Novel two-photon caged GABA compounds

描述(由申请人提供)：新型双光子笼式GABA化合物了解GABA能抑制如何发挥作用，对于破译大脑功能和影响大脑的疾病的病理生理过程是必要的，包括许多癫痫和附加综合征。与此同时，关于GABA能输入到底是如何发挥作用的，并没有统一的共识，甚至关于它们是抑制性还是兴奋性的基本问题也存在活跃的辩论。这些争议的部分原因是抑制输入针对突触后细胞的亚区，而目前还没有好的工具来研究这些输入的高空间分辨率。活神经元受体的局部激活可以通过笼状化合物的双光子光释放来实现。事实上，谷氨酸的双光子去功能化已经彻底改变了目前对哺乳动物神经元兴奋性传递和整合的理解。不幸的是，光化学工具光释放GABA很少，尽管它们对研究GABA能抑制的功能是非常有用的。在这个方案中，我们引入了一类新的双光子笼状GABA化合物。具体地说，我们将测试和表征三种不同化学世代笼中GABAS的双光子释放，并使用最好的化学世代来从小鼠新皮质切片中生成活的锥体神经元上GABA能反应的高分辨率地图。最后，我们将使用一种新型的3D高通量电子显微镜来确认这些图谱的准确性，以识别对称突触。这项拟议的工作将扩大生物包埋的化学工具箱，包括新型高质量笼状GABA化合物，这些化合物可以用双光子激光进行光释放。这些新化合物将使我们能够详细研究GABA能传递对选择性亚细胞室的功能影响，这可能会对我们理解抑制如何改变正常和疾病的大脑功能产生重大影响，因为其中一些化合物可以用来控制癫痫。最后，我们的数据将首次揭示GABA能输入对树突棘的功能影响。由于脊椎介导了大多数兴奋性连接，这些结果也可能改变我们对兴奋性输入是如何整合的理解。 与公共健康相关：新的双光子笼子GABA化合物尽管GABA能回路介导了大脑中的大部分抑制，并在许多大脑病理中受到影响，但它们的功能尚不清楚，部分原因是它们对神经元亚区域具有很大的空间选择性。我们建议开发新的光化学工具，允许以前所未有的空间分辨率光学激活神经元上的GABA能输入，从而能够解剖其功能特性。我们的工作可能导致产生新的光学方法来控制处于高度兴奋病理状态的神经元的活动，例如癫痫。好了！

DESCRIPTION (provided by applicant): Novel two-photon caged GABA compounds Understanding how GABAergic inhibition works is necessary to decipher the function of brain and the pathophysiological processes of diseases that affect it, including many epilepsy and addition syndromes. At the same time, there is no unified agreement as to how exactly GABAergic inputs function and even the basic question of whether they are inhibitory or excitatory is actively debated. Part of the reason for these controversies is the fact that inhibitry inputs target subregions of the postsynaptic cells, and there are not good tools to investigate these inputs with high spatial resolution. Local activation of receptors in living neurons can be achieved by two-photon photorelease of caged compounds. Indeed, two-photon uncaging of glutamate has revolutionized current understanding of excitatory transmission and integration in mammalian neurons. Unfortunately, opto-chemical tools to photorelease GABA are scant, even though they would be extremely useful to study the function of GABAergic inhibition. In this proposal we introduce a novel family of two-photon caged GABA compounds. Specifically, we will test and characterize the two-photon release of three different chemical generations of caged GABAs and use the best ones to generate high-resolution maps of GABAergic responses on living pyramidal neurons from mouse neocortical slices. Finally, we will confirm the accuracy of these maps using a novel 3D high- throughput electron microscope to identify symmetric synapses. The proposed work will expand the chemical toolbox of biological uncaging to include novel high-quality caged GABA compounds that can be photo-released with two-photon lasers. These new compounds will enable the detailed investigation of the functional effects of GABAergic transmission on selective subcellular compartments, something likely to have a major impact on our understanding of how inhibition alters normal and diseased brain function, since some of these compounds can be used to control epilepsy. Finally, our data will reveal, for the first time, the functional effect of GABAergic inputs onto dendritic spines. Since spines mediate most excitatory connections, these results could also alter our understanding of how excitatory inputs are integrated. PUBLIC HEALTH RELEVANCE: Novel two-photon caged GABA compounds Although GABAergic circuits mediate most of the inhibition in the brain, and are affected in many brain pathologies, their function is poorly understood, partly because they act with great spatial selectivity onto subregions of the neurons. We propose the development of novel opto-chemical tools that will allow to optically activate GABAergic inputs onto neurons with unprecedented spatial resolution, thus enabling the dissection of their functional properties. Our work could result in the generation of novel optical methods to control the activity of neurons in hyper-excitable pathological states such as in epilepsy. !