Abstract
The dynamics of postsynaptic density (PSD) formation and remodeling were investigated in live developing hippocampal tissue slices. Time lapse imaging of transfected neurons expressing GFP-tagged PSD95, a prominent PSD protein, revealed that up to 40% of PSDs in developing dendrites are structurally dynamic; they rapidly (<15 min) appear or disappear, but also grow, shrink and move within shafts and spines. New spines containing PSDs were formed by conversion of dynamic filopodia-like spine precursors in which PSDs appeared de novo, or by direct extension of spines or spine precursors carrying preformed PSDs from the shaft. PSDs are therefore highly dynamic structures that can undergo rapid structural alteration within dendrite shafts, spines and spine precursors, permitting rapid formation and remodeling of synaptic connections in developing CNS tissues.
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Acknowledgements
We thank D. Bredt (UCSF) for the PSD95-GFP construct, S. Lee for help with data analysis, J. Rathner for reading the manuscript, and L. Katz, D. Lo and P. Bridgman for discussions on use of the gene gun. This work was supported by grants from the National Institutes of Health (NS37159 to M.E.D.; DC02961 to S.H.G.), Whitehall Foundation (98-6 to M.E.D.), and University of Iowa Biosciences Initiative (M.E.D. and S.H.G.).
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Supplementary Fig. 1.
Quantitative analysis of a neuron co-transfected with PSD95-GFP and DsRed. These analyses demonstrate the minimal effect of z-movement on the size and intensity of PSD95-GFP hotspots during a typical time lapse imaging session, and also show that free DsRed is not significantly 'clumpy.' GFP-PSD95 and DsRed were expressed in single hippocampal pyramidal neurons. Images of GFP-PSD95 and DsRed were captured simultaneously in two different fluorescent channels using a single excitation light source (Argon laser). Twenty optical sections were collected at each time point. The z-step size was 0.9 mm. (a) Selected confocal images of GFP-PSD95 for 8 time points spanning nearly 1 h. Only 10 of the original 20 focal planes are shown. The columns represent focal planes 6 through 15 (f6-f15) through a portion of a dendrite containing PSD95-GFP hotspots. A hotspot within a spine (arrow in f13 at t = 0') is most clearly in focus in different image planes at different time points, indicating z-movement relative to the z-stack of images. In some instances, large focus jumps were intentionally introduced by manually turning the focus knob a random amount between time points. The arrows indicate the optimal (center) image of the spine hotspot at each time point. (b) Analysis of fluorescent intensities in corresponding GFP-PSD95 and DsRed projection images. The projection images represent maximum brightness projections of all captured focal planes (f1-f20). Plots of fluorescent intensity along a line (green and red lines overlaying the projection images) are shown for GFP-PSD95 and DsRed. To account for differences in the level of protein expression and imaging parameters, the PSD95-GFP and DsRed intensity plots were normalized with respect to each other by matching the peak fluorescent intensity values in the shaft region as well as the low intensity values of the non-fluorescent background (as in Fig. 1d). 'Difference' images were computed by mathematically subtracting the DsRed fluorescence signal from the GFP-PSD95 signal. The shaft GFP fluorescence is negated by the DsRed fluorescence (arrowhead), whereas the two PSD95 hotspots (two arrows) are significantly brighter and thus readily apparent in the difference plots at each time point. (JPG 104 kb)
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(c) Peak intensity values at each time point for the shaft and two PSD95 hotspots shown in panel (b). Note that intensities of the shaft and hotspot 1 show little variation over the time period observed, even though the sample drifted wildly within the z-stack of images. Hotspot 2 shows more variation in the fluorescent intensity of GFP-PSD95, but the 'difference' intensity plot that was normalized against DsRed (and thus negates any effect of focus change) closely mimics changes in the raw GFP-PSD95 intensity. These data indicate that z-movement cannot account for the significant changes in hotspot features we present elsewhere in the manuscript. (d) Evidence that the pattern of DsRed expression is not 'clumpy,' and thus DsRed serves as a good volume indicator. Line plots of GFP-PSD95 (green) and DsRed (red) fluorescence intensity in a region of the dendrite shaft devoid of GFP-PSD95 hotspots are shown for the projection images (left) and for a single focal plane through the center of the shaft (right). Note that standard deviations of the pixel intensity values are not significantly different for GFP and DsRed, indicating that the DsRed is no more 'clumpy' than GFP-PSD95 in regions devoid of GFP-PSD95 hotspots. (JPG 38 kb)
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Marrs, G., Green, S. & Dailey, M. Rapid formation and remodeling of postsynaptic densities in developing dendrites. Nat Neurosci 4, 1006–1013 (2001). https://doi.org/10.1038/nn717
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DOI: https://doi.org/10.1038/nn717
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