Highlighting Synaptic Communication in the Enteric Nervous System Pieter Vanden Berghe, Jan Tack, Werend Boesmans  Gastroenterology  Volume 135, Issue 1, Pages 20-23 (July 2008) DOI: 10.1053/j.gastro.2008.06.001 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Immuno double labeling of a mouse myenteric ganglion. The myenteric neurons labeled in green with an antibody against microtubule-associated protein II (MAP-II) are decorated with synaptic contacts, labeled in red with the synaptic vesicle marker synaptophysin. Bar, 20 μm. Gastroenterology 2008 135, 20-23DOI: (10.1053/j.gastro.2008.06.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Imaging of synaptic activity in enteric nerve networks. (A) Ca2+ imaging in neuronal boutons or varicosities. (Top) Representative images of Fluo-4 loaded cultured myenteric neurons from guinea pig either at rest or while active. (Bottom left) Color-coded image representing Fluo-4 loading (green) and maximal “activity over time” (AoT) in red. (Bottom right) Graph showing the relative Fluo-4 fluorescence of 3 neuronal varicosities (color matched with arrows in top images) reflecting their spontaneous activity over time. Bars, 20 μm. (B) Synaptic vesicle recycling in cultured myenteric neurons. (Left) Image of enteric boutons labeled with FM1-43 before destaining protocol. *Neuronal cell bodies. (Right) Representative fluorescence traces of 2 enteric boutons (arrows, in left panel) destained by 3 consecutive stimuli (arrowhead and arrow, 40 and 400 single electrical stimuli; bar, 75 mmol/L K+ application). Bars, 20 μm. (C) SynaptopHluorin visualization in whole mount tissue. (Top) Images of synaptopHluorin in an enteric ganglion at pH 7.4 (left) and treated with NH4Cl (right), to alkalinize all intracellular compartments and reveal the presence of quenched synaptopHluorin molecules. (Bottom left) Immunolabeling of nitric oxide (NOS)–positive neurons, surrounded by synpatopHluorin expressing fibers, in a mouse myenteric ganglion. (Bottom right) Typical traces of single single-release events during which synaptopHluorin is unquenched and causes an increase in fluorescence. Bars, 20 μm. Gastroenterology 2008 135, 20-23DOI: (10.1053/j.gastro.2008.06.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Schematic of the principles and methods for visualizing synaptic vesicle release. (A) Simplified drawing showing the main characteristics of a presynaptic terminal. Upon arrival of an action potential, voltage gated Ca2+ channels open owing to depolarization and allow Ca2+ to flow into the terminal. This triggers the synaptic vesicles to fuse with the presynaptic membrane and release their neurotransmitter contents. (B) Schematic of 3 different optical techniques to visualize synaptic activity. (Left) The Ca2+ indicator Fluo-4 can be used to monitor the increase in intracellular Ca2+ concentration in the terminal. (Middle) FM1-43 intercalates in the membrane and recycles with the exo/endocytic cycle. After washing the excess dye, only the terminals appear labeled. In a last step they can be “destained” to investigate vesicle release kinetics. (Right) Synaptic vesicles of transgenic mice expressing the pH-sensitive probe synaptopHluorin are quenched at rest, because pHluorin is exposed to the inside acidic milieu of the vesicle. Upon vesicle fusion the pHluorin is briefly exposed to the extracellular neutral pH, which can be detected as a subtle increase in fluorescence. Gastroenterology 2008 135, 20-23DOI: (10.1053/j.gastro.2008.06.001) Copyright © 2008 AGA Institute Terms and Conditions