Figure 1.
Loss and Reincorporation Rates of GFP:Synapsin I at Individual Synaptic Boutons
(A) Axons of neurons expressing GFP:Synapsin I. Presynaptic boutons appear as bright puncta along faintly fluorescent axons. The contrast was enhanced nonlinearly in this figure to emphasize axonal fluorescence and demonstrate that the boutons photobleached here did not reside along the same axonal segment. Bar, 10 μm.
(B) Two boutons (arrows) were selectively photobleached by high-intensity laser light, reducing their fluorescence to approximately 30% of their nominal values. Fluorescence recovery at these sites was then followed by time-lapse imaging, initially at 20-s intervals and later at 1-min intervals. GFP:Synapsin I fluorescence levels shown in false color according to color scale near bottom.
(C) At the end of the experiment, presynaptic boutons were labeled (loaded) with FM4–64 by field stimulation (30sec@10hz) followed by unloading (120sec@10hz) to verify the functionality of the photobleached boutons (bottom panels). Note that both photobleached boutons exhibited a capacity for evoked endocytosis and exocytosis of FM4–64. Only boutons that exhibited such a capacity were included in our analysis. Same region as that enclosed in rectangle in (A). Times given in minutes.
(D) Fluorescence recovery time course for photobleached boutons in (A) as well as the mean fluorescence of five nonphotobleached boutons in the same field. Note the gradual reduction of fluorescence in these boutons and its dependence on the sampling rate, indicating that illumination applied during ongoing imaging induces some photobleaching that should be corrected for.
(E) Fluorescence recovery time course after correcting for ongoing photobleaching.
(F) Loss and reincorporation of GFP:Synapsin I molecules at synapses are accelerated by synaptic activity. Neurons were stimulated for 20 s at 20 Hz immediately after collecting the first postbleach images. Despite the brief duration of this stimulation episode, fluorescence recovery was accelerated significantly in comparison to recovery in matched, nonstimulated preparations. Data shown are mean ± standard deviation for all photobleached boutons after normalization as described in Materials and Methods. One-sided error bars only are shown in sake of clarity. The data were fit to a model assuming two pools with different recovery kinetics as described in the text. All experiments were performed in the presence of CNQX (10 μM) and AP-5 (50 μM) to minimize spontaneous activity.
Figure 2.
Synapsin I Lost from One Bouton Is Incorporated into Adjacent Boutons
An axonal segment expressing both CFP (A) and PA-GFP:Synapsin I (B).
(C) Higher magnification of region enclosed in rectangle in (B). PA-GFP:Synapsin I within a single bouton was photoactivated by selective illumination of this bouton at 405 nm (t = 0). Within 10 min of photoactivation, fluorescence at this bouton had decreased, whereas fluorescence at adjacent boutons (distances of 8 to 21 μm) had increased significantly. PA-GFP:Synapsin I fluorescence encoded in pseudo-color as in Figure 1.
(D) Quantification of fluorescence changes following photoactivation at photoactivated and adjacent boutons. Note the rapid reduction of fluorescence at the photoactivated site and the concomitant (but transient) increases recorded at adjacent boutons that were most prominent at boutons nearest to the photoactivation site.
(E) Synapsin lost from one bouton is incorporated into the presynaptic matrix of adjacent boutons. Two presynaptic boutons along an axonal segment expressing PA-GFP:Synapsin I. At time t = 0, PA-GFP:Synapsin I within one bouton (arrowhead) was photoactivated by selective illumination at 405 nm, and the redistribution of the photoactivated material was followed by time-lapse microscopy. At 21 min after photoactivation, a brief (20s@20Hz) stimulation train was delivered, leading to the transient dispersion of PA-GFP:Synapsin I at the photoactivated bouton as well as at the second bouton that had incorporated some of photoactivated PA-GFP:Synapsin I (arrow). PA-GFP:Synapsin I fluorescence encoded in pseudocolor as in Figure 1.
(F) FM4–64 labeling at the end of the experiment confirmed that both sites were functional presynaptic boutons. Bar, 5 μm.
(G) Normalized fluorescence of both boutons before, during, and after the stimulation episode. Note that PA-GFP:Synapsin I in the neighboring bouton exhibited activity induced dispersion just like Synapsin in the photoactivated bouton, indicating that the PA-GFP:Synapsin that had migrated to it had become incorporated into its presynaptic matrix.
Figure 3.
Loss and Reincorporation Rates of GFP:ProSAP2 at Individual Postsynaptic Sites
(A) A fluorescence image of a neuron expressing GFP:ProSAP2 overlaid onto a DIC image of the same region. Postsynaptic sites appear as green puncta. Bar, 10 μm.
