title: Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.
abstract: 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.
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA.
authors.forename: Francheska
authors.initials: F
authors.lastname: Delgado-Peraza
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Nogueras-Ortiz
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA.
authors.forename: Agnes M
authors.initials: AM
authors.lastname: Acevedo Canabal
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Physiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA.
authors.forename: Cristina
authors.initials: C
authors.lastname: Roman-Vendrell
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; Department of Anatomy and Neurobiology, School of Medicine, University of Puerto Rico, San Juan, PR, USA.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Yudowski
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism*; D061251:Primary Cell Culture; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D059010:Single-Cell Analysis
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: Endocytosis; G protein-coupled receptor; Live cell imaging; MOR; Recycling; Resensitization; TIRF