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Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly - PubMed

  • ️Thu Jan 01 2004

Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly

Tal Bresler et al. J Neurosci. 2004.

Abstract

The cellular mechanisms involved in the formation of the glutamatergic postsynaptic density (PSD) are mainly unknown. Previous studies have indicated that PSD assembly may occur in situ by a gradual recruitment of postsynaptic molecules, whereas others have suggested that the PSD may be assembled from modular transport packets assembled elsewhere. Here we used cultured hippocampal neurons and live cell imaging to examine the process by which PSD molecules from different layers of the PSD are recruited to nascent postsynaptic sites. GFP-tagged NR1, the essential subunit of the NMDA receptor, and ProSAP1/Shank2 and ProSAP2/Shank3, scaffolding molecules thought to reside at deeper layers of the PSD, were recruited to new synaptic sites in gradual manner, with no obvious involvement of discernible discrete transport particles. The recruitment kinetics of these three PSD molecules were remarkably similar, which may indicate that PSD assembly rate is governed by a common upstream rate-limiting process. In contrast, the presynaptic active zone (AZ) molecule Bassoon was observed to be recruited to new presynaptic sites by means of a small number of mobile packets, in full agreement with previous studies. These findings indicate that the assembly processes of PSDs and AZs may be fundamentally different.

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Figures

Figure 1.
Figure 1.

Expression pattern of NR1:GFP in cultured hippocampal neurons. A, Hippocampal neuron in primary culture expressing NR1:GFP (9 d in vitro). B, Higher magnification of the region enclosed in a rectangle in A after fixation. C, Same region as in B after immunolabeling against NR1. The expression pattern of NR1:GFP is punctate and suggestive of synaptic localization. Scale bars: A, 10 μm; C, 5 μm.

Figure 2.
Figure 2.

NR1:GFP is targeted correctly to postsynaptic sites. A, Presynaptic boutons were labeled by stimulating neurons to fire action potentials via field stimulation (30 sec at 10 Hz) in the presence of FM4-64 (top left). NR1:GFP (green puncta) were observed to colocalize with FM4-64 puncta (red puncta, right panels) for which the presynaptic identity was confirmed further by unloading the dye with a second train of stimuli (120 sec at 10 Hz). B, The same NR1:GFP clusters were fixed and immunolabeled with an antibody against the NR2A and NR2B subunits of NMDA receptors. Most NR1:GFP clusters colocalized with clusters of NR2A/B. C, Fixed NR1:GFP clusters (green puncta) were immunolabeled against the presynaptic molecule synapsin I (red puncta). Most NR1:GFP clusters were juxtaposed against synapsin I puncta. Arrowheads point to identical locations in all panels of A-C. Scale bars, 5 μm.

Figure 3.
Figure 3.

Gradual formation of new NR1:GFP clusters. A, The formation of two new NR1:GFP clusters (1, 2, arrowheads) is shown in this time-lapse sequence of a dendritic segment of a neuron expressing NR1:GFP (13 d in vitro). Note the gradual increase in the fluorescence intensity of these new clusters (coded according to the pseudocolor look-up table on the right). A quantitative analysis of the fluorescence intensity for these new clusters is shown in Figure 4A. B, FM4-64 labeling performed at the end of the experiment revealed that the new NR1:GFP clusters (green puncta) were juxtaposed against functional presynaptic boutons (red puncta), suggesting that new synapses had formed at these sites. All times are given in minutes. Asterisks mark identical images in A and B. Scale bar, 5 μm.

Figure 4.
Figure 4.

Accumulation of NR1:GFP and GFP:ProSAP1 at new synaptic sites is qualitatively different from GFP:Bsn95-3938 accumulation. Shown is quantification of the fluorescence intensities of the two new NR1:GFP clusters in Figure 3 (A), the two new GFP:ProSAP1 clusters in Figure 6 (B), and the new GFP:Bsn95-3938 cluster in Figure 11 (C). Whereas NR1:GFP and GFP:ProSAP1 show gradual increases in fluorescence intensity over time, GFP:Bsn95-3938 shows stepwise fluorescence intensity changes. Arrows mark the time points at which punctate fluorescence was first observed at these sites. Cluster numbers in A and B match those of Figures 3 and 6, respectively. Horizontal lines in C are averages of fluorescence intensities in each step.

Figure 5.
Figure 5.

GFP:ProSAP1 is targeted correctly to synaptic sites. A, Hippocampal neuron in primary culture expressing GFP:ProSAP1 (9 d in vitro). B, Higher magnification of the region enclosed in the rectangle in A after fixation and immunolabeling against the postsynaptic molecule SAP90/PSD-95 and the presynaptic molecule synapsin I. Most ProSAP1 puncta colocalized with these two synaptic molecules (arrowheads). Scale bars: A, 10 μm; B, 5 μm.

Figure 6.
Figure 6.

Gradual formation of new GFP:ProSAP1 clusters. A, The formation of two new GFP:ProSAP1 clusters (1, 2, arrowheads) is shown in this time-lapse sequence of a dendritic segment of a neuron expressing GFP:ProSAP1 (8 d in vitro). Note the gradual increase in the fluorescence intensity of these new clusters (coded in pseudocolor as in Fig. 3). A quantitative analysis of the fluorescence intensity for these new clusters is shown in Figure 4 B. B, FM4-64 labeling performed at the end of the experiment revealed that the new GFP:ProSAP1 clusters were juxtaposed against functional presynaptic boutons, suggesting that new synapses had formed at these sites. All times are given in minutes. Asterisks mark identical images in A and B. Scale bar, 5 μm.

