Development of kinase biosensors for multiplex neuronal imaging of signaling pathways in behaving mice

项目摘要 大脑中的细胞信号通路是调节大脑活动的复杂系统的重要组成部分 神经网络的协调。在学习和记忆过程中，这些神经网络可以被修改 通过神经元和突触的可塑性过程，其中信息存储在突触网络中。 细胞内信号通路在调节神经元兴奋性和突触强度方面起着关键作用， 从而构成了学习和记忆的细胞和分子机制的重要组成部分。 突触可塑性的适当调节中断涉及许多神经和精神疾病 包括自闭症、精神分裂症和智力残疾在内的障碍。揭示动态的活动和 因此，不同信号通路的相互作用对于理解控制机制至关重要 神经网络在健康和疾病中的作用。然而，在活体中直接询问信号通路的活性 由于缺乏适当的工具，动物一直是具有挑战性的。多条信号转导通路的监测 这样的设置还没有实现。这项研究提案的目标是开发新的工具来 同时监测几个信号通路的活动，并使用快速、灵敏的活体成像 这些信号通路在生理学过程中在活体动物中动态活动的可视化技术 相关的感官体验和学习。大多数现有的信号活动生物传感器都是基于 荧光共振能量转移(FRET)和使用两种不同的荧光蛋白，这限制了 它们用于并行监测多个信号通路。在这项提案中，新的单色荧光 具有高灵敏度和优化的双光子激发特性的蛋白质基激酶生物传感器将被 为参与突触可塑性的信号通路成像而开发，特别是PKA、CaMKII、ERK和 PKC。这项研究计划的最终目标是确定这些新的生物传感器在小鼠身上的使用。 并监测几秒到几分钟量级的信号通路的快速动力学和长时间的 用双光子显微镜研究清醒状态下几周到几个月的信号通路的长期稳定性 表现得像动物一样。这项拟议的项目将是首次对多种神经元活动进行研究。 活体动物体内钙和电压的变化。这些研究将使我们能够检查激酶的调节。 并将有助于阐明突触可塑性过程中信号通路的复杂性 大脑。

Project Summary Cell signaling pathways in the brain are an essential part of a complex system regulating the activity and coordination of neuronal networks. During learning and memory these neuronal networks can be modified through neuronal and synaptic plasticity processes in which information is stored in the synaptic network. Intracellular signaling pathways play critical roles in regulating neuronal excitability and synaptic strength, thereby comprising an important part of the cellular and molecular mechanisms underlying learning and memory. Disruptions in the proper regulation of synaptic plasticity are involved in a number of neurological and psychiatric disorders including autism, schizophrenia, and intellectual disability. Revealing the dynamic activities and interactions of different signaling pathways is therefore crucial for understanding the mechanisms controlling neuronal networks both in health and disease. However, direct interrogation of signaling pathway activity in live animals has been challenging due to a 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 rapid, sensitive in vivo imaging techniques to visualize dynamic activity of these signaling pathways in live animals during physiologically relevant sensory experience and learning. Most existing biosensors for signaling activities are based on fluorescence resonance energy transfer (FRET) and the use of two different fluorescent proteins, which limits their use for monitoring of multiple signaling pathways in parallel. In this proposal, new single-color fluorescent protein-based kinase biosensors with high sensitivity and optimized two-photon excitation properties will be developed for imaging signaling pathways involved in synaptic plasticity, especially PKA, CaMKII, ERK, and PKC. The ultimate goal of this research proposal is to establish the use of these new biosensors in the mouse brain and to monitor both rapid dynamics of signaling pathways on the order of seconds to minutes and the long- term stability of signaling pathways on the order of weeks to months using two-photon microscopy in awake behaving animals. This proposed project will be the first investigation of multiple neuronal activities beyond calcium and voltage changes in live animals. These studies will allow us to examine the regulation of kinase pathways in vivo and will help elucidate the complexity of signaling pathways during synaptic plasticity in the brain.