IMAGING GLUTAMATE IN THE BRAIN USING NOVEL FAST FLUORESCENT PROBES

谷氨酸被认为是中枢神经系统中一种主要的兴奋性神经递质。谷氨酸调节失调涉及焦虑和抑郁等神经精神障碍，以及亨廷顿氏病等急性和慢性神经退行性疾病。基于细菌谷氨酸/天冬氨酸结合蛋白(BP)和环状置换增强型绿色荧光蛋白(CpEGFP)的基因编码谷氨酸传感器iGluSnFR的研制代表着谷氨酸监测方面的重大突破。然而，iGluSnFR的动力学速度太慢，无法跟踪高频动作电位(AP)的放电。我们的新变体iGluu的动力学速度快了5倍，足以显示谷氨酸从AP之间的突触缝隙中清除。在这项提议中，我们将利用这个探针不仅可视化突触谷氨酸的释放，而且还摄取到星形胶质细胞，并使用小鼠模型比较健康和亨廷顿病的动力学(与德国柏林的Rosemarie Grantyn合作)。虽然iGluu是一个有用的工具，但在微秒时间尺度上可视化AMPA受体上谷氨酸的快速结合以及随后的通道开放仍然太慢。我们产生了一种新型的荧光标记谷氨酸传感器，Fl-GluBP，具有亚毫秒级的谷氨酸结合动力学。我们计划将这种传感器定位到膜的胞外位置，以监测与AMPAR反应平行的实时谷氨酸释放(与德国汉堡的Thomas Oertner合作)。此外，我们将开发具有不同性质(动态范围，KD)的类似探针，以匹配不同的谷氨酸水平(星形胶质细胞、突触裂隙、神经元)。谷氨酸从突触间隙的快速清除主要是由位于周围星形胶质细胞质膜上的转运蛋白完成的。在那里，谷氨酸被转化为谷氨酰胺，然后被亚顺序释放，被突触前终末吸收，转化为谷氨酸，并被包装成囊泡(谷氨酸-谷氨酰胺循环)。在细菌谷氨酰胺结合蛋白(GlNBP)的基础上，我们将使用与Fl-GluBP类似的方法来生成谷氨酰胺传感器(Fl-GlNBP)，这将使我们能够通过同时成像脑细胞培养中的星形胶质细胞和神经元来研究谷氨酸-谷氨酰胺循环的不同阶段。此外，Rosemarie Grantyn(德国柏林)将使用我们的探针对HD和正常小鼠纹状体、小脑皮质和丘脑底核中的星形胶质细胞中的谷氨酸和谷氨酰胺进行成像，以确定它们在HD中的作用。

Glutamate is recognised as a major excitatory neurotransmitter in the central nervous system. Dysregulation of glutamate is involved in neuropsychiatric disorders like anxiety and depression, as well as acute and chronic neurodegenerative disorders such as Huntington's disease. The development of a genetically-encoded glutamate sensor, termed iGluSnFR, which is based on the bacterial glutamate/aspartate binding protein (BP) and circular permuted enhanced green fluorescent protein (cpEGFP) represented a major breakthrough in monitoring glutamate. However, the kinetics of iGluSnFR are too slow to track high-frequency action potential (AP) firing. Our new variant iGluu with 5-fold faster kinetics was fast enough to show glutamate clearance from the synaptic cleft in between AP. In this proposal we will make use of this probe to not only visualize synaptic glutamate release but also uptake into astrocytes and compare the dynamics in health and Huntington disease using a mouse model (in collaboration with Rosemarie Grantyn, Berlin, Germany). While iGluu is a useful tool, it is still too slow to visualize the fast binding of glutamate of the AMPA receptor which is followed by the channel opening on the microsecond time scale. We generated a novel fluorescently labelled glutamate sensor, Fl-GluBP, with sub-millisecon glutamate binding kinetics. We plan to target this sensor to the extracellular site of the membrane to monitor real-time glutamate release in parallel to AMPAR response (collaboration with Thomas Oertner, Hamburg, Germany). Furthermore we will develop similar probes with altered properties (dynamic range, Kd) to match different glutamate levels (astrocytes, synaptic cleft, neurons). The fast clearance of glutamate from the synaptic cleft is mainly done by transporters located in the plasma membrane of surrounding astrocytes. There the glutamate gets converted into glutamine and is subquentially release, taken up by the presynaptic terminal, converted into glutamate and packed into vesicles (glutamate-glutamine cycle). On the basis of the bacterial glutamine binding protein (GlNBP) we will use a similar approach as for Fl-GluBP to generate glutamine sensors (Fl-GlNBP), which will allow us to investigate the different stages of the glutamate-glutamine cycle by simultaneous imaging of astrocytes and neurons in brain cell culture. Additionally, Rosemarie Grantyn (Berlin, Germany) will use our probes to image glutamate and glutamine in astrocytes in the striatum, cerebellar cortex and subthalamic nucleus in HD and normal mice to determine their role in HD.