Figure 7. PREM of synapses in control and βIII spectrin knockdown neurons at 17 DIV. A–E, Neurons treated with control shRNA. A, Dendritic spine (yellow) forms a synapse with a presynaptic bouton in the axon (magenta) in nonextracted cells. B, Correlative fluorescence microscopy and PREM of synapses formed on dendritic spines in neurons fluorescently stained with phalloidin (green) and synapsin I (magenta). Fluorescence image is superimposed onto the PREM image in the main panel and shown separately in the inset. C, Synaptophysin immunogold PREM of a dendritic spine (green) making a synapse with a presynaptic bouton (red) labeled with gold particles (pseudocolored in blue). Inset, Overview of the spine. D, Varicosity on a putative axon (magenta) of a control neuron with a collection of vesicles seen through spontaneous perforations of the plasma membrane; it likely corresponds to an orphan bouton. E, Unapposed presynaptic bouton in a control neuron revealed by synaptophysin immunogold PREM. Membranes are perforated due to detergent extraction after glutaraldehyde fixation. F–K, Neurons treated with βIII spectrin shRNA #2. F, A bundle of neurites making extensive lateral contacts in nonextracted cells (individual neurites are shaded in different colors). G–I, Synaptophysin immunogold PREM (gold particles pseudocolored in blue) of a bundle of neurites (shaded in different colors). Boxed regions are zoomed in H (white box) and I (red box). H, Gold particles label an ellipsoid bouton in the putative axon (red-shaded neurite), which makes a shaft synapse with an adjacent putative dendrite. I, Gold-labeled globular varicosity on a putative axon. It may form a shaft synapse with the underlying “dendrite” or correspond to an orphan bouton. J, A putative orphan bouton (magenta) in nonextracted neuron; a collection of vesicles is seen through spontaneous perforations of the plasma membrane. K, An orphan bouton revealed by synaptophysin immunogold PREM (gold particles pseudocolored in blue). Scale bars: A, B, D, F, J, 0.5 μm; G, 1 μm; C, E, H, I, K, 0.2 μm.