Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes

Abstract

Dendritic spines are micron-sized membrane protrusions receiving most excitatory synaptic inputs in the mammalian brain. Spines form and grow during long-term potentiation (LTP) of synaptic strength. However, the source of membrane for spine formation and enlargement is unknown. Here we report that membrane trafficking from recycling endosomes is required for the growth and maintenance of spines. Using live-cell imaging and serial section electron microscopy, we demonstrate that LTP-inducing stimuli promote the mobilization of recycling endosomes and vesicles into spines. Preventing recycling endosomal transport abolishes LTP-induced spine formation. Using a pH-sensitive recycling cargo, we show that exocytosis from recycling endosomes occurs locally in spines, is triggered by activation of synaptic NMDA receptors, and occurs concurrently with spine enlargement. Thus, recycling endosomes provide membrane for activity-dependent spine growth and remodeling, defining a novel membrane trafficking mechanism for spine morphological plasticity and providing a mechanistic link between structural and functional plasticity during LTP.

Figure 1

Recycling Endosomes Localize within or at the Base of Dendritic Spines (A) Distribution of recycling endosomes in dendrites and spines. Hippocampal neurons (DIV17) expressing GFP were incubated with Alexa-Tf (100 μg/ml; Alx-Tf) for 1 hr at 37 °C to label recycling endosomes. Arrows indicate Alexa-Tf-labeled recycling endosomes in dendrites. Note the localization of recycling endosomes either within spines or at the base of spines in the dendritic shaft. Scale bar, 5 μm. (B) Representative images showing the localization of recycling endosomes containing Alexa-Tf, transferrin receptor (TfR), Rab11, or syntaxin13 (Syn13) in spines. Letters indicate the location code illustrated in (C). Scale bars, 2 μm. (C) Proportion of spines containing recycling endosomes in the indicated locations. n = 157, 150, 128, and 182 spines analyzed from 12, 11, 9, and 8 neurons for Alexa-Tf-, TfR-, Rab11-, and syntaxin13-positive compartments, respectively. Note that values do not add up to 100%, as individual spines could fall into more than one category. (D) Top, three-dimensional (3D) reconstruction from sSTEM illustrating endosomal compartments from acute hippocampal slices (PN15). Recycling compartments (RCs; red), multivesicular bodies (MVBs; blue), amorphous vesicular clumps (AVCs; green), and free endosomes (yellow, smooth vesicles; orange, coated vesicles) in spines are illustrated. Postsynaptic densities are labeled in black. Bottom, thin sections through individual spines containing a RC (red arrow; [D1]), an AVC at the base (green arrow; [D2]), or adjacent serial thin sections (D3i and D3ii) through a dendritic spine containing a large RC (red arrow; [D3i]), a MVB (blue arrow; [D3i]), and coated pits both on the head and at the base of the spine (orange arrows; [D3ii]). Arrows are color-coded to match the 3D reconstruction. Scale cube (0.5 μm³) is shown in the upper panel. Scale bar in (D1) applies to (D1)-(D3). (E) Percentage of spines containing endosomal compartments within 0.1 μm of the base (a), in the neck (b) or in the head (c) of spines. n = 14 dendritic segments with an average length of 8.5 μm. (F) Immunogold localization of transferrin receptor (TfR) at dendritic spines in adult rat CA1 hipocampus. Arrows indicate TfR-positive endosomes at the base (Fa), in the neck (Fb), and in the head (Fc) of spines. Scale bars, 200 nm.

Figure 2

The Surface Area of Endosomal Compartments Is a Large Fraction of the Total Spine Surface Area (A and B) Thin section images illustrating how the membrane surface for spines (blue lines in [Aii] and [Bii]), recycling endosomes (red lines in [Aii] and [Bii]), or amorphous vesicular clumps (green lines in [Bii]) was traced to measure membrane surface area. Scale bar in [Bii] applies to all panels. (C) Median with superimposed values of surface areas for individual spines (Spines), recycling endosomes (RC), or amorphous vesicular clumps (AVC). (D) Histograms of spine surface area. Arrows indicate median values. For (C) and (D), n = 14 dendritic segments with an average length of 8.5 μm.

Figure 3

Maintenance of Dendritic Spines Requires Ongoing Transport from Recycling Endosomes (A) Schematic diagram showing pathways of early and late endocytic trafficking between the plasma membrane (PM), early endosomes (EE), recycling endosomes (RE) and late endosomes/lysosomes (LE/Lys). Regulatory proteins and the transport steps they control, are indicated. (B) Bidirectional effect of transport from recycling endosomes on spine formation and loss. Forty-eight hours after transfection with the indicated constructs, neurons were fixed and imaged. Arrows indicate dendritic protrusions. Scale bar, 5 μm. (C) Means ± SEM of the number of protrusions per 10 μm dendrite from (B). n = 36, 21, 28, 15, 18, 21, 18 for GFP, Rme1-WT, Rme1-G429R, Rab11a-WT, Rab11a-S25N, Syn13-WT, Syn13ΔTM, **p<0.05, *p<0.001 relative to GFP; t test. (D and E) Acutely applied cell-permeable TAT-Syn13ΔTM abruptly reduces the number of dendritic protrusions. Twenty-four hours after transfection with tdTomato at DIV17, neurons were imaged before and after applying Alx488-TAT-Syn13ΔTM (3.5 μm). Arrows mark the loss of dendritic protrusions over time. Scale bars, 3 μm in (D) and 2 μm in (E). (F and G) Change in total dendritic protrusion number following application of TAT fusion proteins. N values correspond to the total protrusion number at each time point. N₀ is the average number of dendritic protrusions before application of the indicated TAT fusion proteins. Data indicate means ± SEM, n = 4 and 3 for (F) and (G), respectively.