Figure 7.
Figure 7.

Photobleaching of diffuse GFP:ProSAP1 fluorescence does not reveal a cryptic pool of ProSAP1 transport particles. A, Dendritic segment of a neuron expressing GFP:ProSAP1. Note the diffuse fluorescence as well as the bright puncta of synaptic GFP:ProSAP1. Fluorescence intensity is coded in pseudocolor as in Figure 3. B, The region enclosed by a rectangle in A was scanned by high-intensity 488 nm laser light for ∼20 sec, and images were obtained thereafter at 4.5 sec intervals (only every second image is shown here). No discernible mobile puncta were observed to move into the bleached region from the unbleached segments, but rapid recovery of GFP:ProSAP1 was observed at existing synaptic sites. The recovery time course for the numbered clusters in A (arrowheads) is shown in Figure 9A. The bottom frame is the last image of this time-lapse sequence. The vertical dashed lines demarcate the borders of the bleached region. All times are given in minutes and seconds. Scale bar, 5 μm.

Figure 9.
Figure 9.

Time course of fluorescence recovery at photobleached GFP:ProSAP1 and NR1:GFP clusters. A, Recovery time course of GFP:ProSAP1 clusters shown in Figure 7. B, Recovery time course of NR1:GFP clusters shown in Figure 8. Fluorescence is given as a fraction of the nominal fluorescence of each cluster before the photobleaching procedure. Cluster numbers refer to the labels in Figures 7 and 8, respectively.

Figure 8.
Figure 8.

Photobleaching of diffuse NR1:GFP fluorescence does not reveal a cryptic pool of NR1 transport particles. A, Dendritic segment of a neuron expressing NR1:GFP. Note the diffuse fluorescence as well as the bright puncta of synaptic NR1:GFP. Fluorescence intensity is coded in pseudocolor as in Figure 3. B, The region enclosed by a rectangle in A was scanned by high-intensity 488 nm laser light for ∼20 sec, and images were obtained thereafter at 1 min intervals (for the first 8 min) and at 5 min intervals later on. No discernible mobile puncta were observed to move into the bleached region from the unbleached segments, but a gradual recovery of NR1:GFP was observed at existing synaptic sites. The recovery time course for the numbered clusters in A (arrowheads) is shown in Figure 9B. The bottom frame is the last image of this time-lapse sequence. The vertical dashed lines demarcate the borders of the bleached region. All times are given in minutes and seconds. Scale bar, 5 μm.

Figure 10.
Figure 10.

Expression pattern of GFP:Bsn95-3938. A, GFP:Bsn95-3938 displays a punctate expression pattern in axons. B, Higher magnification of the region enclosed in the rectangle in A. C, D, Same region after the labeling of functional presynaptic boutons with FM4-64. E, Many of the GFP:Bsn95-3938 puncta (green) colocalized with sites of FM4-64 uptake and release (red) as indicated by the arrowheads, indicating that these resided at functional AZs. In addition, a population of smaller GFP:Bsn95-3938 puncta did not colocalize with functional synaptic vesicle release sites (arrows). Many of these were highly mobile, moving at velocities of up to 0.4 μm/sec. Scale bar, 5 μm.

Figure 11.
Figure 11.

Formation of new functional AZs is preceded by the recruitment of mobile GFP:Bsn95-3938 particles. A, Time lapse of an axon of a neuron expressing GFP:Bsn95-3938. At the beginning of the time lapse a single large and static GFP:Bsn95-3938 cluster is seen (arrow) at a site displaying a capacity for activity-evoked FM4-64 uptake and release (B), suggesting that it resides at a functional AZ. Then 6 min into the time lapse a new cluster appears that subsequently breaks up into two clusters (t = 13 min). One of these remains at the original site (arrowheads). At t = 26 min, additional clusters arrive, some of which merge with the first cluster (t = 35 min). A second round of FM4-64 labeling (C) indicates that a new functional presynaptic site has formed at the same location. Quantitative measurements of the fluorescence changes at this site indicate that the formation of the new functional presynaptic site has been preceded by the recruitment of two to three unitary amounts of GFP:Bsn95-3938 (Fig. 4C). All times are given in minutes. Asterisks mark identical images in A and B or in A and C. Scale bar, 5 μm.

Figure 12.
Figure 12.

PSD molecules are recruited to new postsynaptic sites with similar kinetics. The average time course of PSD molecule recruitment to new synaptic sites has been determined by normalizing and pooling together data from all recorded events for NR1:GFP (A), GFP:ProSAP1 (C), and GFP:ProSAP2 (E), as described in Materials and Methods. Data are provided as averages and SDs (error bars) of normalized fluorescence intensities. The solid lines in these panels are single exponentials with time constants of 13, 12, and 14 min for NR1:GFP, GFP:ProSAP1, and GFP:ProSAP2, respectively. Fluorescence intensities of 30 preexisting clusters of NR1:GFP (B), GFP:ProSAP1 (D), and GFP:ProSAP2 (F) measured over similar periods do not show significant changes except for small gradual decreases, probably resulting from photobleaching. G, The average recruitment kinetics for NR1:GFP, GFP:ProSAP1, and GFP:ProSAP2 of A, C, and E plotted together with previously published data for SAP90/PSD-95 (Bresler et al., 2001). Note the very similar recruitment kinetics displayed by all four PSD molecules.

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