Novel two-photon caged GABA compounds

描述（由申请人提供）：新型双光子笼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它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，对于gaba能输入究竟是如何起作用的，甚至是它们是抑制性还是兴奋性的基本问题，也没有统一的意见。这些争议的部分原因是抑制性输入针对突触后细胞的亚区，并且没有很好的工具来研究这些高空间分辨率的输入。通过笼化化合物的双光子光释放，可以实现活神经元中受体的局部激活。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，尽管它们对研究GABA能抑制的功能非常有用。本文介绍了一类新的双光子笼型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中gaba的双光子释放，并使用最好的一代生成高分辨率的gaba能反应图，这些图来自小鼠新皮层切片的活体锥体神经元。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地图的准确性，以识别对称突触。这项工作将扩大生物解锁的化学工具箱，包括可以用双光子激光光释放的新型高质量笼状GABA化合物。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区室的功能影响，这可能对我们理解抑制如何改变正常和患病的大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。由于脊髓介导大多数兴奋性连接，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。

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. !