Optical probes for controlling cellular function

描述（由申请人提供）：自17世纪范·列文虎克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通过实时监测细胞功能，彻底改变了我们对许多细胞功能的理解。笼化化合物（即光敏的、生物惰性的信号分子）配合这种光学技术，因为它们在时间和空间领域提供对细胞化学的控制。这种外源性化学探针的独特优势之一是它们在光解时激活天然膜受体。因此，它们很好地补充了最近开发的通道视紫红质技术。本提案的目标是开发和应用新的发色团，旨在高效地释放神经递质。合成有机化学将用于制造新的化学，光学神经生物学将用于测试探针在脑切片和活体动物中诱导突触可塑性的能力。在过去的十年里，一种新型的固态激光技术（钛：蓝宝石）已经变得很容易获得，它允许红外激发紫外线吸收发色团。使用这种激光器的释放是通过同时吸收两个与一个蓝色光子能量相等的红色光子来产生的。由于激发体积与单个突触的大小大致相同，因此双光子解封是唯一适合于刺激树突表面上选定的突触（一个或多个）的方法。由于神经递质释放仅由光产生，因此刺激的模式可以是任意的，也可以是合理的。具体而言，我们建议制作和测试以下内容：(1)新的笼式谷氨酸探针，可以在活体动物中诱导突触可塑性；(2)用于双色、双光子释放的新型笼状发色团，使谷氨酸和GABA在光谱上独立、同时释放；(3)在更长的波长下激活的新型笼状发色团，使长期的光刺激实验能够降低光毒性；(4)多种其他笼状神经递质（如AMPA、多巴胺、腺苷、血清素、多巴胺和血清素受体的激动剂/拮抗剂等）。中枢神经系统中绝大多数兴奋性突触传递是通过脊柱头部的谷氨酸受体发生的。现在已经确定，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）在某种程度上（大小、数量、分布等）扰乱了脊柱。光学工具能够在单个棘的水平上精确探测突触功能，这对于更充分地了解正常和疾病状态下棘的功能至关重要。我们的建议旨在满足这一需求的一部分。

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.