Optical dissection of glycine and D-serine signalling using newly-developed sensors

兴奋性突触连接强度的持续变化被认为是学习过程的基础。这些变化的诱导通常依赖于谷氨酸受体的一个亚型，即所谓的N-甲基-D-天冬氨酸受体(NMDAR)。NMDAR的激活还需要NMDAR共同激动剂的结合，D-丝氨酸或甘氨酸。因此，它们的可用性控制着突触可塑性的程度。然而，关于D-丝氨酸和甘氨酸在细胞外的空间分布，控制其胞外浓度的机制，以及神经元和星形胶质细胞在其供应中的不同作用，仍存在着激烈的争论。为了直接研究NMDAR共激动剂的信号传递，我们先前设计了新型的光学NMDAR共激动剂传感器。我们已经成功地制作了基于光学FRET的甘氨酸传感器(GlyFS)和D-丝氨酸传感器原型(DSerFS)。使用GlyFS，我们最近可以验证关于海马区静息甘氨酸浓度分布和发展的重要预测，并发现细胞外甘氨酸浓度受可塑性诱导活性控制。然而，甘氨酸的确切机制和细胞来源仍不清楚。在本项目中，我们将进一步开发和完善甘氨酸(GlyFS)和D-丝氨酸(DSerFS)的光学传感器，同时剖析控制NMDR共激动剂水平的机制。对于甘氨酸成像，我们将通过交换FRET对(荧光蛋白)并通过优化FRET供体/受体的方向、距离和相互作用来提炼当前一代的GlyFS。我们还将修改传感器，使其可以针对特定细胞类型和亚域的表面。使用原始的GlyFS和改进的版本，我们将确定网络活动的频率和强度如何决定细胞外甘氨酸水平，哪些机制起作用，以及神经元和星形胶质细胞(在急性海马片中)的相对作用是什么。通过光学记录突触和突触外的甘氨酸浓度，我们将可视化相应NMDAR种群的甘氨酸浓度是如何随活动而变化的。同时，我们将测试目前的D-丝氨酸传感器原型DSerFS，并获得关于D-丝氨酸信号的第一个实验结果。例如，将测试对神经元或星形胶质细胞的直接和单独刺激是否会增加细胞外D-丝氨酸水平。同时，我们将通过使用与GlyFS相同的技术来改善DSerFS的光学特性及其多功能性。我们的工作将为NMDR共激动剂信号转导提供新的重要见解，从而为NMDAR依赖的突触可塑性提供新的重要见解。我们将进一步向神经科学家的工具箱添加两个光学传感器，这对研究记忆形成和大脑中其他依赖NMDAR的过程将是重要的。

Persistent changes of the strength of excitatory synaptic connections are thought to underlie learning processes. The induction of these changes often relies on a subtype of glutamate receptor, the so-called N-methyl-D-aspartate receptor (NMDAR). Activation of NMDARs also requires the binding of an NMDAR co-agonist, either D-serine or glycine. Therefore, their availability controls to what degree synaptic plasticity can occur. There is, however, an ongoing intense debate about the spatial extracellular distribution of D-serine and glycine, the mechanisms that control their extracellular concentration and the differential roles of neurons and astroglia in their supply.To directly study NMDAR co-agonists signalling, we previously designed novel optical NMDAR co-agonist sensors. We have succeeded in generating an optical FRET-based glycine sensor (GlyFS) and a D-serine sensor prototype (DSerFS). Using GlyFS we could recently verify important predictions about the distribution and development of resting glycine concentrations in the hippocampus and discovered that plasticity-inducing activity governs the extracellular glycine concentration. The exact mechanisms and the cellular sources of glycine remained, however, unclear. In the current project, we will further develop and refine optical sensors for glycine (GlyFS) and D-serine (DSerFS) while at the same time dissecting the mechanisms that control NMDR co-agonist levels. For glycine imaging, we will refine the current generation of GlyFS by exchanging the FRET-pair (fluorescent proteins) and by optimization of FRET donor/acceptor orientation, distance and interaction. We will also modify the sensor so it can be targeted to the surface of specific cell types and subdomains. Using the original GlyFS and improved versions we will establish how the frequency and strength of network activity determines extracellular glycine levels, which mechanisms contribute and what the relative role of neurons and astroglial are (in acute hippocampal slices). By optical recording of synaptic and extrasynaptic glycine concentrations we will visualize how the glycine concentration at the corresponding NMDAR populations is changed by activity. In parallel, we will test the current D-serine sensor prototype DSerFS and obtain first experimental results regarding D-serine signalling. For instance, it will be tested if direct and isolated stimulation of neurons or astrocytes increases extracellular D-serine levels. At the same time, we will improve the optical properties of DSerFS and its versatility by using the same techniques as outlined for GlyFS.Our work will provide new and important insights into NMDR co-agonist signalling and thus into NMDAR-dependent synaptic plasticity in the hippocampus. We will further add two optical sensors to the neuroscientist’s toolbox that will be important for studying memory formation and other NMDAR-dependent processes in the brain.