International and trafficking of Mu and Delta opioid receptors in rat primary cortical culture and their response to chronic naloxone and morphine treatment

The Mu and Delta opioid receptors (MOR & DOR) have been shown to undergo complex regulatory changes in response to ligand exposure in transfected cells. However, little is known regarding receptor regulation in neurons. In this study, we have investigated the internalization and trafficking of the MOR and DOR agonists, fluodemorphin (fluo-DRM) and fluo-demorphin-I (fluo-DLT-I), respectively, and the fate of these receptors following ligand binding. Experiments were performed on primary rat cortical neuronal cultures using a ligand binding internalization assay and immunocytochemistry. Incubation of neurons with fluo-DRM and fluo-DLT-I resulted in the specific internalization of the fluo-ligands. The internalization was blocked by phenylarsine oxide, indicating that it is clathrin-mediated. Both fluo-ligands accumulated in the soma while no change was evident in the neuronal processes. Exposure to the microtubule disruptor nocadazole resulted in the accumulation of fluo-ligands in the neuronal processes and decrease in their accumulation in soma, suggesting that fluo-ligands are targeted to the cell soma following internalization. Immunocytochemistry of MOR and DOR revealed that receptor compartmentalization did not change after exposure to their respective opioid agonists suggesting that in contrast to internalized ligands, internalized receptors are recycled locally. The internalization and trafficking of opioid ligands and receptors were also determined after chronic treatment of neuronal cultures with morphine. Chronic morphine treatment led to a profound decrease in fluo-DRM internalization along the processes, but had no effect on the intensity of MOR immunolabeling. However, DOR immunolabeling as well as fluo-DLT-I internalization were greatly increased. This study is the first to demonstrate the internalization and targeting of opioid receptor ligands in neurons. After ligand exposure, MOR and DOR internalize, dissociate from their bound ligands and recyc