Noninvasive bioluminescent imaging of neuronal activity in freely behaving animals

项目总结 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 只包含数千个神经元的神经元投射到整个大脑来控制数十亿个神经元的活动 其他大脑区域。这些神经调节电路，分泌神经调节剂多巴胺，5-羟色胺， 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、进食、交配、 和社交。重要的是，神经调节回路功能的改变是成瘾的基础，例如 多巴胺能、5-羟色胺能和去甲肾上腺素能功能被可卡因和苯丙胺增强，并且 尼古丁的胆碱能功能。然而，很少有人研究神经调节回路是如何反应的。 实时响应环境刺激、行为输出或药物使用。此外，人们对它们是如何形成的知之甚少 随着动物的成熟，神经调节回路也随之发展。 在大脑皮层和海马区，基因编码的钙指示器(GECI)和双光子 显微镜在将特定类型的神经元的活动与不同类型的 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 进行的规模要小得多，主要是因为它们复杂的解剖结构。神经调节的深层定位 邻近大脑其他重要区域的核团使得没有光学透镜或纤维的放置变得非常困难 造成严重的行为或功能缺陷。因此，所需要的，以及不存在的，是一种非 侵入性地记录来自动物外部的特定神经调节网络的总活动. 其长期目标是实现对生活中特定神经元回路的非侵入性活动记录 ，我们建议创建和实现不通过荧光而是通过生物发光来操作的GECI，在 荧光素酶与化学底物反应，以钙依赖的方式产生光。这个 缺乏内源性荧光素酶会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，我们已经产生了一种钙依赖变体，它展示了7倍 对钙的响应增加了光子输出，与广泛使用的荧光报告的响应率相似 GCaMP3。与其他生物发光钙离子记者相比，这位记者产生的数量级更多 600纳米以上的光子，组织对此相对透明。这位记者，我们把它命名为红钙- 调制生物发光指示剂(橙色Cambi)已成功地应用于培养的神经元。 我们现在建议开发非侵入性可视化特定神经调节活性的方法。 自由活动小鼠的环路，通过(1)测试Cambi在整个大脑和底物中表达的能力 静脉注射，腹膜内注射，脑室注射，在一项活动中从大脑产生发光- 依赖方式，(2)制作经颅头盔光学记录装置，并测试其检测能力 神经调节回路中的活动，以及(3)进一步改善Cambi的亮度和钙的反应性。

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.