Abstract The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact in both basic bioscience and in future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (AMPARs/NMDARs, known as iGluRs) are involved in neuron-to-neuron communication across synapses, where these receptors contribute to learning and memory, and when dysregulated, to neurodegenerative diseases including Alzheimer's, Parkinson's and complications from strokes. A critical mechanistic event is the transport of iGluRs into and out of synapses (or parts of synapses) in a dynamic process called synaptic plasticity. A revolution is underway because of the recent ability to resolve these events at the nanometer-scale using fluorescence super-resolution microscopy (FSRM). However significant inherent problems with this technology have led to confounding results and misinformation. The biggest problem has been with the fluorescent probes used to image receptors: conventional organic fluorescent probes last only a few seconds; commercial (and big) quantum dots (bQDs), despite their exceptional brightness and photostability, are over 20 nm in diameter and are too large to fit inside the synaptic cleft where iGluRs are active. We recently overcame this problem through an R21, which enabled us to develop small quantum dots (sQDs) that are <10 nm in diameter. They specifically label iGluRs in the synaptic cleft, which is just ~20-30 nm wide. The sQDs do this with tremendous brightness and stability, resulting in FSRM images in 3-dimensions with 100 ms time-resolution for greater than 2 minutes of continuous excitation. In contrast, bQD-labeled AMPARs are predominantly stuck in the extra-synaptic space because steric hindrance prevents them from going inside. We have recently extended these findings with a newer sQD that is completely stable, and with small organic fluorophores that we now show are stable enough, on live neurons (which previously had been too photolabile for such measurements.) Our findings, some of which have been published in 3 papers resulting from our R21 grant, may have tremendous implications for basic science and health: the surface mobility and trafficking of iGluRs, which depend on the ease of diffusion inside and outside of synapses, regulates synaptic efficacy. Here we wish to understand the distribution and dynamics of iGluRs, both within the synapses and between synapses, using our new sQDs and other new photoactivatable fluorescent proteins and some organic fluorophores. For this, a number of new advances in optics, probe design, and care with receptor monovalency are necessary. After these technical problems are solved (which will be useful to answer many different biological questions), we will validate the biology that we have observed, and to apply these to proof-of-principle experiments involved in two key biological questions: 1) In what way do receptors move into and around the synapse during homeostatic and synaptic plasticity? 2) Do endocytosed receptors communicate with each other between synapses on the same neuron?

Project Narrative: AMPA- and NMDA-type glutamate receptors are neurotransmitter receptors that regulate learning, memory, and numerous neurodevelopmental and neurodegenerative diseases. We will develop new microscopy tools, probes, and methods to visualize their trafficking into and out of synapses to help understand these processes, exemplified by some “proof-of-principle” experiments.