Figure 4

Blocking Recycling Endosome Transport Abolishes LTP-Induced New Spine Formation and Preexisting Spine Growth (A) Glycine stimulation induces new spine formation (arrows) and preexisting spine growth (arrowheads). Neurons expressing tdTomato as a cell fill (DIV 14-18) were stimulated with glycine (200 μm, 3 min), and then imaged for 30 min after glycine stimulation. Scale bar, 20 μm. (B) Formation and growth of dendritic protrusions following glycine stimulation (arrow) in neurons expressing tdTomato (top, Control) or GFP-Rab11a-S25N (bottom) to block recycling endosome transport. tdTomato is expressed as a cell fill. Times are indicated in min:sec. Scale bar, 3 μm. See Movie S1. (C) Quantitative analysis of spine formation following glycine stimulation. N, number of dendritic protrusions per 10 μm at the indicated time. N₀,average number of dendritic protrusions per 10 μm before application of glycine or AP5. Glycine (200 μm) and D-AP5 (100 μm) were applied at the times indicated by bars. n = 5 each. Data represent means ± SEM. (D) Glycine-stimulated spine formation is blocked by expression of Rme1-G429R or Rab11a-S25N. Twenty-four to 48 hr after transfection, neurons were imaged before and after glycine treatment. n = 5 each. Analysis as in (C). (E) Acute application of Alexa 488-TAT-Syn13ΔTM prevents LTP-induced formation of new dendritic protrusions. Neurons expressing tdTomato were preincubated with either Alexa-488-TAT-Syn13ΔTM or Alexa-488-TAT-Syn7ΔTM (3.5 μm each) for 2 hr and imaged before and after glycine stimulation. n = 6 and 4 for TAT-Syn13ΔTM and TAT-Syn7ΔTM, respectively. Analysis as in (C). (F) Growth of preexisting spines following glycine stimulation in neurons expressing tdTomato either alone (left panels, Control) or with GFP-Rab11a-S25N (right panels, Rab11a-S25N). Times are indicated in min:sec. Scale bar, 1 μm. See Movie S2. (G) Quantitative analysis of spine growth following glycine stimulation. Spine area (A) indicates the spine area of the individual spine at the indicated time. A₀ indicates the average of spine areas before application of glycine or AP5. Glycine (200 μm) and D-AP5 (100 μm) were applied at the times indicated by bars. n = 15 spines on 3-4 neurons each. Data represent means ± SEM. (H) Glycine-stimulated preexisting spine growth is blocked by expression of Rme1-G429R or Rab11a-S25N. Experiments as in (D). n = 15, 15, 14 spines on 3-4 neurons each for Control, Rme1-G429R, Rab11a-S25N. Analysis as in (G). (I) Acute application of Alexa 488-TAT-Syn13ΔTM prevents LTP-induced growth of preexisting spines. Experiments as in (E). n = 16 and 10 spines on 2 neurons each for TAT-Syn13ΔTM and TAT-Syn7ΔTM, respectively. Analysis as in (G).

Figure 5

Recycling Endosomes Move into Spines in Response to LTP-Inducing Stimuli (A) TfR-GFP labels recycling endosomes in hippocampal neurons. Alexa-transferrin (Alx-Tf) was applied to live neurons expressing TfR-GFP for 1 hr at 37 °C to functionally label recycling endosomes. Neurons were fixed and imaged. Arrows indicate the colocalization of TfR-GFP and Alexa-Tf. Scale bars, 20 μm in the large panels and 3 μm in the insets. (B) Neurons were cotransfected with TfR-GFP to label recycling endosomes and mRFP as a cell fill. Before glycine stimulation, recycling endosomes (TfR-GFP) are present in the dendritic shaft at the base of spines (left panels). Following glycine stimulation, recycling endosomes coalesce in the spine neck and physically extrude into spines (right panels). Scale bar, 2 μm. (C) Proportion of spines containing TfR-GFP positive endosomes at the indicated locations. n = 50 spines from 3 neurons. (D) Time lapse imaging of recycling endosomes mobilized into spines following glycine stimulation. Neurons expressing TfR-GFP and mRFP were imaged every 15-30 s before and after glycine stimulation (200 μm, 3 min). Two examples are shown (Da and Db). Times are indicated in min:sec. Arrows indicate the movement of large endosomes into the spine from the shaft. Arrowheads indicate vesicles or tubules emanating from larger TfR-containing endosomes. Scale bars, 2 μm in (Da), 1 μm in (Db). See Movies S3A and S3B. (E) Line-scan analysis of TfR-GFP fluorescence through individual dendritic spines and the corresponding apposed segment of the dendritic shaft. Two examples are shown (Ea and Eb). TfR-GFP fluorescence intensity along the white dotted lines in inset images is plotted at various times before (dotted lines) and after (solid lines) glycine stimulation. D and S indicate dendrite and spine, respectively. Times are indicated in min:sec. (F) Integrated intensity of TfR-GFP fluorescence was measured both in individual spines (black circles) and at the base of spine in the shaft (open circles) over time. Two examples are shown (Fa and Fb). The black bar indicates the duration of glycine application. The increase of TfR-GFP in spines occurs simultaneous with the decrease in the adjacent dendritic shaft.

