Howard Hughes Medical Institute Boston, MA, USA ; Department of Neurobiology, Harvard Medical School Boston, MA, USA.
Department of Neurobiology, Harvard Medical School Boston, MA, USA.
Front Pharmacol. 2014 Apr 2;5:56. doi: 10.3389/fphar.2014.00056. eCollection 2014.
Neuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation.
神经调质对行为有深远的影响，但它们的细胞内效应器的动态仍然不清楚。大多数神经调质通过 G 蛋白偶联受体 (GPCR) 发挥作用。理解神经调质作用的一个主要挑战是缺乏对 GPCR 激活产生的生化信号的动态读出。环腺苷酸/环 AMP/蛋白激酶 A (PKA) 模块是这种生化信号的核心组成部分。这个模块受几种行为相关的重要神经调质受体的调节。此外，PKA 活性对于许多形式的突触可塑性的诱导以及长时记忆的形成是必要的。为了监测脑组织中的 PKA 活性，我们开发了一种 2 光子荧光寿命成像显微镜 (2pFLIM) 兼容的 PKA 传感器，称为 FLIM-AKAR，它基于比率型 FRET 传感器 AKAR3。FLIM-AKAR 具有大的动态范围和较小的 pH 敏感性。此外，它是一种快速扩散的细胞质蛋白，能够在原位特异性地报告净 PKA 活性。FLIM-AKAR 通过多种转染方法在各种脑区中表达稳健，可以靶向遗传鉴定的细胞类型，并对内源性 GPCR 的激活和谷氨酸的时空特异性传递做出反应。初步实验揭示了神经调质输入对细胞内不同亚区 PKA 活性的不同调节。因此，报告基因 FLIM-AKAR 与 2pFLIM 结合使用，能够研究大脑中遗传鉴定的神经元对神经调质输入的 PKA 活性的反应，并揭示内源性 GPCR 激活的细胞内动力学。
