Novel two-photon caged GABA compounds

描述（由申请人提供）：新型双光子笼状GABA化合物了解GABA能抑制如何起作用对于破译大脑功能和影响大脑的疾病（包括许多癫痫和加法综合征）的病理生理过程是必要的。与此同时，关于GABA能输入究竟如何起作用，甚至它们是抑制性还是兴奋性的基本问题也没有统一的共识。这些争议的部分原因是，神经元的输入目标的突触后细胞的亚区，并没有很好的工具来调查这些输入与高空间分辨率。活神经元中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，谷氨酸的双光子释放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，光释放GABA的光化学工具很少，即使它们对研究GABA能抑制功能非常有用。在这个建议中，我们介绍了一个新的家庭的双光子笼状GABA化合物。具体来说，我们将测试和表征三种不同化学代的笼状加巴斯的双光子释放，并使用最好的生成高分辨率的映射GABA能反应的活锥体神经元从小鼠新皮层切片。最后，我们将使用一种新的三维高通量电子显微镜来确认这些地图的准确性，以确定对称的突触。拟议的工作将扩大生物解开的化学工具箱，包括新的高质量的笼状GABA化合物，可以用双光子激光光释放。这些新化合物将使GABA能传递对选择性亚细胞区室的功能影响的详细研究成为可能，这可能对我们理解抑制如何改变正常和患病的脑功能产生重大影响，因为其中一些化合物可用于控制癫痫。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。由于棘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如何整合的理解。 公共卫生相关性：新型双光子笼状GABA化合物虽然GABA能回路介导大脑中的大部分抑制，并且在许多大脑病理中受到影响，但它们的功能知之甚少，部分原因是它们以很大的空间选择性作用于神经元的子区域。我们建议开发新的光化学工具，以前所未有的空间分辨率将GABA能输入光学激活到神经元上，从而能够解剖其功能特性。我们的工作可能会导致产生新的光学方法来控制神经元的活动在过度兴奋的病理状态，如癫痫。!

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. !