Molecular mechanisms underlying the transmitter release at the serotonergic axon terminals

我们正在应用共聚焦显微镜，以便我们能够表征5-羟色胺能终末神经递质释放的特性和机制。每四个成年人中就有一个在任何一年中至少经历过一次可诊断的精神疾病。在英国和全世界，精神疾病是导致残疾的最大原因。5-羟色胺系统是治疗精神障碍最广泛使用的靶点，选择性5-羟色胺再摄取抑制剂是最常用的抗抑郁药。尽管5-羟色胺很重要，但我们仍然对大脑中参与5-羟色胺释放的分子角色知之甚少。与此同时，还有其他神经递质，如谷氨酸，可以与5-羟色胺一起从同一神经中释放。我们不知道神经做出的决定背后的机制，也不知道个体发射器何时以及如何释放。在这项研究中，我们计划回答这些问题。最近，科学家们发现，他们可以用生物素标记感兴趣分子的邻近蛋白质，然后用质谱仪鉴定这些标记的蛋白质。我们使用这一策略来定位参与调节5-羟色胺和其他位于同一神经中的递质释放的蛋白质。为了研究这些蛋白质的功能，我们建立了一个3D模型，模仿可以放在盘子里的含有5-羟色胺的神经的投影。这是通过使用有机体和组装体技术实现的，从小鼠胚胎的特定脑区培养细胞，形成类似于大脑中神经元连接的结构。我们可以使用分子工具来表达或敲除3D模型中神经元的某些基因。这些工具包括光遗传学、基因编码的荧光传感器和CRISPR-CAS9基因编辑系统。光遗传工具是一种光敏蛋白质，当它们被光触发时，可以调节神经元的活动。新开发的基因编码的5-羟色胺传感器一旦检测到5-羟色胺就会产生荧光信号。我们将在5-羟色胺能细胞中表达光遗传工具，并通过照射多束激光来激活神经中的几个点，并通过成像表达5-羟色胺传感器的周围细胞来监测这些神经中5-羟色胺的释放。我们还可以使用谷氨酸传感器来检测来自相同神经的谷氨酸释放。同时，我们将对成像的神经元进行电生理记录，以检测这些递质造成的影响。该实验要求具有较高的时间和空间分辨率，以及多点刺激系统、成像系统和电生理系统之间的协调。我们将通过建立一个带有高端激光扫描共焦的多功能实时成像设备来实现这一点。这将使我们能够表征5-羟色胺能神经末梢的递质释放特性。然后，我们将使用CRISPR-Cas9策略敲除编码我们定位的蛋白质的基因，并使用我们的多功能实验装置来测试这些蛋白质的移除如何影响释放特性。我们的结果将从根本上更深入地理解5-羟色胺能终末共同传递背后的分子机制。他们还将发现5-羟色胺传递的新的分子和生理特征，这可能为治疗精神障碍提供全新的药物靶点。

We are applying for a confocal microscope so that we can characterize the properties and the mechanisms of neurotransmitter release at the serotonergic terminals. One in four adults experiences at least one diagnosable mental illness in any given year. Mental illnesses represent the largest single cause of disability in the UK and worldwide. The serotonin system is the most widely used target for treating mental disorders, and selective serotonin reuptake inhibitors are the most commonly prescribed antidepressants. Despite its importance, we still know very little about the molecular player involved in serotonin release in the brain. Meanwhile, there are other neurotransmitters, such as glutamate, that can be released together with serotonin from the same nerve. We don't know the mechanism behind the decision made by the nerve, about when and how the individual transmitter is released. In this study, we plan to answer these questions. Recently, scientists find that they can label the neighbouring proteins of an interesting molecule with biotin and then identify these labelled proteins by mass spectrometry. We use this strategy to map the proteins that are involved in the regulation of the release of serotonin and other transmitters localized in the same nerve. To study the functions of these proteins, we have established a 3D model mimicking the projection of the nerves containing serotonin that can be put in a dish. This is achieved by using organoid and assembloid technologies, culturing cells from certain brain regions of mouse embryos to form structures resembling neuronal connections in the brain. We can use molecular tools to express or knock out certain genes from neurons in our 3D model. These tools include optogenetics, genetically encoded fluorescent sensors, and CRISPR-Cas9 gene-editing system. Optogenetic tools are light-sensitive proteins that can modulate neurons' activity when they are triggered by light. The newly developed genetically encoded serotonin sensor will generate a fluorescent signal once it detects serotonin. We will express optogenetic tools in the serotonergic cells and activate several spots in the nerves by shining multiple beams of laser, and monitor serotonin release from these nerves by imaging the surrounding cells that express serotonin sensors. We can also use a glutamate sensor to detect glutamate release from the same nerves. At the same time, we will perform electrophysiological recording from the imaged neurons to examine the effects caused by these transmitters. This experiment requires high resolution in both time and space, as well as coordination among the multi-point stimulation system, the imaging system and the electrophysiological system. We will achieve this by setting up a multi-functional live imaging rig with a high-end laser scanning confocal. This will allow us to characterize the properties of transmitter release at the serotonergic nerve terminals. We will then use CRISPR-Cas9 strategy to knock out the genes encoding the proteins we mapped and use our multi-functional rig to test how the removal of these proteins influences the release properties. Our results will provide a fundamentally deeper understanding of the molecular mechanism behind co-transmission at the serotonergic terminals. They will also uncover new molecular and physiological signatures of serotonin transmission that could provide entirely novel drug targets for treating mental disorders.