AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK-dependent manner during long-term potentiation

Abstract

The exocytosis of AMPA receptors is a key step in long-term potentiation (LTP), yet the timing and location of exocytosis and the signaling pathways involved in exocytosis during synaptic plasticity are not fully understood. Here we combine two-photon uncaging with two-photon imaging of a fluorescent label of surface AMPA receptors to monitor individual AMPA receptor exocytosis events near spines undergoing LTP. AMPA receptors that reached the stimulated spine came from a combination of preexisting surface receptors (70-90%) and newly exocytosed receptors (10-30%). We observed exocytosis in both the dendrite and spine under basal conditions. The rate of AMPA receptor exocytosis increased approximately 5-fold during LTP induction and decayed to the basal level within approximately 1 min, both in the stimulated spine and in the dendrite within approximately 3 microm of the stimulated spine. AMPA receptors inserted in the spine were trapped in the spine in an activity-dependent manner. The activity-dependent exocytosis required the Ras-ERK pathway, but not CaMKII. Thus, diffusive Ras-ERK signaling presumably serves as an important means for signaling from synapses to dendritic shafts to recruit AMPA receptors into synapses during LTP.

Fig. 1.

SEP-GluA1 is recruited to spines after stimulation. (A) Images of a dendritic segment of a neuron transfected with mCherry (Left) and SEP-GluA1 (Right) before (Top), immediately after (Middle), and 30 min after (Bottom) single-spine stimulation. (Scale bar, 1 μm.) (B) Time course of mCherry (red) and GFP-GluA1 (green) fluorescence increase after single-spine stimulation. Stimulated spines (filled circles) increase in size transiently before plateauing. Adjacent spines do not grow (open circles). Fluorescence is normalized to three reference images before uncaging. n = 8 for stimulated spines, 17 adjacent spines, 3 cells. (C) Time course of mCherry and SEP-GluA1 fluorescence after single-spine stimulation. (Red fluorescence)^2/3 shown as blue dotted line. n = 34 for stimulated spines, 16 adjacent spines, 21 cells. (D) Time course of mCherry and membrane-tagged YFP-CD8 fluorescence after single-spine stimulation. (Red fluorescence)^2/3 shown as blue dotted line. n = 13 spines, 4 cells. (E) Time course of fluorescence increase during uncaging, normalized to peak increase. (F) Time course of mCherry fluorescence under control, MEK inhibitor U0126 (open circles, n = 18 spines, 7 cells), and CaMKII inhibitor KN62 (open triangles, n = 17 spines, 7 cells) conditions. (G) Time course of SEP-GluA1 fluorescence under drug conditions.

Fig. 2.

Newly exocytosed AMPARs are recruited to stimulated spines. (A) Images of mCherry (Left) and SEP-GluA1 (Right) fluorescence before bleaching (Top), after bleaching (Middle), and after uncaging (Bottom). (B) Fluorescence time course of mCherry (Left) and SEP-GluA1 (Right) for the spine and dendrite shown in A. (C) Population data of mCherry (Left) and SEP-GluA1 (Right) fluorescence during bleaching protocol. SEP normalized to prebleach fluorescence. n = 18 stimulated spines, 9 adjacent spines, and 16 neurons. (D) mCh (Left) and SEP fluorescence (Right) in the presence of TeTX (black crosses), KN62 (open blue squares), and U0126 (filled triangles). n = 10 spines, 6 cells for TeTX; 10 spines, 6 cells for U0126; and 15 spines, 5 cells for KN62. (E) mCh (Left) and SEP (Right) fluorescence after lamp bleaching and uncaging. Only the stimulated spine (filled square) recovered SEP fluorescence, whereas the dendrite (filled triangles) and adjacent spines (dotted line, cross) did not. TeTX blocked SEP-GluA1 fluorescence recovery in stimulated spines (open circles). n = 9 spines, 6 cells for lamp bleaching; 6 spines, 3 cells for TeTX under lamp bleach.

Fig. 3.

Kinetics of exocytosis events. (A) mCherry (Upper) and SEP-GluA1 images (Lower) of spines undergoing stimulation. Filtered spatially (0.75 μm) and temporally (1.25 s). Exocytosis is measured as a sharp increase in spine fluorescence. Stimulated spine shown by open arrowhead. Exocytosis shown by closed arrowhead. (Left) Example of transient spine exocytosis. (Right) Example of sustained spine exocytosis. Numbers indicate time after starting uncaging(s). (Scale bar 1 μm.) (B) mCherry and SEP-GluA1 fluorescence during dendritic exocytosis. (Left) Example in dendrite immediately beneath spine, showing movement of fluorescence into the stimulated spine (40–41 s). (Right) Example that is 2 μm away. (C) Fluorescence time course for region of interest (ROI) shown as yellow circle in A (filtered with 1.25-s window). (D) Fluorescence time course for ROI shown in B. (E) Average of all unstimulated spine exocytosis events (thick green line) and four example exocytosis events (thin lines). n = 25 events. Average was taken of unfiltered data, whereas individual traces have been filtered (1.25 s). (F) Average of all stimulated exocytosis events (thick green line), trace examples in A (black lines), and four other example exocytosis time courses. n = 46 events. (G) Average fluorescence time course for unstimulated dendritic exocytosis (thick green) and four individual example time courses. (H) Average time course of stimulated dendritic exocytosis events (thick green line), examples from B (black lines), and three other example time courses. n = 134 events.

Fig. 4.

Location and timing of activity-dependent exocytosis. (A) Time course of exocytosis in spine. Fifty-second movies were taken and split into 25-s epochs. (B) Time course of dendritic exocytosis within 2.5 μm of stimulated spine. (C) Distance from stimulated spine exocytosis occurred before (open circles), during (filled triangles), and after (gray squares, filled diamonds) stimulation. (D) (Left) Exocytosis rate in spine during stimulation (0–50 s). Same color scheme as in A. *Significant differences from control rate (ANOVA, P < 0.05). (Right) Exocytosis rate in the dendrite, near (0–2.5 μm) and away from (3.5–6.5 μm) the stimulated spine during stimulation (0–50 s). n = 112 spines/dendrites from 31 cells for control; 39 spines, 7 cells for U0126; 41 spines, 9 cells for KN62; 25 spines, 6 cells for tetanus toxin (TeTX); and 49 spines, 12 cells for dnRas.