Imaging of Free Cytosolic Calcium Dynamics in Spines

树突棘是兴奋性突触的接收端，是微小的膜性隔室。脊椎含有突触受体、通道、信号分子、细胞骨架蛋白和平滑内质网。脊椎表达的突触可塑性可能是某些类型记忆形成的基础，许多形式的精神障碍与脊柱病理有关。特别令人感兴趣的是脊椎中的[钙]信号。脊椎将进入脊椎细胞质的钙离子分隔开来。脊髓钙离子通过调节触发的突触后酶，在大多数形式的突触可塑性的诱导中起着关键作用 突触强度的快速修改，也激活转录因子，促进这些修改的长期维持。一个重要的问题是，钙离子如何以任何一种特异性编码所有这些功能？答案肯定在于细节：不同幅度、不同时间段或不同位置的[Ca2+]信号对细胞具有不同的生化意义。我们将测量脊柱[Ca~(2+)]信号和塑造它们的机制，重点是Ca~(2+)来源和挤出机制。我们将测量试验与试验之间的钙离子内流波动，并计算作为钙来源的钙通道和突触受体。使用血管紧张素调素(CAM)激活的荧光指示剂 我们将根据其激活CaM的能力来表征[Ca2+]升高的模式，从而构建详细的CaM原位激活的动力学模型。我们还将研究钙-钙调素依赖性激酶(CaMKII)介导的脊髓钙通道的可塑性。从我们的测量中获得的参数将被合并到脊椎中的[Ca2+]信号和CaMKII的定量模型中，该模型以Mcell编码。此外，我们的测量结果将作为该模型的重要基准。Mcell模型将允许我们在超出我们目前的实验方法的分辨率下探索塑造[Ca2+]动力学的机制。 我们将使用Mcell模型来了解钙通道依赖于[Ca~(2+)]的可塑性，这种可塑性可能是由高[Ca~(2+)]微区触发的，而不是光学技术所能分辨的。

Dendritic spines are tiny membranous compartments that are the receiving ends of excitatory synapses. Spines contain synaptic receptors, channels, signaling molecules eytoskeletal proteins, and smooth endoplasmic reticulum. Synaptic plasticity expressed at spines may underlie the formation of some kinds of memories, and many forms of mental disorders are associated with spine pathologies. Of particular interest is [Ca 2+] signaling in spines. Spines compartmentalize Ca 2+ ions that enter the spine cytoplasm. Spine Ca 2+ plays a crucial role in the induction of most forms synaptic plasticity by regulating postsynaptic enzymes that trigger rapid modifications of synaptic strength, and also to activate transcription factors that facilitate long-term maintenance of these modifications. An important question is how Ca 2+ can encode all of these functions with any kind of specificity? The answer must lie in the details: [Ca 2+] signals with different amplitudes, time courses or in different locations will have distinct biochemical meanings for the cell. We will measure spine [Ca 2+] signals and the mechanisms that shape them, focusing on Ca 2+ sources and extrusion mechanisms. We will measure the trial-to-trial fluctuations in Ca 2+ influx and count Ca 2+ channels and synaptic receptors that serve as Ca 2+ sources. Using fluorescent indicators of ealmodulin (CAM) activation we will characterize patterns of [Ca 2+] elevation in terms of their ability to activate CaM, allowing us to construct a detailed kinetic model of CaM activation in situ. We will also study plasticity of Ca 2+ channels in spines mediated by Ca2+-CaM dependent kinase (CaMKII). Parameters derived from our measurements will be incorporated into a quantitative model of [Ca 2+] signaling and CaMKII in spines, coded in MCell. In addition, our measurements will serve as important benchmarks for the model. The MCell model will allow us to explore the mechanisms shaping [Ca 2+] dynamics at resolutions beyond our current experimental methods. We will use the MCell model to gain an-understanding of [Ca 2+] dependent plasticity of Ca 2+ channels, which may be triggered by microdomains of high [Ca2+], beyond the resolution of optical techniques.