The same synaptic vesicles drive active and spontaneous release

Abstract

Synaptic vesicles release neurotransmitter both actively (upon stimulation) and spontaneously (at rest). It has been long assumed that identical vesicles use both modes of release; however, recent evidence has challenged this view. Using several assays (FM dye imaging, pHluorin imaging and antibody-labeling of synaptotagmin), in neuromuscular preparations from Drosophila, frog and mouse as well as rat cultured neurons, we suggest that the same vesicles participate in active and spontaneous release.1 Synaptic vesicles fuse with the neuronal plasma membrane to release their neurotransmitter contents both upon the arrival of action potentials (active release) and at rest (spontaneous release). It has been assumed for almost six decades that identical vesicles exocytose in both cases, an assumption which has been used to generate the quantal (vesicular) release theory 1 . This assumption is in agreement with the fact that prolonged active 2 or spontaneous release 3 can both release essentially all vesicles (see also 4,5 ). The issue of whether the same vesicles can be released both spontaneously and actively has been tested recently by investigating the loading and unloading of styryl (FM) dyes during vesicle recycling 6 . In cultured hippocampal neurons, the vesicles loaded with FM dyes during active or spontaneous recycling were unloaded efficiently only by the same release paradigm. Also, inhibiting synaptic vesicle re-acidification (and therefore refilling with neurotransmitter) by use of folimycin during spontaneous release specifically depleted the spontaneous pool 6 . Surprisingly, both of these findings have been later contested in similar experiments -FM dyes taken up either at rest or during stimulation could be released with identical kinetics, and the inhibition of reacidification during depolarization led to a cessation of spontaneous release 7 (see also 5 ). However, later studies have again found different FM dye release kinetics when using different loading paradigms 8,9 , and labeling experiments using the expression of a biotinylated variant of the synaptic vesicle marker VAMP2 (synaptobrevin) also supported the hypothesis that spontaneously and actively recycling vesicles are different 10 .We tested here this controversial issue by combining several fluorescence imaging assays. We sought to reproduce the FM dye loading/unloading paradigms used in the past. A number of FM dyes have been used, including FM 1-43 and FM 2-10 6-9 ; both dyes have provided evidence for a difference between the spontaneously and actively recycling vesicles 8,9 . It has been recently suggested that FM 2-10 reports this difference better than FM 1-43 9 . However, as FM 2-10 is less bright when compared to FM 1-43, it is typically used at very high concentrations (400 µM; ~4-fold higher than its membrane dissociation constant 11 ). Since the FM dyes have a detergent-like structure, they inhibit vesicle recycling at high concentrations 11 . We therefore decided to employ FM 1-43 in our work (the concentration typically used,...

Independent Vesicle Pools Underlie Different Modes of Release during Neuronal Development

“…To bypass some of the inherent technical problems of using these dyes, we previously used the biosyn technique to permanently label vesicle pools and demonstrated that spontaneous and evoked release derive from separate pools (Fredj and Burrone, 2009), but this view has recently been challenged (Hua et al, 2010;Wilhelm et al, 2010). However, technical issues with the techniques used suggest that there may be inherent problems with the methods of studying vesicle cycling.…”

“…In one paper (Wilhelm et al, 2010), most of the experiments use FM dyes, with issues that have been discussed previously (Fredj and Burrone, 2009). In a key experiment, Wilhelm et al (2010) use the pH-sensitive exocytosis reporter, synaptopHluorin, to demonstrate similar levels of exocytosis due to both active and spontaneous cycling following labeling of the activitydependent pool.…”

“…In a key experiment, Wilhelm et al (2010) use the pH-sensitive exocytosis reporter, synaptopHluorin, to demonstrate similar levels of exocytosis due to both active and spontaneous cycling following labeling of the activitydependent pool. However, the authors removed the protonpump inhibitor folimycin from the medium so that, over the 15 min period used to show exocytosis of the evoked pool by spontaneous release, reacidification might occur due to potential reversibility of these drugs (Sankaranarayanan and Ryan, 2001;Chang and Mennerick, 2010), which could account for the observed change in fluorescence.…”

“…Vesicle fusion occurs both in response to depolarization of the presynaptic membrane, following the arrival of an action potential, and spontaneously. Recent work has described the identity of the pools of vesicles responsible for each form of release, although debate has surrounded the issue of whether spontaneous vesicular release at mature synapses derives from the same pool of vesicles as evoked release (Groemer and Klingauf, 2007;Hua et al, 2010;Wilhelm et al, 2010) or constitutes a separate pool that is mobilized independently (Sara et al, 2005;Atasoy et al, 2008;Fredj and Burrone, 2009;Chung et al, 2010).…”

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

“…Typically in such experiments, both staining and destaining are triggered by extensively stimulating the neurons (evoked activity). Recently, however, synaptic vesicle recycling in response to weak stimulation has also been analyzed, as has recycling in the absence of an external stimulus (spontaneous and miniature synaptic activity) 9,[17][18][19] . Spontaneous and miniature synaptic activities are defined as those that occur in the absence of external stimuli, with the former involving the spontaneous firing of action potentials (Figure 3).…”