(B) Two postsynaptic sites (arrows) were selectively photobleached by high-intensity laser light, reducing their fluorescence to approximately 30% to 40% of their nominal values. Fluorescence recovery at these sites was then followed by time-lapse imaging. Same regions as those enclosed in rectangles in (A). GFP:ProSAP2 fluorescence levels shown in false color as in Figure 1.
(C and D) At the end of the experiment, presynaptic boutons in the field were labeled with FM4–64 (as described for Figure 1) to verify the synaptic identity of the photobleached GFP:ProSAP2 puncta. Note that the photobleached puncta (green) were juxtaposed to presynaptic boutons (red) that exhibited a capacity for evoked endocytosis (C) and exocytosis (D) of FM4–64. Only puncta with functional presynaptic counterparts were included in our analysis.
(E) Fluorescence recovery time course for the photobleached GFP:ProSAP2 clusters shown in (B) as well as the mean fluorescence of five nonphotobleached clusters in the same field.
(F) Fluorescence recovery time course after correcting for ongoing photobleaching.
Figure 4.
GFP:ProSAP2 Loss and Reincorporation Rates Are Accelerated by Synaptic Activity
(A) A neuron (23 d in vitro) expressing GFP:ProSAP2 that was maintained for 7 d in glutamate receptor blockers (10 μM CNQX and 50 μM AP-5). Four PSDs (blue arrows at bottom of image) were photobleached and the recovery of fluorescence at these sites was monitored (B). Then, the preparation was switched to blocker-free solution and four other PSDs (red arrows at top of image) were photobleached. After collection of the first set of postphotobleach images, the preparations were stimulated at 20 Hz for 20 s every 3 min while monitoring the recovery of the photobleached PSDs (C). A comparison of mean recovery kinetics for both sets of photobleached PSDs (D) reveals that recovery kinetics were greatly accelerated by the stimulation protocol, indicating that the loss and reincorporation kinetics of ProSAP2 at individual PSDs are accelerated by synaptic activation. Note that PSD 4 from (C) was not included here as its final fluorescence exceeded its original level, indicating that the size of this PSD may have changed during this experiment. All data are shown after correcting for ongoing photobleaching as in Figure 3. Bar in (A), 20 μm.
Figure 5.
Synaptic Activity Accelerates GFP:ProSAP2 Loss and Reincorporation Rates
(A) Mean recovery time course for all photobleached puncta in three separate experiments identical to that shown in Figure 4. Data shown are mean ± standard deviation for all photobleached puncta.
(B) The data were fit according to a model that assumed two GFP:ProSAP2 pools with different recovery kinetics as described in the text.
(C) Extrapolation of recovery time courses using the recovery rates and relative pool sizes that provided the best fit to the data.
Figure 6.
ProSAP2 Lost from Dendritic Spine Heads Is Incorporated into PSDs of Adjacent Spines
A dendritic segment expressing both CFP (A) and PA-GFP:ProSAP2 (B). At time t = 0, PA-GFP:ProSAP2 within the region enclosed in rectangles was photoactivated by selective illumination at 405 nm. With time from photoactivation, fluorescence at tips of remote spines increased, whereas spine head fluorescence within the photoactivated region diminished. The contrast in (B) was enhanced linearly to emphasize fluorescence changes in remote spines, resulting in the apparent saturation at photoactivated spines. Spatial relationships between spines and PA-GFP:ProSAP2 puncta before and 29 min after photoactivation are shown in (C) and (D), respectively. In these images, PA-GFP:ProSAP2 fluorescence data were overlaid onto the CFP images after rendering the latter with the “emboss” filter of Adobe Photoshop. Note that PA-GFP:ProSAP2 fluorescence is restricted to spine heads, with little fluorescence observed in spine necks or the dendrite shaft. This distribution indicates that the PA-GFP:ProSAP2 that migrated to adjacent spines had become integrated into the PSD at these sites. Bar, 10 μm. Quantification of fluorescence changes at photoactivated (E) and neighboring spine heads (F) reveals a gradual decrease of fluorescence in the photoactivation region concomitant with fluorescence increases at nearby spines, most prominent at spines nearest to the photoactivation site. Values are normalized to prephotoactivation fluorescence levels.
Figure 7.
Incorporation Rates of Synapsin I from Somatic Sources at Remote Presynaptic Boutons
(A) Low-magnification composite image of a neuron expressing CFP and PA-GFP:Synapsin I. Only CFP fluorescence is shown here. Parts of the axonal arbor are marked with arrowheads. Fluorescence is encoded using an inverted gray scale to increase detail clarity.
