Noninvasive bioluminescent imaging of neuronal activity in freely behaving animals

自由行为动物神经元活动的无创生物发光成像

• 批准号: 9906190
• 负责人: Michael Z. Lin
• 金额: $ 19.81万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906190
• 关键词: Acetylcholine; Address; Aequorin; Affect; Amphetamines; Anatomy; Animals; Behavior; Behavioral; Bioluminescence; Brain; Brain region; Calcium; Cell Nucleus; Cells; Chemicals; Cocaine; Collection; Detection; Devices; Disease; Dopamine; Drug Addiction; Drug usage; Eating; Energy Transfer; Engineering; Enzymes; Esthesia; Fiber; Fire - disasters; Fluorescence; Future; Genetic; Goals; Green Fluorescent Proteins; Head; Hippocampus (Brain); Histamine; Image; In Vitro; Intravenous; Intraventricular; Learning; Light; Location; Luciferases; Measures; Mental Depression; Methods; Microscope; Motivation; Mus; Names; Neurologic Deficit; Neuromodulator; Neurons; Nicotine; Non-Invasive Cancer Detection; Norepinephrine; Operative Surgical Procedures; Optics; Oranges; Output; Oxytocin; Partner in relationship; Photons; Physiological; Play; Population; Production; Proteins; Renilla Luciferases; Reporter; Reporting; Resolution; Role; Route; Schizophrenia; Serotonergic System; Serotonin; Signal Transduction; Socialization; Specificity; Stimulus; System; Testing; Time; Tissues; Ultrasonography; Variant; Vasopressins; Work; addiction; calcium indicator; cholinergic; cranium; detector; dopamine system; dopaminergic neuron; experience; feeding; free behavior; improved; intraperitoneal; lens; light emission; luminescence; magnetic resonance imaging/electroencephalography; mature animal; nanoluciferase; neuronal circuitry; neuroregulation; noradrenergic; photonics; response; two photon microscopy; unpublished works; willingness

PROJECT SUMMARY Small numbers of neurons can play outsized roles in brain function. For example, neuromodulatory circuits that contain only thousands of neurons project throughout the brain to control the activity of billions of neurons in other brain regions. These neuromodulatory circuits, which secrete the neuromodulators dopamine, serotonin, norepineprhine, acetylcholine, and histamine, regulate essential brain functions such as learning, eating, mating, and socialization. Importantly, alteration of neuromodulatory circuit function underlies addiction, e.g. dopaminergic, serotonergic, and noradrenergic functions are potentiated by cocaine and amphetamines, and cholinergic functions by nicotine. However, very little work has addressed how neuromodulatory circuits respond in real time to environmental stimuli, behavioral output, or drug use. In addition, little is known about how these neuromodulatory circuits develop as animals mature. In the cortex and hippocampus, genetically encoded calcium indicators (GECIs) and two-photon microscopy together have been enormously successful in relating activities of specific types of neurons to various stimuli or behavioral outputs. In contrast, efforts to apply calcium imaging to neuromodulatory circuits have proceeded on a far smaller scale, mostly due to their difficult anatomy. The deep locations of neuromodulatory nuclei near other vital brain regions make the placement of optical lenses or fibers very difficult to perform without creating severe behavioral or functional deficits. Thus, what is needed, and what does not exist, is a way to non- invasively record the total the activity of a specific neuromodulatory network from outside the animal. With the long-term goal of enabling noninvasive activity recording of specific neuronal circuits in living animals, we propose to create and implement GECIs that operate not via fluorescence but via bioluminescence, in which a luciferase enzyme reacts with a chemical substrate to produce light in a calcium-dependent manner. The lack of endogenous luciferases leads to background that is negligible, so high contrast can be achieved even with external detectors. In preliminary work, we have generated a calcium-dependent variant that demonstrates 7-fold increase in photonic output in response to calcium, a similar response ratio to the widely used fluorescent reporter GCaMP3. Compared to other bioluminescent calcium reporters, this reporter produces orders of magnitude more photons above 600 nm, to which tissue is relatively transparent. This reporter, which we name red calcium- modulated bioluminescent indicator (orange CaMBI), has been used successfully in cultured neurons. We now propose to develop methods to non-invasively visualize the activity of specific neuromodulatory circuits in freely behaving mice, by (1) testing the ability of CaMBI expressed throughout the brain and substrates injected intravenously, intraperitoneally, intraventricularly to produce luminescence from the brain in an activity- dependent manner, (2) making a transcranial head-mounted optical recording device, and test the ability to detect activity in neuromodulatory circuits, and (3) futher improving CaMBI for brightness and calcium responsiveness.

项目概要 少量的神经元可以在大脑功能中发挥巨大的作用。例如，神经调节电路 仅包含数千个神经元的项目遍布整个大脑，以控制数十亿个神经元的活动 其他大脑区域。这些神经调节回路分泌神经调节剂多巴胺、血清素、 去甲肾上腺素、乙酰胆碱和组胺调节基本的大脑功能，如学习、饮食、交配、 和社会化。重要的是，神经调节回路功能的改变是成瘾的基础，例如 可卡因和安非他明可增强多巴胺能、血清素能和去甲肾上腺素能功能，并且 尼古丁的胆碱能功能。然而，很少有工作涉及神经调节回路如何响应 实时了解环境刺激、行为输出或药物使用。此外，人们对于这些是如何进行的还知之甚少。 神经调节回路随着动物的成熟而发育。 在皮层和海马体中，基因编码钙指示剂 (GECI) 和双光子 显微镜技术在将特定类型神经元的活动与各种神经元的活动联系起来方面取得了巨大的成功。 刺激或行为输出。相比之下，将钙成像应用于神经调节回路的努力已经取得了进展。 其规模要小得多，主要是因为它们的解剖结构很困难。神经调节的深层位置 其他重要大脑区域附近的核团使得光学透镜或纤维的放置非常困难，如果没有 造成严重的行为或功能缺陷。因此，需要什么和不存在什么，是一种非 侵入性地记录动物体外特定神经调节网络的总活动。 长期目标是实现对活体中特定神经元回路的无创活动记录 动物，我们建议创建并实施 GECI，其不通过荧光而是通过生物发光进行操作， 荧光素酶与化学底物发生反应，以钙依赖性方式产生光。这 缺乏内源性荧光素酶导致背景可以忽略不计，因此即使使用 外部探测器。在初步工作中，我们已经生成了一种钙依赖性变体，其表现出 7 倍 对钙的响应增加光子输出，与广泛使用的荧光报告剂的响应比相似 GCaMP3。与其他生物发光钙报告基因相比，该报告基因的产生量要高出几个数量级 600 nm 以上的光子，组织对其相对透明。这位记者，我们称之为红钙—— 调制生物发光指示剂（橙色 CaMBI）已成功用于培养的神经元。 我们现在建议开发方法来非侵入性地可视化特定神经调节的活动 通过 (1) 测试 CaMBI 在整个大脑和底物中表达的能力，在自由行为的小鼠中建立电路 静脉内、腹膜内、心室内注射，以在活动中从大脑产生发光- (2)制作经颅头戴式光学记录装置，并测试检测能力 (3) 进一步改善 CaMBI 的亮度和钙反应性。