IMAGING GLUTAMATE IN THE BRAIN USING NOVEL FAST FLUORESCENT PROBES

点击翻译按钮获取中文摘要

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.