Fig. 9.

Low to high-magnification images of cross-fractured mixed synapse in matched double-replicas from RSN (from the red line labeled “9A” in Fig. 6B. (A) Low magnification image of RSN (blue overlay) and surrounding neuropil. RSN nucleus is to the right (pink overlay); axon terminals (several delineated by green overlays) surround the entire neuronal soma. (B) Magnified image of the boxed area in A, showing a portion of RSN cytoplasm (blue overlay) and five of >30 axon terminals (green overlays). (C,D) Higher magnification stereoscopic image of boxed area in B, shown as matched complementary replicas, wherein P-faces in (C) are matched by E-faces in (D), and vice versa. A cross-fractured gap junction is double-labeled in both images, with Cx35 (10-nm gold beads) in the axon terminal cytoplasm in both images (blue strip overlays), and Cx34.7 (5-nm gold beads; arrowheads) in the postsynaptic cytoplasm in both stereoscopic images (yellow strip overlays). Because of the 28-nm radius of immunogold labeling [“radius of uncertainty”(Fujimoto, 1995; Fujimoto, 1997; Kamasawa et al., 2006)], the area of potential overlap of immunogold labeling is indicated by intervening green strip overlays. Lavender overlays indicate matching structural details in the two images, including several round/hemispherical synaptic vesicles, which combined with the gap junction, positively identify this as an excitatory (probably glutamatergic) mixed synapse. Calibration bars are 0.1 μm.