Multiplex in vivo imaging of cell-specific and circuit-specific signaling pathways during synaptic plasticity

项目摘要 大脑中的细胞信号传导通路是调节活动的复杂系统的重要组成部分， 神经回路的协调。在学习和记忆过程中，突触可塑性过程调节神经元的活动。 突触连接的强度和修改的神经元回路。细胞内的信号通路起着至关重要的作用 在调节突触强度中的作用，并且是潜在的分子机制的重要部分。 学习和记忆。激酶信号传导通路在整合神经调节和 兴奋性和抑制性突触输入，以控制神经元和神经元回路在行为过程中如何适应。 然而，直接询问活的清醒动物中的信号传导通路一直具有挑战性，这是由于缺乏 合适的工具。在这样的环境中监测多个信号通路尚未实现。目标 这项研究的一个重要目的是开发新的工具，同时监测几种信号的活动。 并使用体内成像技术来可视化这些通路的细胞特异性和回路特异性活性。 在生理相关的感觉体验和学习过程中，活体动物的动态信号通路。在 初步研究使用基因编码的生物传感器将用于监测体内信号通路 使用双光子显微镜对小鼠躯体感觉皮层中现有的生物传感器进行成像。新单曲- 具有比现有的基于FRET的传感器更大的动态范围的基于彩色荧光蛋白的生物传感器 将被开发用于成像信号通路（PKA，CaMKII，ERK和mTOR复合物1）， 突触可塑性这项研究提案的最终目标是将这些新的生物传感器引入到 小鼠大脑，并监测信号传导途径的快速动态（秒至分钟）， 使用双光子显微镜观察信号通路的长期稳定性， 清醒的动物这个拟议的项目将是第一次调查细胞特定和电路， 在活体动物中除了钙和电压变化之外的特定神经元信号传导。这些研究将使我们能够 揭示信号通路调节的潜在机制，并将揭示 在体内突触可塑性的信号通路。

Project Summary Cell signaling pathways in the brain are an essential part of a complex system regulating the activity and coordination of neuronal circuits. During learning and memory synaptic plasticity processes regulate the strength of synaptic connections and modify neuronal circuits. Intracellular signaling pathways play critical roles in regulating synaptic strength and are an important part of the molecular mechanisms underlying learning and memory. Kinase signaling pathways play a central role integrating neuromodulatory and excitatory and inhibitory synaptic inputs to control how neurons and neuronal circuits adapt during behavior. However, direct interrogation of signaling pathways in live awake animals has been challenging due to lack of appropriate tools. Monitoring of multiple signaling pathways in such a setting has not been achieved. The goal of this research proposal is to develop novel tools to simultaneously monitor the activity of several signaling pathways and to use in vivo imaging techniques to visualize cell-specific and circuit-specific activity of these dynamic signaling pathways in live animals during physiologically relevant sensory experience and learning. In initial studies the use of genetically encoded biosensors will be used to monitor signaling pathways in vivo using two-photon microscopy to image an existing biosensor in the somatosensory cortex of mice. New single- color fluorescent protein based biosensors with a greater dynamic range than existing FRET-based sensors will be developed for imaging signaling pathways (PKA, CaMKII, ERK, and mTOR complex 1) involved in synaptic plasticity. The ultimate goal of this research proposal is to introduce these new biosensors into the mouse brain and to monitor both rapid dynamics of signaling pathways on the order of seconds to minutes and long-term stability of signaling pathways on the order of weeks to months using two-photon microscopy in awake behaving animals. This proposed project would be the first investigation of cell-specific and circuit- specific neuronal signaling beyond calcium and voltage changes in live animals. These studies will allow us to uncover mechanisms underlying the regulation of signaling pathways and will shed light on the complexity of signaling pathways during synaptic plasticity in vivo.