神经科学

Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with isolating single proteins within a biological system. Here we provide a detailed protocol to conduct ex vivo single molecule imaging where single transmembrane proteins are isolated by rapidly extracting nanovesicles containing receptors of interest from different regions of the brain and subjecting them to single molecule study by using total internal reflection fluorescence (TIRF) microscopy. This protocol discusses the isolation and separation of brain region specific nanovesicles as well as a detailed method to perform TIRF microscopy with those nanovesicles at the single molecule level. This technique can be applied to study trafficking and stoichiometry of various transmembrane proteins from the central nervous system. This approach can be applied to a wide range of animals that are genetically modified to express a membrane protein-fluorescent protein fusion with a wide range of potential applications in many aspects of neurobiology.

Graphic abstract:

EX vivo single molecue imaging of membrane receptors

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria. As a potent GABA_A receptor agonist, muscimol suppresses the activity of the central nervous system, reduces anxiety and induces sleep. We investigated the effects of muscimol on Drosophila behavior. Drosophila behavioral assays are powerful tools that are used to assess neural functions by focusing on specific changes in selected behavior, with the hypothesis that this behavioral change is due to alteration of the underlying neural function of interest. In this study, we developed a comparatively simple and cost-effective method for feeding adult flies muscimol, a pharmacologically active compound, and for quantifying the phenotypes of “resting” and “grooming+walking”. This protocol may provide researchers with a convenient method to characterize small molecule-induced behavioral output in flies.

Detecting protein-protein interactions by co-immunoprecipitation provided a major advancement in the immunology research field. In the G-protein-coupled receptors (GPCRs) research field, colocalization and co-immunoprecipitation were used to detect interactions, but doubts arose due to specificity of the antibodies (monoclonal in the case of receptors related to immunology and polyclonal in the case of GPCRs) and due to the possibility of false positive due to the potential occurrence of bridging proteins. Accordingly, new methodological approaches were needed, and energy transfer techniques have been instrumental to detect direct protein-protein, protein-receptor or receptor-receptor interactions. Of the two most relevant methods (Förster, or fluorescence resonance energy transfer: FRET and Bioluminescence energy transfer: BRET), the protocol for BRET is here presented. BRET has been instrumental to detect direct interactions between GPCRs and has contributed to demonstrate that GPCR dimers/oligomer functionality is different from that exerted by individual receptors. Advantages outweigh those of FRET as no fluorescence source is needed. Interestingly, BRET is not only useful to validate interactions detected by other means or hypothesized in the basis of indirect evidence, but to measure signal transduction events. In fact, BRET may, for instance, be used to assess β-arrestin recruitment to activated GPCRs.