Fig 1: AVP–VGLUT2 immunopositive (AVP+/VGLUT2+) fibers establish Gray type I (asymmetric) synapses onto tyrosine hydroxylase immunopositive (TH+) dendrites within the LC. (A,B) Confocal photomicrographs showing AVP+ fibers making contacts with TH+ dendrites (A) and the co-localization of AVP+/VGLUT2+ segments (B, two Herring body-like bodies were indicated by arrow heads). (A) AVP reaction was made using the antibody gifted from H. Gainer laboratory and image was taken at 0.5 AU (Airy Units, using Leica SP5 confocal system) optical section thickness of around 0.596 µm. This measure helps to improve the optical resolution to visualize the contact points in a strongly labeled tissue. The panel B was from mouse LC tissue using a different antibody (Abcam ab39363) and taken at 1 AU (optical section thickness was around 0.892 µm). (C) Photomicrograph taken from a pre-embedding immunoreaction of trimmed resin capsule surface (the “pyramid,” osmicated losing the purple color under LM), containing LC prepared for electron microcopy (EM), using DAB/VIP (Very Intense Purple) double peroxidase-chromogen immunostaining for electron microscopy. AVP+ fibers were evidently making contact with TH+ dendritic segments indicated by arrowheads. The inserts are TEM micrographs from serial samples of the region indicated by rectangle area in C. The image shows an AVP+ axon with a terminal (depicted in four serial sections) containing AVP+ dense-core vesicles (dcv, indicated with green arrowhead), establishing a Gray-type I synapse onto a TH+ dendrite (TH is demonstrated by granular labeling produced by VIP reagent at electron microscopy level, yellow arrowheads). Postsynaptic density (PSD), a TEM feature of a Gray type I synapse, which is generally indicative of a glutamatergic synapse, are indicated by green arrows. (D) TEM micrograph showing one segment of the same AVP+ profile (DAB-nickel labeling) coursing in parallel with two TH+ (VIP labeling) dendrites. Note that the VIP labeling for dendrite (white arrowhead) was weaker than for axonal segment (black arrowhead). (E) An Herring body-like body (large axonal varicosity), an anatomical feature of the vasopressinergic magnocellular neurosecretory neurons (white asterisks of panel B,E) examined under EM. Scale bars: 400 nm unless stated otherwise.