Optical probes for controlling cellular function

用于控制细胞功能的光学探针

• 批准号: 8296478
• 负责人: Graham Ellis-Davies
• 金额: $ 32.7万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8296478
• 关键词: AMPA Receptors; Action Potentials; Adenosine; Alzheimer' s Disease; Animal Behavior; Animals; Area; Biological Factors; Brain; Capsaicin; Cell physiology; Cells; Chemicals; Chemistry; Color; Communication; Complement; Complex; Development; Disease; Dopamine; Dopamine Antagonists; Down Syndrome; Equilibrium; GABA Receptor; Generations; Glutamate Receptor; Glutamates; Goals; Head; Human; In Situ; In Vitro; Individual; Laser Scanning Confocal Microscopy; Lasers; Life; Light; Membrane; Methods; Microscope; Microscopy; Monitor; N-Methyl-D-Aspartate Receptors; Neurobiology; Neurons; Neurotransmitters; Optics; Organic Chemistry; Pattern; Photons; Phototoxicity; Physiology; Political Systems; Property; Sapphire; Serotonin; Serotonin Agonists; Signaling Molecule; Slice; Structure; Surface; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Technology; Testing; Time; Vertebral column; Vesicle; Work; absorption; chromophore; design; gamma-Aminobutyric Acid; improved; in vivo; interest; light microscopy; nervous system disorder; neurotransmitter release; novel; photolysis; postsynaptic; presynaptic; public health relevance; receptor; research study; serotonin receptor; solid state; synaptic function; tool; two-photon

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.

描述（由申请人提供）：自从货车列文虎克在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已经彻底改变了我们对许多细胞功能的理解，使细胞功能的监测在真实的时间。笼状化合物（即光敏的、生物惰性的信号分子）与这样的光学技术合作，因为它们在时间和空间域中提供细胞化学的控制。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。该提案的目标是开发和应用新的生色团，用于高效释放神经递质。合成有机化学将用于制造新的化学物质，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。在过去的十年中，一种新型的固态激光器技术已经变得容易获得（钛：蓝宝石），其允许紫外线的红外激发。吸收发色团。使用这种激光器的解除锁定是通过同时吸收两个与一个蓝光子能量相等的红光子来产生的。由于激发体积与单个突触的大小大致相同，因此2-光子撑开唯一适合于允许刺激树突表面上的选定突触（一个或多个）。由于神经递质的释放是由光单独产生的，刺激的模式可以是任意的和合理的。具体来说，我们建议制作和测试：（1）新的笼状谷氨酸探针，将允许在活体动物中诱导突触可塑性;（2）新的笼状生色团，用于双色，双光子解开，使光谱独立，同时光释放谷氨酸和GABA;（3）在较长波长下被激活的新型笼状发色团，从而使得能够进行具有降低的光毒性的长期光刺激实验;（4）多种多样的其它笼状神经递质（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）。中枢神经系统中绝大多数的兴奋性突触传递是通过棘头上的谷氨酸受体进行的。现在已经很好地确定，许多神经系统疾病（例如阿尔茨海默氏症、唐氏症、亨廷顿氏症等）以某种方式扰乱棘（大小、数量、分布等）。光学工具，使突触功能的精确探测在单个棘的水平是必不可少的一个更全面的了解棘在正常和疾病状态下的功能。我们的建议旨在满足这一需求的一部分。 人类大脑是我们所知道的最复杂的结构，拥有超过一万亿个突触。阿尔茨海默氏病、唐氏综合症和亨廷顿氏病是极度衰弱和致命的疾病，这些疾病涉及我们大脑正常功能的深刻变化。这些疾病的目标是最小的功能性神经元结构，即单个突触。我们的工作旨在制作探针，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。