Multiplex interrogation of neuromodulatory signaling in behaving animals with enhanced depth and resolution

项目摘要 哺乳动物大脑对环境变化的动态适应性是显着的，因为它是复杂的 神经元网络的基础上的操作，允许这种适应。虽然我们有一些 尽管我们了解了这一点的解剖学和功能基础，但我们仍然缺乏一个详细的图片， 神经元活动的调节起作用。这些神经调质释放的时间和位置是什么， 与兴奋/抑制回路的关系神经调节剂电路如何完成调节 神经元的放电和突触特性填补这些知识空白将促进我们的 了解神经调质生物学的所有方面，并允许发现新的治疗策略。到 为了缩小这一差距，我们使用了创造性的方法来开发基因编码， 报告行为触发和调制的神经调质释放，包括5-羟色胺（5-HT），多巴胺（DA） 和去甲肾上腺素（NE）。我们已经将这些指标传播给神经科学界， 新的机制调节神经调节剂释放的动机和成瘾的重大发现。 在这一初步成功的基础上，我们建议进一步扩大这种NM传感器工具箱的有效性， 能够在深度和亚细胞分辨率下成像稀疏释放。我们的具体目标是：（1）提高 我们目前的传感器的灵敏度，使稀疏神经调节剂释放的强大成像，推动他们的空间 分辨率到亚细胞水平，并在较低浓度下增加响应的线性;（2）扩大其 光谱范围扩展到红光/远红光，以增强成像深度、SNR和体内多路测量， 使用两种或三种不同的颜色来操纵多个电路组件，以及（3）表征可能的 干扰电流传感器与内源性信号传导，并系统地验证新兴的传感器， 广泛的显微镜方法在体内。我们的策略依赖于传感器之间的动态协作 设计团队和最终用户获得持续的反馈，以实现对传感器的有效改进。 我们的目标是迅速传播广泛的特征鲜明、高度敏感的指标， 神经科学社区将被用来研究行为小鼠，鱼，苍蝇和蠕虫，以丰富我们的知识， 神经调节剂在大脑回路中的功能作用。

Project Summary The dynamic adaptability of the mammalian brain to environmental changes is remarkable, as it is the complexity of the networks of neurons underlying the operations that allow for such adaptations. Although we have some understanding of the anatomical and functional basis of this, we are still lacking a detailed picture of how the modulation of neuronal activity works. What is the timing and locations of these neuromodulator release and relationship with excitatory/inhibitory circuits? How does the neuromodulators circuitry accomplish the regulation of firing and synaptic properties of targeted neurons? Filling these gaps in knowledge would advance our understanding of all aspects of neuromodulator biology and allow discovery of new therapeutic strategies. To help close this gap, we have used creative approaches to the development of genetically encoded to directly report behaviorally triggered and modulated neuromodulator release including serotonin (5-HT), dopamine (DA) and norepinephrine (NE). We have disseminated these indicators to the neuroscience community and spurred major discoveries of novel mechanisms regulating neuromodulator release underlying motivation and addiction. Build on this initial success, we propose to further expand the effectiveness of this toolbox of NM sensors to enable imaging sparse release at depth and subcellular resolution. Our specific goals are to (1) improve the sensitivity of our current sensors to enable robust imaging of sparse neuromodulator release, push their spatial resolution to the subcellular level and increase linearity of response at lower concentrations; (2) expand their spectral range to red/far-red to enhance imaging depth, SNR and in vivo multiplex measurement and manipulation of multiple circuit components using two or three distinct colors, and (3) characterize the possible interference of current sensors with endogenous signaling and systematically validate emerging sensors with a wide-ranging microscopy approaches in vivo. Our strategy relies on a dynamic collaboration between the sensor design team and end users to obtain continuous feedback to implement efficient improvements to the sensors. It is our goal to rapidly disseminate a wide range of well-characterized, highly sensitive indicators for the neuroscience community to be employed to study behaving mice, fish, flies and worms, to enrich our knowledge on the functional roles of neuromodulators in the brain circuitry.