Figure 1. Enrichment and stability of synaptoneurosomes.

Microscopy: A) Nuclear contamination of the homogenate (2 µl smears) (DAPI fluorescence) decreases after sequential passage through mesh screens with B) the post- 10 µm screen synaptoneurosome pellet (100 µl volume) still retaining a few nuclei (Bar = 20 µm). C) Intact synaptoneurosomes are detected by phase contrast in smears of pelleted post-10 µm screen synaptoneurosome as typical “snowman” pre- and postsynaptic profiles (arrowheads). Larger, empty, membranous structures are also observed. (Bar = 5 µm). D) Electron microscopy reveal “snowman” profiles with apparent postsynaptic densities (arrowheads); (Bar = 300 nm). E) At higher magnification, presynaptic terminals containing synaptic vesicles, 20 nm in size (white arrows) are indicated (Bar = 100 nm). Immunoblots: F) Homogenates (black bars) and synaptoneurosomes (white bars) were compared for stability and enrichment of synaptic proteins and contamination with other cellular debris. Densitometric comparison on immunoblots of postsynaptic proteins PSD-95, NMDAR1 and GluR2 in synaptoneurosomes shows more than a two-fold enrichment compared to homogenates, an increase in presynaptic protein SNAP-25 but a 40% decrease in glial protein GFAP. Cytoplasmic proteins β-tubulin and PKCα show little change. The ratio of each protein to GAPDH is given in arbitrary units based on the integrated density value which is the sum of all pixel values after background correction (IDV). G) In synaptoneurosomes, de novo synthesis of several proteins is observed as newly translated, biotinylated proteins detected with streptavidin (lane 1), and one at 50 kD co-migrates with a band detected with antibody to αCAMKII (lane 4,arrowhead). Actinomycin D, a transcription inhibitor, does not reduce the protein band profile (lane 2). Protein synthesis is inhibited by the translation inhibitor, anisomycin (lane 3), although endogenously biotinylated proteins present in synaptoneurosomes are still seen at ∼140 and 75 kD (asterisks).