Optical probes for controlling cellular function

用于控制细胞功能的光学探针

• 批准号: 7861240
• 负责人: Graham Ellis-Davies
• 金额: $ 33.37万
• 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/7861240
• 关键词: 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; Figs - dietary; GABA Receptor; Generations; Glutamate Receptor; Glutamates; Goals; Hand functions; 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van Leeuwenhoek在C17th中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重要技术。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来彻底改变了我们对许多细胞功能的理解。笼子化合物（即光敏的，生物学上的惰性信号分子）伴侣在时间和空间结构域中可以控制细胞化学的光学技术。这种外源化学探针的独特优势之一是它们在光解时活跃的天然膜受体。因此，他们很好地补充了最近开发的ChannelRhopoptin技术。该提案的目的是开发和应用新的发色团，设计用于高效的神经递质的衰老。合成有机化学将用于制造新的化学化学和光学神经生物学，以测试探针诱导脑切片和活体动物突触可塑性的功率。在过去的十年中，新型的固态激光技术已经很容易获得（TI：Sapphire），该技术允许对U.V.-Absorbing发色团进行红外激发。使用这种激光器的脱离是通过同时吸收两个等效能量与一个蓝色光子的红色光子来产生的。由于激发体积大小与单个突触大致大致相同，因此2光子的不老龄是唯一适合允许在树突状表面刺激选定的突触（一个或多个）。由于神经递质的释放是单独创建的，因此刺激的模式既可以是任意的又可以是理性的。具体而言，我们建议制作和测试以下内容：（1）新的笼谷氨酸探针，该探针将允许在活动物中诱导突触可塑性； （2）双色，2光块未约会的新笼发色团，使谷氨酸和GABA的频谱独立，同时的光弹性； （3）在更长的波长上激活的新型笼子发色团，从而可以降低光毒性，从而实现长期的光学刺激实验； （4）其他笼中的神经递质（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）各种各样的阵列。中枢神经系统中的绝大多数兴奋性突触传播是通过脊柱头部的谷氨酸受体发生的。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，亨廷顿等）以某种方式（大小，数字，分布等）扰动刺。能够在单个棘水平上进行精确探测突触功能的光学工具对于对正常和疾病状态中棘的功能的充分了解至关重要。我们的建议旨在满足这一需求的一部分。 公共卫生相关性：人脑是我们知道的最复杂的结构，拥有超过1万亿的突触。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疾病的例子，涉及我们大脑正常功能的深刻变化。这种疾病靶向最小的功能性神经元结构，即单个突触。我们的工作旨在进行探针，使我们能够使用现代激光技术研究正常和疾病状态中一个或多个突触的精确功能。