An increase in the number of AMPA-type glutamate receptors (AMPARs) is critical for long-term potentiation (LTP), synaptic plasticity regarded as a basal mechanism of learning and memory. However, when and how each type of AMPAR reaches the postsynaptic membrane remain unclear. We have developed experimental methods to form postsynaptic-like membrane (PSLM) on a glass surface to precisely visualize the location and movement of receptors. We observed fluorescence-labeled AMPAR subunits (GluA1-3) around PSLM with total internal reflection fluorescence microscopy. The increases of GluA1, 2, and 3 in PSLM showed different time courses after LTP induction. GluA1 increased first, and was exocytosed to the periphery of PSLM soon after LTP induction. GluA2 and GluA3 initially decreased, and then increased. Exocytosis of GluA2 and GluA3 occurred primarily in non-PSLM, and later than exocytosis of GluA1. Thus, GluA1-3 appear to increase in the postsynaptic membrane through distinct pathways during LTP.

This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein-coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.

Synaptic plasticity such as long-term depression (LTD) has been regarded as a cellular mechanism of learning and memory. LTD is expressed by the decrease in number of postsynaptic AMPA-type receptor (AMPAR) at glutamatergic synapses. Although endocytosis is known to play an essential role in the decrease in AMPAR on postsynaptic membrane, the difficulty to detect individual endocytic events hampered clarification of AMPAR dynamics around synapses. Previously, we developed a method to induce formation of postsynaptic-like membrane (PSLM) on the glass surface and observed pHluorin-tagged AMPAR around PSLM with total internal reflection fluorescence microscopy. By this method, individual exocytosis of AMPAR-pHluorin was recorded in both PSLM and non-PSLM. In other studies, endocytic vesicles containing pHluorin-tagged receptors were visualized by changing extracellular pH. Here, we have combined PSLM formation method and rapid pH change method, and detected individual endocytic events of AMPAR around PSLM with high spatial and temporal resolutions. Endocytic events of AMPAR were characterized by comparison with those of transferrin receptor. Constitutive endocytosis of AMPAR was not dependent on clathrin and dynamin in contrast to that of transferrin receptor. However, AMPAR endocytosis triggered by LTD-inducing stimulation was clathrin-and dynamin-dependent.

The decrease in number of AMPA-type glutamate receptor (AMPAR) at excitatory synapses causes LTD, a cellular basis of learning and memory. The number of postsynaptic AMPARs is regulated by the balance of exocytosis and endocytosis, and enhanced endocytosis of AMPAR has been suggested to underlie the LTD expression. However, it remains unclear how endocytosis and exocytosis of AMPAR change during LTD. In this study, we addressed this question by analyzing exocytosis and endocytosis of AMPAR by imaging super-ecliptic pHlorin (SEP)-tagged AMPAR around postsynaptic structure formed directly on the glass surface in the hippocampal culture prepared from rat embryos of both sexes. Contrary to a prevailing view on the LTD expression by endocytosis enhancement, the LTD induction by NMDA application only transiently enhanced endocytosis of SEP-tagged GluA1 subunits of AMPAR, which was counteracted by simultaneous augmentation of exocytosis. As a result, soon after the start of the LTD induction (∼1 min), the surface AMPAR did not markedly decrease. Thereafter, the surface GluA1-SEP gradually decreased (2-5 min) and kept at a low level until the end of observation (>30 min). Surprisingly, this gradual and sustained decrease of surface AMPAR was accompanied not by the enhanced endocytic events of GluA1, but by the suppression of exocytosis. Together, our data highlight an unprecedented mechanism for the LTD expression by attenuation of exocytosis of AMPAR, but not by enhanced endocytosis, together with a reduction of postsynaptic AMPAR scaffolding protein PSD95. It has been generally assumed that LTD is expressed by enhancement of AMPAR endocytosis. Previous studies reported that endocytosis-related protein was involved in LTD and that significant amount of cell-surface AMPAR moved into intracellular compartments during LTD. Here, we report changes of cell-surface amount of AMPAR, and where and when individual exocytosis and endocytosis occurred during LTD. Cell-surface AMPAR gradually decreased in synchrony with suppression of exocytosis but not with enhancement of endocytosis. These results suggest that the decrease of cell-surface AMPAR amount during LTD was caused not by enhancement of endocytosis but rather by suppression of exocytosis, which revises current understanding of the expression mechanism of LTD.

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17–0.19 μm2/s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.