CRCNS: Quantitative description of initial biochemical steps in LTP

LTP由钙离子通过NMDAR进入触发；随后，钙离子激活钙调蛋白(CaM)，这是 然后激活CaMKIl。尽管广泛的研究表明CaMKIl在LTP和LTP中的关键作用 记忆，其在活细胞中激活的机制尚不清楚。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椎和复杂的建模框架下进行模拟 这些反应。因为实验和计算方法以前是不可用的，所以 这将是第一次尝试解释活细胞中一种酶的活性。 目标1.将测量体系的关键定量性质，包括游离CaMKIl， Cam，Ng和CaMKil。这将使用校准的光学方法来完成。 目的2.要模拟CaM的激活，需要了解脊椎中钙离子的空间/时间梯度。 谷氨酸的双光子去功能化将被用来以受控的方式激活NMDAR；由此产生的Ca^* 将测量大段脊柱细胞质的高度。 目的3.用荧光寿命法(FLIM)研究单细胞内CaMKI1激活的时间过程 脊椎将被测量。然后将使用计算机模拟来预测Ca^*的高度如何，AS 在目标2中确定，导致CaMKI1激活。这需要在目标1中确定的参数 计算。将预测的CaMKIl激活与测量的激活进行比较。 目的4.神经颗粒素(Ng)是一种丰富的突触后蛋白，与CaM结合，可能在 控制可用于激活CaMKIl的凸轮。将研究Ng基因敲除的影响。 相关性(请参阅说明)： 这项拟议的研究与成瘾有关，成瘾涉及突触强度的持续变化。这个 CaMKIl在突触强度的持续性中的作用最近被证明；特别是生化 CaMKIl的攻击逆转了突触强度。因此，有可能是攻击的特工 CaMKIl可以用来逆转成瘾背后的突触变化。

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.