Abstract：
: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.
journal_name
J Neurosci Methodsjournal_title
Journal of neuroscience methodsauthors
Clayton EL,Cousin MAdoi
10.1016/j.jneumeth.2009.09.016subject
Has Abstractpub_date
2009-12-15 00:00:00pages
76-81issue
1eissn
0165-0270issn
1872-678Xpii
S0165-0270(09)00507-Xjournal_volume
185pub_type
杂志文章abstract：:Adult rat spinal cords were perfused intracardially with aldehyde fixatives containing either sodium cacodylate or sodium phosphate buffer for EM studies. The perfusion flow rate of 24 cc/min for the first 3 min and 33 cc/min for completion of prefixation was found to be optimal. Ultrastructural examination of the L-1...
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