Optical probes for controlling cellular function

描述(申请人提供)：自从Van Leeuwenhoek在C17使用他的手工显微镜以来，光学显微镜一直是研究活细胞的基本技术。最近，激光扫描共聚焦显微镜的发展使我们能够实时监测细胞功能，从而彻底改变了我们对许多细胞功能的理解。笼状化合物(即光敏的、生物惰性的信号分子)与这种光学技术合作，因为它们在时间和空间域提供了对细胞化学的控制。这种外源化学探针的独特优势之一是它们在光解过程中激活了天然的膜受体。因此，它们很好地补充了最近开发的通道视紫红质技术。这项提议的目标是开发和应用新的生色团，旨在高效地去除神经递质。合成有机化学将被用来制造新的化学和光学神经生物学，将被用来测试在脑片和活动物中诱导突触可塑性的探针的能力。在过去的十年里，一种新型的固体激光技术已经变得容易获得(钛：蓝宝石)，它允许紫外光吸收发色团的红外激发。使用这种激光器的色散是由两个能量相等的红光子同时吸收到一个蓝光子而产生的。由于兴奋体积与单个突触的大小大致相同，因此双光子去势特别适合于刺激树突表面的选定突触(一个或多个)。由于神经递质的释放仅由光产生，刺激的模式既可以是任意的，也可以是理性的。具体地说，我们建议制作和测试以下内容：(1)新的笼式谷氨酸探针，将能够诱导活体动物的突触可塑性；(2)新的笼式发色团，用于双色、双光子覆盖，使谷氨酸和GABA能够在不依赖光谱的情况下同时释放；(3)新型笼状发色团，可以在更长的波长激活，从而能够在降低光毒性的情况下进行长期的光刺激实验；(4)各种笼式神经递质(例如AMPA、多巴胺、腺苷、5-羟色胺、多巴胺和5-羟色胺受体的激动剂/拮抗剂等)。中枢神经系统中的兴奋性突触传递绝大多数是通过脊椎头部的谷氨酸受体进行的。现在公认许多神经系统疾病(如阿尔茨海默氏症、唐氏症、亨廷顿氏症等)以某种方式扰乱脊椎(大小、数量、分布等)。能够在单个脊椎水平上精确探测突触功能的光学工具对于更全面地了解脊柱在正常和疾病状态下的功能是必不可少的。我们的建议旨在满足这一部分需求。 与公共健康相关：人脑是我们所知的最复杂的结构，有超过1万亿个突触。阿尔茨海默病、唐氏综合症和亨廷顿病都是极其虚弱和致命的疾病，涉及到我们大脑正常功能的深刻变化。这种疾病的目标是最小的功能性神经元结构，即个体突触。我们的工作旨在制造探测器，使我们能够使用现代激光技术研究一个或多个突触在正常和疾病状态下的精确功能。

DESCRIPTION (provided by applicant): Light microscopy has been the essential technique for studying living cells since van Leeuwenhoek used his hand-made microscopes in the C17th. More recently, the development of laser-scanning confocal microscopy has revolutionized our understanding of many cellular functions by enabling the monitoring of cellular function in real time. Caged compounds (i.e. photosensitive, biologically inert signaling molecules) partner such optical techniques, as they provide control of cellular chemistry, in both temporal and spatial domains. One of the distinctive strengths of such exogenous chemical probes is that they active native membrane receptors upon photolysis. Thus, they nicely complement the recently developed channelrhodopsin technique. The goal of this proposal is the development and application of new chromophores designed for highly efficient uncaging of neurotransmitters. Synthetic organic chemistry will be used to make new chemical and optical neurobiology will be used to test the power of the probes for induction of synaptic plasticity in brain slices and living animals. During the past ten years a new type of solid-state laser technology has become readily available (Ti:sapphire), which allows for infra-red excitation of u.v.-absorbing chromophores. Uncaging using such lasers is produced by the simultaneous absorption of two red photons of equivalent energy to one blue photon. Since the excitation volume is approximately the same size as single synapse, 2-photon uncaging is uniquely suited to allow stimulation of selected synapses (one or many) on a dendritic surface. Since neurotransmitter release is created by light alone, the pattern of stimulation can be both arbitrary and rational. Specifically, we propose to make and test the following: (1) new caged glutamate probes that will allow the induction of synaptic plasticity in living animals; (2) new caging chromophores for dual-color, 2-photon uncaging, enabling spectrally independent, simultaneous photorelease of glutamate and GABA; (3) novel caging chromophores that are activated at longer wavelengths so enabling long-term optical stimulation experiments with reduced phototoxicity; (4) a diverse array of other caged neurotransmitters (e.g. AMPA, dopamine, adenosine, serotonin, agonists/antagonists of dopamine and serotonin receptors, etc.). The vast majority of excitatory synaptic transmission in the CNS occurs via glutamate receptors at spine heads. It is now well established that many neurological diseases (e.g. Alzheimer's, Down's, Huntingdon's, etc.) perturb spines in some way (size, number, distribution, etc.). Optical tools that enable the precise probing of synaptic function at the level of single spines are essential for a fuller understanding of the function of spines in normal and disease states. Our proposal is designed to fill part of this need. PUBLIC HEALTH RELEVANCE: The human brain is the most complex structure we know, having over one trillion synapses. Alzheimer's disease, Down's syndrome, and Huntingdon's disease are examples of extremely debilitating and deadly diseases that involve profound changes in the normal function of our brains. Such diseases target the smallest functional neuronal structures, namely individual synapses. Our work is designed to make probes that will allow us to study the precise function of one or many synapses in normal and disease states using modern laser technology.