Unraveling AMPA receptor subpopulations nanoscale organization and traffic – AMPAR_SubPopTraffic

The plasticity of excitatory transmission in the brain is the cellular basis of learning. Its dysfunction is at the origin of neurodevelopmental and neurodegenerative diseases. Despite decades of work on the molecular mechanism behind synaptic plasticity, many of its aspects remain unknown. A major mechanism for modifying the efficiency of excitatory transmission is the regulation of the number and traffic of the AMPA subtype of glutamate receptors (AMPAR). Our project aims to solve one of the main mysteries of synaptic plasticity mediated by these receptors, which lies in the respective roles of the major subclasses of these receptors, and in particular those that are either permeable (CP-AMPAR) or impermeable (CI-AMPAR) to calcium. The presence of either of the receptor subtypes has very important functional consequences. This will be achieved through the development of innovative labeling and imaging tools, specific to receptor subpopulations, and combined with physiology. This project brings together cell biologists, structural biologists and imagers. We will break one of the essential barriers to our understanding of the mechanisms of synaptic plasticity.
Our objectives are:
1. Leverage our development of monovalent ligands specific to GluA1-3 subunits to measure their specific organization.
2. Implement a synthetic antibody discovery stream to develop ligands of different AMPAR populations.
3. Image nanoscale organization and surface traffic of endogenous AMPAR subunits and subpopulations in cultured neurons and brain slices.
4. Study the recruitment of different AMPAR populations during short- and long-term plasticity in brain slices. Derive multivalent ligands to specifically disrupt synaptic function.
To achieve these goals, we will combine existing and novel ligands with dynamic super-resolution imaging methods and physiology techniques.
The collaboration between a structural biologist with unique capabilities to develop new ligands specific to molecular assemblies - and thus to specific complexes of AMPA receptors, and a cellular neurobiologist with expertise in nanoscopic imaging of AMPA receptors and their physiology, provides an exceptional opportunity to address a fundamental question about the mechanism of glutamatergic synapse function that remains unanswered due to the lack of adequate tools.
Through the use and development of specific ligands for the extracellular domains of calcium-permeable and non-permeable AMPA receptors, combined with the use of STORM imaging, STED, expansion microscopy and single molecule tracking, we will for the first time determine the dynamic nanoscopic organization of these different receptor subtypes around and within the synapse. We will use both dissociated culture models of mouse hippocampal neurons and organotypic brain slices. We will be able to directly visualize the dynamic reorganizations of the different receptor subcategories during synaptic plasticity processes in order to better understand their functional roles.
Finally, by exploiting our recent developments in functional modification of multivalent ligands using optogenetics and suicide substrates, we will be able to modify with light both the trafficking and the existence of specific receptor subcategories in order to analyze their respective contributions to different stages of synaptic plasticity and neuronal physiology.