Novel Optogenetic Tools for Controlling Neuronal Signaling in Living Brain

研究目标：开发和优化一套光遗传学工具，允许光对细胞内cAMP和cGMP信号的时空操纵。*** ***背景/理由：cAMP和cGMP是细胞内信号传递必不可少的第二信使，介导信息的神经转导及其调节。药理学试剂和遗传操作已被用于研究它们的功能，然而，这些方法在空间和时间特异性方面受到限制。在这里，我们建议建立一套光遗传学工具，通过使用光激活酶来快速增加/减少cAMP/cGMP信号传导。******方法：cAMP和cGMP是由ATP/GTP由腺苷酸/鸟苷酸环化酶（AC/GC）合成的，ATP/GTP随后被细胞内的磷酸二酯酶（PDEs）降解。我们已经开始开发或优化一组光活化催化酶，使用基因工程光活化酶来增加或降低cAMP/cGMP水平，这些酶参与cAMP/cGMP的合成（AC/GC）和降解（PDEs）。对于光活化AC/GC，我们利用了从细菌中分离出来的内源性蓝光活化形式的AC。我们将验证/优化绿光或远红光活化的AC/GC，以提高cAMP/cGMP。为了补充光活化环化酶，我们将设计光活化PDE4和PDE5来特异性降解cAMP和cGMP。为了双向增加/减少cAMP和cGMP，我们将验证/优化蓝光或绿光活化AC/GC和远红光敏感PDE4/PDE5的组合。此外，为了针对大脑深部组织中单突触水平（~1µm3）的光激活，我们将利用双光子激光激发，并利用不同波长的双光子激发光将该技术扩展为双光激活。******目的：***1)通过建立一套光激活酶来开发/优化cAMP/cGMP水平的光依赖性调节***2)建立双光子光遗传学方法来控制cAMP/cGMP激活******光遗传学工具将允许光对cAMP和cGMP水平进行无创的时空操纵，并以前所未有的方式提供新的强大工具来研究活体脑内cAMP/cGMP的快速交互功能

RESEARCH GOAL: To develop and optimize a set of optogenetic tools that allow spatiotemporal manipulation of intracellular cAMP and cGMP signaling by light.*** ***BACKGROUND/RATIONALE: cAMP and cGMP are essential second messengers for intracellular signaling that mediate the neuro-transduction of information and its modulation. Pharmacological reagents and genetic manipulations have been used to study their functions, however, these approaches are limited in spatial and temporal specificity. Here we propose to establish a set of optogenetic tools to rapidly increase/decrease second messenger signaling by employing photoactivatable enzymes for cAMP/cGMP signaling.******METHODOLOGY: cAMP and cGMP are synthesized by adenylyl/guanylyl cyclase (AC/GC) from ATP/GTP which is subsequently degraded by phosphodiesterases (PDEs) within cells. We have begun the development or optimization of a panel of photoactivatable catalytic enzymes to increase or decrease cAMP/cGMP level using genetically engineered photoactivatable enzymes that are involved in their synthesis (AC/GC) and their degradation (PDEs). For photoactivatable AC/GC, we have utilized an endogenous blue-light activatable form of AC that has been isolated from bacteria. We will validate/optimize green or far-red light activatable AC/GC to increase cAMP/cGMP. To complement the photoactivatable cyclases, we will design photoactivatable PDE4 and PDE5 to specifically degrade cAMP and cGMP. To bidirectionally increase/decrease cAMP and cGMP, we will validate/optimize the combination of blue or green light activatable AC/GC and far-red light sensitive PDE4/PDE5. Furthermore, for targeting the photoactivation at the single synapse level (~1 µm3) within deep brain tissues, we will utilize two-photon laser light excitation and expand this technique for dual photoactivation using different wavelengths of two-photon excitation light.******AIMS:***1) Development/optimization of light-dependent regulation of cAMP/cGMP levels by establishing a set of photoactivatable enzymes***2) Establish two-photon optogenetic approaches to control cAMP/cGMP activation******The optogenetic tools will allow for non-invasive spatiotemporal manipulation of cAMP and cGMP levels by light and provide novel powerful tools to study the rapid interactive cAMP/cGMP functions within living brain in an unprecedented way.**