions. Because the experimental and computational methods were not previously available, this will be the first attempt to account for the measured activation of an enzyme in a living cell. Aim 1. Measurements will be made of critical quantitative properties of the system, including free CaMKIl, CaM, Ng and CaMKIl. This will be done used calibrated optical methods. Aim 2. To model CaM activation requires information about the spatial/temporal gradients of Ca^* in spines. 2-photon uncaging of glutamate will be used to activate NMDARs in a controlled way; the resulting Ca^* elevation in the bulk spine cytoplasm will be measured. Aim 3. Using fluorescence lifetime methodology (FLIM)), the time course of CaMKIl activation in single spines will be measured. Computer simulations will then be used to predict how the elevation of Ca^*, as determined in Aim 2, leads to CaMKIl activation. The parameters determined in Aim 1 are needed for this calculation. This predicted CaMKIl activation will be compared to the measured activation. Aim 4. Neurogranin (Ng) is an abundant postsynaptic protein that binds CaM and may be important in controlling the CaM that is available to activate CaMKIl. The effects of Ng knockout will be studied. RELEVANCE (See instructions): The proposed research is relevant to addiction, which involves persistent changes in synaptic strength. The role of CaMKIl in the persistence of synaptic strength has recently been demonstrated; notably biochemical attack of CaMKIl has reversed synaptic strength. There is therefore the possiblity that agents that attack CaMKIl can be used to reverse the synaptic changes that underlie addiction.