Development of Fluorescent False Neurotransmitters

总结 荧光假神经递质的研究进展 单胺神经传递在调节兴奋性和抑制性突触中起重要作用， 中枢神经系统，从而提供响应于变化的行为的必要微调。 环境因此，单胺神经传递的异常是许多神经和 神经精神障碍，包括帕金森病、精神分裂症、ADHD、抑郁症和药物依赖性精神障碍。 成瘾我们建立了一个跨学科的研究计划，重点是开发新的 在突触水平上研究单胺神经传递的分子探针。具体来说，我们介绍了 一种概念上新的探针，称为“荧光假神经递质”（FFN）， 多巴胺和去甲肾上腺素的示踪剂FFNs提供了第一个成像突触囊泡的实验手段 在脑组织和活体动物中，在单个突触前静脉曲张处的内容物摄取和释放。使用 FFN和多光子显微成像，我们已经取得了一些意想不到的发现，包括发现 沉默的多巴胺能和去甲肾上腺素能突触，突触囊泡的活性依赖性超充电， 和对精神兴奋剂药物的机械见解。在这次续期申请中，我们建议扩大 FFN的范围，以血清素突触，第三个主要的单胺系统，在概念验证的基础上， 问题研究我们还将开发新的成像和光子计数方法的基础上组合使用 FFNs作为突触前探针，具有作为突触后传感器的新兴遗传编码单胺报告物 提供活体小鼠的多参数突触读数。除了补充每个FFN类， 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， 每个传感器用于不同的单胺，将能够同时检查两种神经递质 系统.这反过来将使复杂过程的研究，如共传递和神经递质 在突触水平上切换。我们还将研究单胺释放剂对突触的影响， 多巴胺、去甲肾上腺素和5-羟色胺轴突释放位点的神经递质动力学，以探测两者 活动性和静止性静脉曲张中的囊泡池以及这些精神活性药物的作用机制。 该申请解决了在开发用于成像的分子工具和方法中的主要未满足的需求 在活体动物中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.