Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.

Total internal reflection fluorescence (TIRF) microscopy allows probing the cellular events occurring close and at the plasma membrane. Over the last decade, we have seen a significant increase in the number of publications applying TIRF microscopy to unravel some of the fundamental biological questions regarding G protein-coupled receptors (GPCRs) function such as the mechanisms controlling receptor trafficking, quaternary structure, and signaling among others. Most of the published work has been performed in heterologous systems such as HEK293 and CHO cells, where the imaging surface available is higher and smoother when compared with the narrow processes or the smaller cell bodies of neurons. However, some publications have expanded our understanding of these events to primary cell cultures, mostly rat hippocampal and striatal neuronal cultures. Results from these cells provide a bona fide model of the complex events controlling GPCR function in living cells. We believe more work needs to be performed in primary cultures and eventually in intact tissue to complement the knowledge obtained from heterologous cell models. Here, we described a step-by-step protocol to investigate the surface trafficking and signaling from GPCRs in rat hippocampal and striatal primary cultures.

Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures.

Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.