Fig. 5.

Relationship of PSD-95+ puncta to VGluT2+ and Geph+ puncta to VGAT+ BDA-labeled varicosities in 3D rendered confocal images. A,B: Large images (8 and 12 serial 0.33-μm-thick optical sections) of BDA-labeled axons and varicosities (solid arrowheads, pseudo-color green, Cy2) that are immunopositive for VGluT2 (pseudo-color blue, Cy3), face PSD-95+ profiles (facing pointers, pseudo-color red, Cy5) and featured in the zoomed images on the right (5 and 12 serial 0.33-μm-thick optical sections). Note also, in the large images, the frequent association of VGluT2+ varicosities with PSD-95+ puncta (pointers). C: Image (eight serial sections) of a BDA+ varicosity (open arrowhead, pseudo-color green, Cy2) that is immunonegative for VGluT2 (pseudo-color blue, Cy3) and does not face a PSD-95+ profile, as is also evident in the zoom images on the right (10 serial sections). Note VGluT2+ varicosities in the vicinity that are associated with PSD-95+ puncta (pointer in large image). D,E: Images (eight and seven serial sections, respectively) of BDA-labeled axons and varicosities (solid arrowheads, pseudo-color green, Cy2) that are positive for VGAT (pseudo-color blue, Cy3) and face Geph+ profiles (facing pointers, pseudo-color red, Cy5), as featured in the zoomed images on the right (nine and seven serial sections). In the large image of D, VGAT+/Geph+ elements appear to surround the unlabeled soma of a neuron with the VGAT+ varicosities outside and the Geph+ puncta (pointers) inside, presumably on the cell membrane. Note in the large panels that many VGAT+ varicosities are associated with Geph+ puncta (pointers). F: Image (seven serial sections) of a BDA+ varicosity (open arrowhead, pseudo-color green, Cy2) that is immunonegative for VGAT (pseudo-color blue, Cy3) and is not associated with any Geph+ puncta, as is also evident in the zoomed images on the right (five serial sections). Note, in contrast, that other VGAT+ varicosities in the vicinity are associated with Geph+ puncta (pointer). Deconvolution was applied to all images with a confidence limit of 95% (see Materials and Methods). For abbreviations, see list. Scale bar = 5 μm in F (applies to A–F, large images; 1 μm in F, lower right corner (applies to A–F, small images).