Real-time imaging of mu opioid receptors by total internal reflection fluorescence microscopy.

Receptor trafficking and signaling are intimately linked, especially in the Mu opioid receptor (MOR) where ligand-dependent endocytosis and recycling have been associated with opioid tolerance and dependence. Ligands of MOR can induce receptor endocytosis and recycling within minutes of exposure in heterologous systems and cultured neurons. Endocytosis removes desensitized receptors after their activation from the plasma membrane, while recycling promotes resensitization by delivering functional receptors to the cell surface. These rapid mechanisms can escape traditional analytical methods where only snapshots are obtained from highly dynamic events.Total internal reflection fluorescence (TIRF) microscopy is a powerful tool that can be used to investigate, in real time, surface trafficking events at the single molecule level. The restricted excitation of fluorophores located at or near the plasma membrane in combination with high sensitivity quantitative cameras makes it possible to record and analyze individual endocytic and recycling event in real time. In this chapter, we describe a TIRF microscopy protocol to investigate in real time, the ligand-dependent MOR trafficking in Human Embryonic Kidney 293 cells and dissociated striatal neuronal cultures. This approach can provide unique spatio-temporal resolution to understand the fundamental events controlling MOR trafficking at the plasma membrane.

Fast modulation of µ-opioid receptor (MOR) recycling is mediated by receptor agonists.

The µ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.