(B) Higher magnification of rectangle marked as “B” in (A). At time t = 0, PA-GFP:Synapsin I within the cell body was photoactivated by selective illumination of the soma at 405 nm. Within minutes, photoactivated PA-GFP:Synapsin was observed to spread out into the axon and, much later, to appear at presynaptic boutons in the same field of view.
(C) Higher magnification of rectangle marked as “C” in (A), enclosing a group of relatively remote presynaptic boutons. Note the gradual increase in fluorescence of these remote boutons over time.
(D) Quantification of fluorescence changes at remote boutons following photoactivation of somatic PA-GFP:Synapsin. Boutons were grouped according to distance from the soma as shown in (A). Fluorescence data for each bouton were normalized to prephotoactivation fluorescence levels at the same bouton. Bars: (A), 50 μm; (B and C), 20 μm.
Figure 8.
Incorporation Rates of ProSAP2 from Somatic Sources into Remote PSDs
(A) Composite image of a neuron expressing CFP and PA-GFP:ProSAP2. Only CFP fluorescence is shown here.
(B) PA-GFP:ProSAP2 fluorescence for same region as in (A). At time t = 0, PA-GFP:ProSAP2 within the cell body was photoactivated by selective illumination of the soma at 405 nm. With time, photoactivated PA-GFP:ProSAP2 migrated to PSDs along the dendrites, initially to proximal PSDs and later to more distal ones. During the time-lapse session, recurrent, low-level photoactivation of the soma was performed, to maintain a constant level of somatic, photoactivated PA-GFP:ProSAP2.
(C) Quantification of fluorescence changes at the soma.
(D) Quantification of fluorescence changes at PSDs, grouped according to distance from the soma. Fluorescence data for each PSD were normalized to prephotoactivation fluorescence levels for the same PSD. Bar, 20 μm.
Figure 9.
Effects of Cycloheximide and MG132 on Steady State Levels and Exchange Rates of GFP:Synapsin I and GFP:ProSAP2
(A) Long-term imaging of axons of neurons expressing GFP:Synapsin I, before and after the addition of cycloheximide (CHX). CHX was added to attain a final concentration of 100 μM 1 h 12 min after the beginning of the experiment.
(B) Mean fluorescence levels of all GFP:Synapsin puncta recorded in this experiment normalized to their mean fluorescence intensities at the beginning of the experiment (five cells, 13 fields of view, 197 synaptic boutons).
(C) A similar experiment in which MG132 was added 2 h 29 min after the beginning of the experiment to attain a final concentration of 20 μM. Fluorescence levels were normalized to fluorescence intensities at the beginning of the experiment (four cells, ten fields of view, 225 synaptic boutons).
(D) Paired FRAP experiments were performed in the same preparations of neurons expressing GFP:Synapsin I, first in the absence and then in the presence of cycloheximide (three experiments, 12 boutons before cycloheximide addition, 11 boutons after cycloheximide addition).
(E) Paired FRAP experiments as in (D) but with MG132 instead of cycloheximide (four experiments, 13 boutons before MG132 addition, 16 boutons after MG132 addition). All experiments in (A–E) were performed in CNQX and AP-5 to avoid the confounding effects of spontaneous activity.
(F) Long-term imaging of dendrites of neurons expressing GFP:ProSAP2, before and after the addition of cycloheximide (at 2 h 36 min after the beginning of the experiment).
(G) Mean fluorescence levels of all GFP:ProSAP2 puncta recorded in this experiment normalized to their mean fluorescence intensities at the beginning of the experiment (five cells, five fields of view, 678 PSDs).
(H) A similar experiment in which MG132 was added 3 h 4 min after the beginning of the experiment (seven cells, eight fields of view, 574 PSDs).
(I) Paired FRAP experiments were performed in neurons expressing GFP:ProSAP2, first in the absence and then in the presence of cycloheximide (three experiments, ten PSDs before cycloheximide addition, eight PSDs after cycloheximide addition).
(J) Paired FRAP experiments as in (I) but with MG132 instead of cycloheximide (three experiments, nine PSDs before MG132 addition, nine PSDs after MG132 addition). Data in FRAP experiments were not corrected for photobleaching during the recovery phase to avoid canceling out general changes in puncta fluorescence related to cycloheximide or MG132 addition. For sake of clarity, only one-sided error bars are shown in (D, E, I, J). Bars (A, F), 10 μm.