Physiopathological regulation of single NMDA receptor dynamics in the hippocampus – DynHippo

Most of the maturation and plasticity of glutamatergic synapses rely on the postsynaptic activation of N-Methyl-D-Aspartate receptor (NMDAR). In addition, a dysregulation of the NMDAR signaling has been proposed to play a direct role in the emergence of neuropsychiatric disorders, such as schizophrenia. Thus, understanding the physiopathological regulation of the NMDAR signaling has captured a lot of attention and major advances have been made in the recent years. Among these, the set-in-stone dogma that NMDAR are stable in synapses has simply shattered, opening a new conceptual framework. These seminal studies unveiled that NMDAR surface trafficking is regulated by molecular pathways located in the intracellular compartment, within the plasma membrane, as well as in the extracellular environment. However, our current understanding comes from in vitro investigations using mostly primary cultures of neurons. Central questions related to the complexity and regulation of NMDAR surface distribution and dynamics thus urgently need to be tackled in intact brain tissue. This represents a particularly obvious challenge since the interplay between the extracellular environment and the NMDAR dynamics now needs to be explored. Accessing this process in physiology will also allow us to directly address one of the hypothesis for schizophrenia proposing that NMDAR transmission deficit on pyramidal and/or GABAergic interneurons is due to an altered extracellular environment, such as the reduced extracellular matrix and perineuronal net observed in schizophrenic postmortem brains. Thus, understanding how membrane NMDAR are organized and trafficked in principal cells and interneurons in health and schizophrenia will surely break our current frontier knowledge.

The main objective of this proposal is to measure NMDAR surface dynamics in intact tissue and define the regulatory properties of the extracellular environment on this cellular pathway in health and models of schizophrenia. We hypothesize that the spatio-temporal dynamics of NMDAR and extracellular environment components are intimately entangled and play a pivotal role both in physiological and schizophrenic brains. Understanding this complex dynamic interplay requires first dedicated investigation strategies. An ultimate strategy for imaging and understanding biological phenomena in living cells or cell assemblies relies on single-molecule microscopy since it has the advantage to extract and identify the molecular behavior of receptor sub-populations while retrieving molecule localizations with sub-wavelength precision. Based on our past experience and preliminary data, we are now in a unique position to track NMDAR subtypes at the surface of pyramidal cells and interneurons in hippocampal acute brain slices using single nanoparticle (QD) tracking. To complement this first approach to study single NMDAR dynamics, we will also apply super-resolution approaches (uPAINT and STORM) in order to provide the first high-density quantitative map of surface NMDAR subtypes in pyramidal cells and interneurons from brain slices. With these, we will study the physiological impact of the extracellular environment on the NMDAR dynamic, both in pyramidal cells and interneurons. Then, we will use rodent models of schizophrenia to determine the alteration of NMDAR surface distribution, dynamics and transmission in this disorder. Ultimately, we will manipulate the NMDAR surface dynamics in schizophrenic models in order to restore the proper transmission and prevent the behavioural deficits. Performing single molecule tracking deep in the brain tissue, or even in living animals, is one of our future aims so we will develop new classes of ultra-bright nanolabels to be imaged with two-photon microscopy at the single molecule level. Altogether, this interdisciplinary project will tackle a fundamental and clinical question in neuroscience by bringing together world-leader experts in optics and nanolabel chemistry.