Ultra-High Resolution Imaging of the Dynamic Nature of Post-Synaptic Molecules

Abstract

The spatial and temporal regulation in the composition of the postsynaptic membrane of synapses participates in the different forms of synaptic plasticity that trigger the cellular processes of memory formation, consolidation, and retrieval. Neurotransmitter receptors move rapidly in and out of synapses by lateral diffusion. This mobility is crucial to control the number of receptors present at a given synapse. Thus, the equilibrium between the synaptic and extra synaptic AMPA receptor number is crucial in controlling basal transmission and synaptic plasticity. This balance is regulated by the subunit composition of these receptors and by the interaction of intracellular scaffold proteins. However, how the trafficking of receptors and the scaffolding molecules in and out synapse is controlled remains unknown. Here we attempt to determine the relative distribution and trafficking properties of AMPA receptors and various scaffold proteins at unprecedented spatial (< 40nm) and temporal resolution (>50 Hz) using a variety of novel ultra-high resolution fluorescence imaging approaches. We combine Single Particle Tracking (SPT) and Photo Activation Localization Microscopy (PALM) to map trajectories at the level of individual molecules. Here we describe the implementation of a multimodal microscope along with the development of a new dedicated analysis for single molecule segmentation and tracking. Furthermore we will discuss the application of SPT-PALM experiments on living neurons. With this novel approach, we expect to comprehend the motilities of receptors or scaffolding proteins when they traffic between the submicron sized molecular zones of dendritic spines. The combination of this type of detection and analysis will provide the information from thousands of discrete trajectories from a single cell with which it would be possible to appreciate finer details of versatile molecular mechanisms pertinent in the functioning of an excitatory synapse.