Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface

Abstract

Regulation of exo- and endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) plays a critical role in the expression of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) at excitatory central synapses. Enhanced AMPAR exocytosis or endocytosis has been suggested to contribute to LTP or LTD, respectively. However, several unsettled fundamental questions have remained about AMPAR exo- and endocytosis in the basal condition and during synaptic plasticity: (1) Does the size of each exo- or endocytosis event, and/or do the frequencies of these events change during LTP or LTD? If they change, what are the time courses of the respective changes? (2) Where does the exo- or endocytosis preferentially occur in each condition: inside or in the vicinity of postsynaptic membrane, or in the extrasynaptic membrane? (3) Do different types of AMPAR, such as GluA1 homo-tetramer, GluA1/2 hetero-tetramer and GluA2/3 hetero-tetramer, show distinct exo- and endocytosis changes? To address these questions, we developed new methods to observe individual events of AMPAR exo- or endocytosis with a high signal to noise (SN) ratio in a culture preparation using total internal reflection fluorescence microscopy (TIRFM). In these studies, hippocampal neurons were cultured on a neurexin (NRX)-coated glass coverslip, which induced formation of postsynaptic-like membrane (PSLM) directly on the glass surface. Then, a super-ecliptic pHluorin (SEP)-tagged AMPAR subunit such as GluA1 (GluA1-SEP) was expressed in neurons and its fluorescence changes during LTP induced by high frequency electrical field stimulation were observed with TIRFM, which showed different time courses of exocytosis changes of GluA1-, GluA2-, or GluA3-SEP in and around PSLM. In addition, a new method to detect individual endocytosis events of AMPAR was developed by combining TIFRM observation of GluA-SEP around PSLM with a rapid extracellular pH exchange method using a U-tube. Recent results on exo- and endocytosis changes of GluA-SEP during N-methyl-D-aspartate (NMDA)-induced LTD suggested that suppression of AMPAR exocytosis rather than enhancement of AMPAR endocytosis primarily contributes to LTD expression, although the NMDA application transiently enhances clathrin-dependent endocytosis of GluA1-containing AMPAR.

Keywords: AMPA receptor; LTD; LTP; endocytosis; exocytosis; hippocampus; live-cell imaging; total internal reflection fluorescence microscopy.

Figure 1

Formation of postsynaptic-like membrane (PSLM). NRX, Neurexin; NLG, Neuroligin; BSA, Bovine serum albumin; Fc, fragment crystallizable of immunoglobulin. This figure is newly drawn based on our previous publications (Tanaka and Hirano, ; Tanaka et al., 2014), and copyright permission is not required.

Figure 2

PSLM and normal synapses observed with total internal reflection fluorescence microscopy (TIRFM) or with conventional epi-fluorescence. (A) Scheme of PSLM and a normal synapse on NRX-coated glass. Excitation light (yellow) reaches only PSLM and the lower parts of dendrites in TIRFM (left), whereas it covers the whole area under epi-fluorescence (right). At a normal synapse, postsynaptic PSD95 signal is apposed to presynaptic vglut1 signal. (B) PSD95-EGFP signal (green) and vglut1 signal (magenta) recorded with TIRFM (left) or with epi-fluorescence (right), respectively. Arrows indicate PSLMs that are clearly observed with TIRFM and are not accompanied by vglut1 signals, and arrowheads indicate normal synapses which are not clearly observed with TIRFM. These figure panels were first published in Tanaka et al. (2014), and copyright permission was obtained.

Figure 3

Changes of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) subunit number by long-term potentiation (LTP)-inducing stimulation. (A–C) Averaged time courses of GluA1–3 fluorescence intensity in PSLM (red) and in non-PSLM (black) measured every 4 min before and after the field stimulation (arrows). Data in the presence of APV (+APV) are also shown (dotted lines). Error bars indicate SEM. *p < 0.05, **p < 0.01 and ***p < 0.001. (D–F) GluA-super-ecliptic phluorin (SEP) signals (green) and PSD95-RFP signal (magenta) are shown. PSD95-RFP was recorded before the stimulation, and images of the two signals were overlaid. GluA-SEP signals in PSLM and non-PSLM are indicated by arrows and arrowheads, respectively. Scale bar, 2 μm. These figure panels were first published in Tanaka and Hirano (2012), and copyright permission is not necessary.

Figure 4

Two examples of GluA1-SEP (green) exocytosis (arrows) shown together with PSD95-RFP (magenta). The numbers indicate time (seconds) after the field stimulation. These figure panels were first published in Tanaka and Hirano (2012), and copyright permission is not necessary.

Figure 5

Scheme of exocytosis changes of each AMPAR subtype during LTP expression. In a basal condition (Basal), exo- and endocytosis of GluA1/GluA2 and GluA2/GluA3 hetero-tetramer are in equilibrium. Soon after the high frequency electrical stimulation (Transient, around 1 min after the stimulation), exocytosis of GluA1 homo-tetramer occurs in the periphery of PSLM (red arrow), and exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). In the following period (Intermediate, about 3–10 min after the stimulation), exocytosis of GluA1/GluA2 increases outside PSLM (blue arrow). Finally (Late, about 20 min after the stimulation) exocytosis of GluA2/GluA3 increases outside PSLM (blue arrow). Some AMPAR exocytosed outside PSLM are likely to move into PSLM by lateral diffusion on the plasma membrane. This figure is newly drawn based on our previous publication (Tanaka and Hirano, 2012), and copyright permission is not required.

Figure 6

Rapid extracellular pH exchange method using a U-tube and detection of individual AMPAR endocytosis. When a bulb on a U-tube is open (Bulb open), the pH 6.0 solution flows inside U-tube and the extracellular pH 7.3 solution is also soaked into the U-tube. When the bulb is closed (Bulb closed), the intra U-tube pH 6.0 solution leaks out to the extracellular solution. In this pH 6.0 condition, only SEP signals from intracellular vesicles with near-neutral luminal pH such as those immediately after endocytosis can be detected. This figure is newly drawn based on our previous publication (Fujii et al., 2017), and copyright permission is not required.

Figure 7

Scheme of exo- and endocytosis changes of GluA1-containing AMPAR during long-term depression (LTD) expression. In a basal condition (Basal) exo- and endocytosis of GluA1-containing AMPAR are in equilibrium. Immediately after the N-methyl-D-aspartate (NMDA) application (Transient, around 1 min after the onset of NMDA application), clathrin-dependent endocytosis and exocytosis of AMPAR increase in the periphery of PSLM. In the following period (Intermediate, about 3–10 min), AMPAR exocytosis is suppressed. Finally (Late) exo- and endocytosis of AMPAR go into equilibrium in a low level. How each type of AMPAR changes during LTD remains to be clarified. This figure is newly drawn based on our previous publications (Fujii et al., 2017, 2018), and copyright permission is not required.