Inhibitory neurotransmitter release and receptor dynamics during synaptic transmission: real-time imaging at single molecule level – SynTrack

The aim of this project is to understand at the microscopic and molecular level the relationship between inhibitory neurotransmitter release and receptor dynamics during synaptic transmission. To this aim we will make use of real-time super-resolutive microscopy.

Actually, transfer of information requires the precise alignment between the releasing sites of neurotransmitters in presynaptic terminals and the locations of activated receptors in postsynaptic differentiations. Recently, super-resolution microscopy has allowed to define at excitatory synapses the “nano-columns” which are substructures allowing the apposition of presynaptic release sites and postsynaptic receptors-enriched nano-domain. Although inhibitory synapses regulate the excitability in the network accurately and rapidly, the functional alignment between releasing sites of inhibitory synaptic vesicles and inhibitory receptors has not been studied yet. One reason is the difficulties of real-time imaging of individual vesicles and receptors whose sizes are smaller than the optical resolution of classical fluorescence microscopes. Our objective is to investigate simultaneously exocytoting presynaptic vesicles and the diffusion and stabilization dynamics of apposing receptors in living neurons, thus providing a functional counterpart to nano-column. Understanding intimately this dynamic phenomenon will be of fundamental importance to unravel mechanisms implicated in short term plasticity. Furthermore it will provide new insights for the comprehension of mechanisms and the development of drugs acting on inhibitory synapses.

This project will be possible thanks to recent progress in single-molecule fluorescence technique. More precisely: 1) Prof. Hyokeun Park at HKUST (in Hong-Kong) built a state-of-the-art real-time nanometer-accuracy three-dimensional (3D) tracking microscopy setup, which enables to track an individual synaptic vesicle (40 nm in diameter) with better accuracy than its diameter; 2) Prof. Antoine Triller at École Normale Supérieure (ENS, Paris) tracked individual postsynaptic receptors in live neurons for the first time and found dynamics of receptors in postsynaptic compartments. He recently developed single particle tracking (20 nm pointing accuracy) using super-resolution PALM (Photo-Activated Localization Microscopy). He showed an activity dependent stabilization of both GABA and glycine receptors by the scaffolding protein (gephyrin) at synapses.

Our joint team will provide a unique combination of skills for this project. We will study the specific properties of GABAergic and glycinergic nano-columns in cultured striatal and spinal cord neurons, respectively. To this aim, we will first develop microscopic and molecular tools to access simultaneously the release of neurotransmitters and diffusion behavior of receptors. We will then be able to investigate how neuronal activity control exocytosis and its consequences on apposed receptors. This will lead us to propose a framework for the molecular control of functional nano-column at inhibitory synapses. Ultimately, we will model the consequences of this coordinated pre-and post-synaptic activity on the dynamic equilibrium ensuring the stability and short-term plasticity of synapses.

This research will allow, for the first time, to provide a functional counterpart to inhibitory nano-column by accessing simultaneously single-vesicle exocytosis and the mobility of receptors. Furthermore, this research will provide a new framework for the pharmacology at synapses and possible pathogenic mechanisms for neurological disorders caused by the imbalance of excitation and inhibition.