Development of Fluorescent False Neurotransmitters

摘要 荧光假性神经递质的研究进展 单胺类神经递质在兴奋性和抑制性突触的调节中起重要作用 中枢神经系统，从而提供必要的行为微调，以响应变化 环境。因此，单胺类神经传递的异常是许多神经学和 神经精神障碍，包括帕金森氏症、精神分裂症、多动症、抑郁症和药物 上瘾。我们已经建立了一个跨学科的研究计划，专注于开发新的 在突触水平上研究单胺类神经传递的分子探针。具体地说，我们已经推出了 一种概念上的新类型的探针，被称为“荧光假神经递质”(FFN)，其作用是光学 多巴胺和去甲肾上腺素的示踪剂。FFN提供了第一个成像突触小泡的实验手段 脑组织和活体动物个体突触前曲张的内容摄取和释放。vbl.使用 FFNS和多光子显微镜成像我们有了几个意想不到的发现，包括这个发现 无声的多巴胺能和去甲肾上腺素能突触，突触小泡的活性依赖超负荷， 以及对精神刺激药物的机械洞察力。在这份续期申请中，我们建议扩大 在概念验证的基础上，将FFN的范围扩大到第三大单胺系统--5-羟色胺突触 学习。我们还将开发基于组合使用的新的成像和光子计数方法 FFN作为突触前探针，新出现的单胺报告基因作为突触后传感器 在活体小鼠身上提供多参数突触读数。除了补充每个FFN类别之外 相应的传感器(例如，多巴胺FFN和多巴胺传感器)、混合和匹配FFN和 分别用于不同单胺的传感器可以同时检测两种神经递质 系统。这反过来将使研究复杂的过程，如共同传递和神经递质 开启突触水平。我们还将检查单胺释放剂对突触的影响 多巴胺、去甲肾上腺素和5-羟色胺轴突释放部位的神经递质动力学研究 静脉曲张和活动性静脉曲张的囊泡池以及这些精神活性药物的作用机制。 该应用解决了在开发用于成像的分子工具和方法方面尚未满足的主要需求 活体动物的多种神经递质系统，具有足够的空间和时间分辨率。

SUMMARY Development of Fluorescent False Neurotransmitters Monoamine neurotransmission plays important roles in modulation of excitatory and inhibitory synapses in the central nervous system and thus provides essential fine tuning of behavior in response to a changing environment. Consequently, aberrations in monoamine neurotransmission underlie many neurological and neuropsychiatric disorders including Parkinson’s disease, schizophrenia, ADHD, depression, and drug addiction. We have established an interdisciplinary research program focused on development of new molecular probes to study monoamine neurotransmission on a synaptic level. Specifically, we have introduced a conceptually new class of probes, termed “fluorescent false neurotransmitters” (FFNs), that act as optical tracers of dopamine and norepinephrine. FFNs provide the first experimental means to image synaptic vesicle content uptake and release at individual presynaptic varicosities in brain tissue and in living animals. Using FFNs and multiphoton microscopy imaging we have made several unexpected findings, including the discovery of silent dopaminergic and noradrenergic synapses, activity-dependent super-charging of synaptic vesicles, and mechanistic insights on psychostimulant drugs. In this renewal application, we propose to expand the scope of FFNs to serotonin synapses, the third major monoamine system, on the basis of proof-of-concept studies. We will also develop new imaging and photon-counting methods based on the combinatorial use of FFNs as presynaptic probes with emerging genetically encoded monoamine reporters as postsynaptic sensors to provide multi-parameter synaptic readouts in living mice. In addition to complementing each FFN class with the corresponding sensor (for example, dopamine FFN and dopamine sensor), mixing and matching FFNs and sensors each for a different monoamine will enable simultaneous examination of two neurotransmitter systems. This in turn will enable the study of complex processes such as co-transmission and neurotransmitter switching on a synaptic level. We will also examine the effect of monoamine releasing agents on synaptic neurotransmitter dynamics at the dopamine, norepinephrine, and serotonin axonal release sites to probe both the vesicular pools in active and silent varicosities and the mechanisms of action of these psychoactive drugs. This application addresses a major unmet need in developing molecular tools and methods for imaging multiple neurotransmitter systems in living animals with adequate spatial and temporal resolution.