The relationship between PSD-95 clustering and spine stability in vivo

Abstract

The appearance and disappearance of dendritic spines, accompanied by synapse formation and elimination may underlie the experience-dependent reorganization of cortical circuits. The exact temporal relationship between spine and synapse formation in vivo remains unclear, as does the extent to which synapse formation enhances the stability of newly formed spines and whether transient spines produce synapses. We used in utero electroporation of DsRedExpress- and eGFP-tagged postsynaptic density protein 95 (PSD-95) to investigate the relationship between spine and PSD stability in mouse neocortical L2/3 pyramidal cells in vivo. Similar to previous studies, spines and synapses appeared and disappeared, even in naive animals. Cytosolic spine volumes and PSD-95-eGFP levels in spines covaried over time, suggesting that the strength of many individual synapses continuously changes in the adult neocortex. The minority of newly formed spines acquired PSD-95-eGFP puncta. Spines that failed to acquire a PSD rarely survived for more than a day. Although PSD-95-eGFP accumulation was associated with increased spine lifetimes, most new spines with a PSD did not convert into persistent spines. This indicates that transient spines may serve to produce short-lived synaptic contacts. Persistent spines that were destined to disappear showed, on average, reduced PSD-95-eGFP levels well before the actual pruning event. Altogether, our data indicate that the PSD size relates to spine stability in vivo.

Figure 1.

A, Mixture of CAG-PSD-95-eGFP-WPRE and CAG-DsRedExpress-WPRE plasmids was injected in the lateral cerebral ventricle of E16 embryos in utero. Neurons were electroporated using electrode tweezers. B, Coronal section showing a large group of labeled L2/3 pyramidal cells in the barrel cortex located under the craniotomy. C, In vivo 2PLSM image of the transfected area (scale bar, 50 μm), showing a partial colocalization of the two fluorescent proteins. D, Timeline of the imaging sessions. Boxes indicate days, ticks indicate multiple imaging sessions per day.

Figure 2.

A, Dendritic branch imaged at high magnification (scale bar, 5 μm). Three types of structure are visible: #1, a long thin spine without a PSD-95-eGFP punctum (SpO); #2, a long thin spine with a PSD-95-GFP punctum (SpP); #3, a mushroom-like spine with a PSD-95-eGFP punctum (SpP); and #4, a PSD-95-eGFP punctum in the dendritic shaft (PO). For the sake of visualization, both channels are presented with different lookup tables. B, Procedure to determine the presence of PSD-95-eGFP puncta. PSD-95-eGFP fluorescence (right) in the spine head (gray arrowhead) was determined by integrating pixel values over an area smaller than the spine head (Sp_G). This value was divided by the average of the integrated pixel values of several (3–4) equally sized areas (D_G) in the dendritic shaft (black arrowhead), carefully avoiding apparent PSD-95-eGFP puncta (open arrowhead) in the shaft. C, Left, Distribution of the fraction of PSD-95-eGFP bound in spine heads (n = 217) as measured according to Otmakhov et al. (2004). Right, Result of a k-means cluster analysis (k = 2) of the spine population from the left graph. Rapid puncta-based scoring results shown in color. Note that our scoring results nearly match the clustering. Arrowheads, two SpOs from Figure 3; arrow, smallest SpP from Figure 3.

Figure 3.

A, Dendritic branch of a neuron that was electroporated using a patch pipette in the adult cortex. Left, dsRedExpress. Right, PSD-95-eGFP (scale bar, 5 μm) B, FIBSEM reconstruction of the boxed region of the dendrite in A. All spines and PSDs match; #1, big mushroom SpP; #2, small mushroom SpP; #3, SpP with the smallest PSD in the reconstruction; #4, PSD on the shaft (PO); #5 and #6, thin spines without PSDs (SpOs). C, Relationship between the size of the PSDs in the EM reconstruction and the normalized fluorescence intensities of PSD-95-eGFP in vivo. D, FIBSEM micrographs of two spines (#1 and #2). E, High magnifications of structures #3, #4, and #5 in the red and green channel (images are filtered several times for optimal visualization; scale bar, 1 μm).

Figure 4.

A, D, Fraction of spines with a PSD-95-eGFP punctum (SpP, dark gray), spines without a PSD-95-eGFP punctum (SpO, light gray), and PSD-95-eGFP puncta in the dendritic shaft (PO, white). C, D, Fraction of new spines with a PSD-95-eGFP punctum (SpP, dark gray) and new spines without a PSD-95-eGFP punctum (SpO, light gray) for spines appearing over 6, 18, and 96 h intervals (left) and spines disappearing over 6, 18, and 96 h intervals (right). A, C, Averages over mice (*p < 0.05; ***p < 0.005; paired t test). B, D, Structures from all mice pooled (***p < 0.005, χ²).

Figure 5.

A, Time-lapse image of a dendritic region containing the following: a SpP disappearing after day 13 (#1); a PO disappearing after the second imaging session on day 0 (#2); a transient SpO appearing and disappearing within 24 h after the first imaging session (#3); and a new spine appearing as a SpO on day 6 and turning into a SpP on day 7 (#4). Scale bar, 1 μm. B, Fraction of new spines (<24 h old) that survives over the next 24 h. The fraction of new SpPs that is still present after 24 h is larger than the fraction of SpOs (***p < 0.005). C, Fraction of new spines (<6 h old) that survives over the next 18 h. The fraction of new SpPs that is still present after 18 h is larger than the fraction of SpOs (***p < 0.005). D, New spines that show a PSD-95-eGFP punctum at least once during their lifetime have a higher probability of surviving for >96 h (***p < 0.005). E, Fractions of lost SpPs that maintain or lose their PSD-95-eGFP punctum before disappearance. F, Left, Fraction of new SpOs with lifetimes of more or <96 h. Right, Fraction of SpPs that continue as SpOs for more or <96 h before disappearing (***p < 0.005). G, New SpO that is present over two time points (#6). Scale bar, 1 μm. H, Disappearing spine that loses its PSD-95-eGFP punctum before pruning (#5). Scale bar, 1 μm.

Figure 6.

A, Examples of SpPs with small (left) and large (right) volumes. B, Procedure for measurement of DsRedExpress and PSD-95-eGFP fluorescence intensities. In brightest optical section of each channel, background-corrected pixel intensities are integrated over an area spanning the spine head (circles). Background pixel values are taken from nearby regions (rectangles). In each region of interest, spine values are normalized to the average maximum pixel values of neighboring areas in the dendritic shaft (jagged lines). C, Linear correlation between green and red fluorescence intensities in a random set of SpPs at t = 0 h. D, New SpO (#1) and a new SpPs (#2 and #3). E, Linear fits of red and green fluorescence for new SpPs (blue), new SpOs (red), and spines from C (black). Inset shows the smallest data points. F, Cumulative distribution of red (continuous lines) and green (dotted lines) fluorescence intensities for all spines from E and C. Scale bars, 1 μm.

Figure 7.

A, Relationship between changes in red and green fluorescence intensities. Dotted lines represent the measurement error. B, New spines display an increase in both green and red fluorescence over the first two time points after their appearance compared with persistent spines (***p < 0.005). Lost spines display a decrease in both green and red fluorescence over the last two time points before their disappearance compared with persistent spines (***p < 0.005). C, Absolute value of fluorescence intensity ratios over a random set of two consecutive time points for persistent, new, and preexisting disappearing spines. New and lost spines display increased fluctuations (***p < 0.005). D, Example of a time interval used in B. E, Example of time intervals used in C.