Figure 6

Plasticity of Endosomes in Spines during LTP in Hippocampal Slices (A and B) Dendritic segments at 5 min (A) and 30 min (B) after LTP induction by theta-burst stimulation (TBS). Endosomal compartments including recycling compartments (RCs, red), amorphous vesicular clumps (AVCs, green), multivesicular bodies (MVBs, blue), and free endosomes (yellow or orange) are illustrated. Synapses are marked with black. Scale cube, 0.5 μm³. (C and D) Electron micrographs from dendritic segments at 5 min (C) and 30 min (D) after LTP induction. A smooth vesicle (yellow arrow) apparently inserting at a spine head (C1), a large vesicle in the left spine head ([C2i], left green arrow), and an AVC in the right spine head from the same dendrite ([C2i], right green arrow) are shown. Four serial sections later (C2iv), the AVC is still present in the spine head (green arrow). Adjacent serial sections (Di and Dii) show a RC with multiple tubules and vesicles (red arrows) in a large spine head. Scale bar in (C2i) is for all images in (C) and (D). (E) Spines contain more endosomes within minutes after TBS. The graph shows relative change in the percentage of spines with or without endosomes at 5 and 30 min after TBS. (F) Quantitative analysis of endosome area in spines following LTP. The graph shows changes in the surface area (SA) of RCs and AVCs at 5 and 30 min after TBS. Values in (E) and (F) are relative to average values of all control spines. n = 14 dendritic segments each whose average length was 8.5 μm for control and 8.0 μm for LTP; a total of 199, 89, and 91 dendritic spines were analyzed in the control, in the 5 min LTP condition, and in the 30 min LTP condition, respectively. See Experimental Procedures for details.

Figure 7

Exocytosis from Recycling Endosomes on Dendrites and Spines following LTP (A) Visualization of externalized recycling cargo using transferrin receptor-superecliptic pHluorin (TfR-pHluorin). Fluorescence signal from lumenally tagged TfR-pHluorin in a stretch of dendrite was monitored during brief exposures to alkaline solution (NH₄ Cl, black bar in lower panel) and to acidic solution (pH 5.5, hatched bar in lower panel). The graph shows a quantitative analysis of fluorescence intensity. Times in the images are indicated in min:sec. Scale bar, 3 μm. (B) Exocytosis of TfR-pHluorin following glycine-induced LTP. TfR-pHluorin fluorescence was imaged before (top) or 20 min after (bottom) glycine stimulation. Arrows and arrowheads indicate growing and newly formed dendritic protrusions, respectively. A pseudocolor intensity scale bar is shown. Scale bar, 10 μm. See Movie S4. (C) Quantitative analysis of surface appearing TfR-pHluorin over hippocampal neuron dendrites. Data represent means ± SEM of the change in fluorescence intensity (ΔF) normalized to the average of initial fluorescence intensity prior to glycine stimulation (F₀). The black bar indicates the period of glycine application. n = 12 dendritic segments from 6 neurons (1090 μm in dendritic length analyzed) and 10 dendritic segments from 3 neurons (894 μm in dendritic length analyzed) for Gly and Basal, respectively. (D) Local exocytosis from recycling endosomes in spines. TfR-pHluorin was imaged before and after glycine stimulation as indicated in the two examples (Da and Db). Note the appearance of externalized TfR-pHluorin within the spine head (arrowheads). Times in the images are indicated in min:sec. Pseudocolor intensity scale bars are shown. Scale bars, 3 μm each. See Movie S5. For more examples, see Movie S6. (E) Data represent means ± SEM of ΔF/F₀ from spines (dark circles; n = 15) and segments of the dendritic shaft (open circles; n = 12). The black bar indicates the period of glycine application. Note the ordinate scale difference compared to (C).

Figure 8

LTP-Induced Spine Growth Is Simultaneous with Exocytosis from Recycling Endosomes (A and B) Quantitative changes in spine area (red line) and TfR-pHluorin fluorescence (black line) over time following glycine stimulation (black bar) during growth of existing spines (A) or new spine formation (B). The empty bar indicates application of pH 5.5 solution.

Figure 9

Schematic Model for Recycling Endosome Transport in Spine Morphological Plasticity Intracellular organelles filled in black are recycling endosomes. PSD, postsynaptic density; ExoZ, exocytic zone; EZ, endocytic zone. See text for details.