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

Total internal reflection fluorescence (TIRF) microscopy allows probing the cellularevents occurring close and at the plasma membrane. Over the last decade, we have seen asignificant increase in the number of publications applying TIRF microscopy to unravelsome of the fundamental biological questions regarding G protein-coupled receptors(GPCRs) function such as the mechanisms controlling receptor trafficking, quaternarystructure, and signaling among others. Most of the published work has been performed inheterologous systems such as HEK293 and CHO cells, where the imaging surfaceavailable is higher and smoother when compared with the narrow processes or the smallercell bodies of neurons. However, some publications have expanded our understanding ofthese 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 controllingGPCR function in living cells. We believe more work needs to be performed inprimary cultures and eventually in intact tissue to complement the knowledge obtainedfrom heterologous cell models. Here, we described a step-by-step protocol to investigatethe surface trafficking and signaling from GPCRs in rat hippocampal and striatal primarycultures.