The efficacy of synaptic transmission changes depending on the neuronal activity. Such synaptic plasticity has been regarded as a basal mechanism of learning and memory. During expression of synaptic plasticity, the number of neurotransmitter receptors is dynamically controlled. These receptors show substantial mobility such as lateral diffusion, exocytosis and endocytosis, which seem to change during synaptic plasticity. In the last decade, researchers have attempted to reveal trafficking of AMPA receptor (AMPAR), which plays a major role in excitatory synaptic transmission in the central nervous system. The advances in imaging techniques have made it possible to observe lateral diffusion of AMPAR using quantum dot, and discrete exocytosis event using Total Internal Reflection Fluorescence Microscopy (TIRF). However, it has been difficult to observe individual endocytosis event of AMPAR.
We use pH-sensitive GFP variant, super ecliptc pHluorin (SEP)-tagged AMPAR and TIRF, both of which have been commonly used for observation of exocytosis. Moreover, in order to visualize individual endocytosis event, we changed extracellular pH intermittently to visualize vesicles containing AMPAR. In this method, AMPARs internalized just before the pH change to 6 were visualized. At pH6, the fluorescent signal from SEP fused to AMPAR on the cell surface became undetectable, and only signals from SEP in intracellular vesicles with neutral pH were detected. Further in this study, we formed postsynaptic-like membrane (PSLM) on a glass surface using neurexin-coated glass (Tanaka et al., 2014 Nature Protocols), and succeeded in detecting endocytosis around PSLMs.
Using this method, we observed stimulus-triggered endocytosis of AMPAR. Bath application of NMDA enhanced endocytosis of AMPAR around PSLM, which might be related to long-term depression. Thus, a new methods to record individual endocytosis event of postsynaptic AMPAR has been established, which will contribute to study regulation mechanisms of the number of postsynaptic receptors both in a basal condition and during synaptic plasticity.