Figure 7.
Ultrastructural localization of Chmp2b in dendritic spines. A, Electron micrograph of Chmp2b immunoreactivity in the mouse stratum radiatum, detected by pre-embedding immunogold labeling. D, Dendrites; S, spines; at, axon terminals. B, High-magnification views of typical dendritic spines. Chmp2b usually appears on the periphery of the PSD or on the neck of the spine, beneath the plasma membrane. Bottom right, Labeled MVB in a dendritic shaft. C, Quantitative analysis of immunogold labeling in the stratum radiatum. The graph shows mean density of gold particles in three neuronal compartments. The ultrastructural localization of 2100 gold particles was analyzed in 30 fields similar to that shown in A. Chmp2b density was significantly higher in spines than in shafts or axonal boutons (p = 0.0498, n = 3 animals, 3 compartments and 10 fields per animal, Friedman test). D, Distribution of gold particle positions inside dendritic spines (n = 335 particles, 3 animals). Top, Histogram of normalized positions along the spine radius. Inset, Coordinate 0 corresponds to the spine center, and 1.0 corresponds to the plasma membrane. Particles concentrate in the subplasmalemmal cortex. Bottom, Histogram of normalized particle distances from the PSD. Inset, Coordinate 0 corresponds to the PSD edge, and 1.0 corresponds to the point equidistant from both edges of the PSD. Particles concentrate in the proximity of the PSD and become scarcer further away, with a slight rebound in frequency at the usual position of the neck. E, 3D reconstruction of a typical spine using immuno-electron micrographs of serial sections. The plasma membrane is in gray, PSD in green, and particles are marked by dots. Particles appear beneath the perisynaptic membrane around the PSD and also in the neck. Left, View from above the PSD; right, lateral view. An animated version of the 3D model appears in Movie 1 (see Notes). F, Quantitation of immunogold labeling in the stratum radiatum of WT and KD siblings. The density of gold particles in the three neuronal compartments is much lower in the KD animals (p < 10−5, n = 3 animals per genotype, Kruskal–Wallis test, density vs genotype). G, Comparison of Chmp2b amounts in WT and KD brains. Total brain extract was obtained from three animals of each genotype and analyzed by SDS-PAGE and Western immunoblotting with anti-Chmp2b antibody. Actin was used as a loading control. Quantitation of the Chmp2b band in each lane was obtained by densitometry and normalized to the respective actin control. KD brain still expressed 30% of the wild-type amount. H, Western blot used for the quantitation in G.