Quantitative monitoring of activity-dependent bulk endocytosis of synaptic vesicle membrane by fluorescent dextran imaging.
Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) retrieval mode in central nerve terminals during periods of intense neuronal activity. Despite this fact there are very few real time assays that report the activity of this critical SV retrieval mode. In this paper we report a simple and quantitative assay of ADBE using uptake of large flourescent dextrans as fluid phase markers. We show that almost all dextran uptake occurs in nerve terminals, using co-localisation with the fluorescent probe FM1-43. We also demonstrate that accumulated dextran cannot be unloaded by neuronal stimulation, indicating its specific loading into bulk endosomes and not SVs. Quantification of dextran uptake was achieved by using thresholding analysis to count the number of loaded nerve terminals, since monitoring the average fluorescence intensity of these nerve terminals did not accurately report the extent of ADBE. Using this analysis we showed that dextran uptake occurs very soon after stimulation and that it does not persist when stimulation terminates. Thus we have devised a simple and quantitative method to monitor ADBE in living neurones, which will be ideal for real time screening of small molecule inhibitors of this key SV retrieval mode.
Clayton
Emma Louise
EL
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, Scotland, UK.
Cousin
Michael Alan
MA
D000818:Animals; D000831:Animals, Newborn; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004705:Endocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D018377:Neurotransmitter Agents / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D011726:Pyridinium Compounds / Q000737:chemistry / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure
D003911:Dextrans; C073804:FM1 43; D005456:Fluorescent Dyes; D018377:Neurotransmitter Agents; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds