Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Abstract

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity. Video LinkThe video component of this article can be found at

Differentiation of Spiral Ganglion-Derived Neural Stem Cells into Functional Synaptogenetic Neurons

“…FM1-43 has been used for vesicle recycling including synaptic vesicle recycling [28]. In this study, synaptic vesicles of ScNs were monitored by FM1-43 synaptic vesicle staining using passage 4-5 ScNs cultured in the BDNF-containing differentiation medium.…”

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

“…Functional nerve terminals (boutons) were stained by labeling recycling synaptic vesicles with FM1-43 (T35356; Thermo Fisher Scientific, Waltham, Massachusetts, USA) under a condition promoting high neuronal activity (Iwabuchi et al, 2014a;Kakazu et al, 2012a), off-stage, at room temperature (~21°C). The coverslips were immersed in 10 μM FM1-43 in a high-K + solution (45-mM KCl) for 1 min.…”

“…Neurons were loaded with a green-emitting [Ca 2+ ]c indicator Fluo-5F-AM (F14222; Thermo Fisher Scientific) (Iwabuchi et al, 2013a;Iwabuchi et al, 2013b). Functional nerve terminals were loaded with FM4-64 (T13320; Thermo Fisher Scientific), a red-shifted version of FM1-43, by spontaneous synaptic activities (Iwabuchi et al, 2014a;Kakazu et al, 2012a;Kakazu et al, 2012b). These were achieved by immersing the coverslips in prewarmed Minimum Essential Medium (MEM, 51200-038; Thermo Fisher Scientific) containing 1 μM Fluo-5F-AM and 2.5 μM FM4-64, in the culture incubator at 37°C for 10 min.…”

“…• Selection of FM dyes and Ca 2+ indicator dyes o Representative dyes for labeling the recycling synaptic vesicles include greenemitting FM1-43 and red-emitting FM4-64 (Iwabuchi et al, 2014). In the partial onstage ICC protocol, either FM dye can be used interchangeably, by using proper light sources and fluorescent filters.…”

“…An example is found in the study of presynaptically silent synapses. A certain proportion of nerve terminals do not release neurotransmitter even when neurons are stimulated extensively (Crawford and Mennerick, 2012;Moulder et al, 2004): these presynaptically silent synapses do not take up FM dye, a fluorescent marker of the synapticvesicle recycling (Iwabuchi et al, 2014a;Kavalali and Jorgensen, 2014;, although those nerve terminals are morphologically mature. Thus, such nerve terminals lack fluorescent FM signals yet are positive for ICC signals for structural markers of nerve terminals.…”