Figure 5.

Chronic activation with ephrinB2 reduces levels of EphB2 and synaptic-associated proteins in ENS cultures.A–C, primary cultures of E15 rat intestines were grown on glass coverslips and fully matured into an enteric-like neuronal network with ganglia interconnected with neural fibers at 12 DIV. Cultures were then treated with control-Fc (Control) or ephrinB2-Fc ligand (ephrinB2) for 5 consecutive days. Cultures were fixed and double-immunostained for EphB2 (A), synapsin I (B), synaptophysin (C) and pan-neuronal marker HuC/D (A–C). Images represent ganglia depicting neuronal cell bodies and processes. D–F, quantitations of images represented in (A–C) for HuC/D-positive neurons (D), and neuronal fiber density of EphB2 (E), synapsin I (F) or synaptophysin (G). Note that chronic exposure of neurons to ephrinB2 decreased EphB2 expression within neuronal processes without affecting overall neuronal density. Synapsin I and synaptophysin were reduced within neuronal processes. H, images represent ganglia depicting neuronal cell bodies (HuC/D staining) and neuronal processes (Tuj-1 staining). I and J, quantitations of images represented in (H) for the number of neuronal processes/ganglionic fiber bundles (I) and overall fiber density (J). (n = 10–15 ganglia per condition from at least three independent cultures, unpaired t test). ∗∗∗p ≤ 0.0001.