Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin

Abstract

SummaryGlycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca v 2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca 2+ oscillations sensitive to the inhibitor -conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.

Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion

“…After 5 min stimulation, the nerve terminals exhibited a regularly spaced pattern of labeling (Figure 6B) characteristic of the recycling vesicle organization previously reported at the frog NMJ [27]. Motor nerve terminals stimulated at 20 Hz for 10 min, with a pulse of FM1-43 applied during the last 5 min of stimulation, revealed the appearance of recyclosomes (Figure 6C), as previously described in Figure 1 [7]. The same experiments were carried out on NMJ preparations pre-treated with dyngo-4a (30 µM) for 40 min.…”

“…This maturation process might be triggered by the longer duration of our stimulation (20 Hz for 10 min) intended to deplete both the RRP and RP of synaptic vesicles [32]. Recently, similar donut-like structures were detected in presynaptic nerve terminals treated with the stimulatory neurotoxin glycerotoxin [7].…”

Synaptic Vesicle Endocytosis

“…Bulk endocytosis leads to the generation of endocytic vacuoles that are subsequently converted into synaptic vesicles by a mechanism that remains elusive (Fig. 1A) (Miller and Heuser 1984;Holt et al 2003;Paillart et al 2003;Wu and Wu 2007;Hayashi et al 2008;Meunier et al 2010). A third form of endocytosis, the rapid closure of a fusion pore without collapse of the vesicle, termed "kiss and run," remains an attractive model to explain electrophysiological and dynamic imaging data that are difficult to reconcile with other endocytic mechanisms (Fig.…”

Structure of the neuromuscular junction: function and cooperative mechanisms in the synapse

“…1). Although the molecular basis of vesicle retrieval (including kiss‐and‐run recycling 7 and bulk endocytosis 8 ) remains debated, the synapse may use these two alternative or cooperative modes as an adaptive response to synaptic fatigue. The influx of external Ca 2+ required for the Ca 2+ ‐triggered exocytosis and the fast‐mode of endocytosis is promoted by the activation of presynaptic M1‐type muscarinic AChR (M1 mAChR) 9 as well as the interaction of brain‐derived neurotrophic factor (BDNF) with receptor tyrosine kinase B (TrkB) 10 .…”