Acute Dynamin Inhibition Dissects Synaptic Vesicle Recycling Pathways That Drive Spontaneous and Evoked Neurotransmission

Abstract

Synapses maintain synchronous, asynchronous, and spontaneous forms of neurotransmission that are distinguished by their Ca 2ϩ dependence and time course. Despite recent advances in our understanding of the mechanisms that underlie these three forms of release, it remains unclear whether they originate from the same vesicle population or arise from distinct vesicle pools with diverse propensities for release. Here, we used a reversible inhibitor of dynamin, dynasore, to dissect the vesicle pool dynamics underlying the three forms of neurotransmitter release in hippocampal GABAergic inhibitory synapses. In dynasore, evoked synchronous release and asynchronous neurotransmission detected after activity showed marked and unrecoverable depression within seconds. In contrast, spontaneous release remained intact after intense stimulation in dynasore or during prolonged (ϳ1 h) application of dynasore at rest, suggesting that separate recycling pathways maintain evoked and spontaneous synaptic vesicle trafficking. In addition, simultaneous imaging of spectrally separable styryl dyes revealed that, in a given synapse, vesicles that recycle spontaneously and in response to activity do not mix. These findings suggest that evoked synchronous and asynchronous release originate from the same vesicle pool that recycles rapidly in a dynamin-dependent manner, whereas a distinct vesicle pool sustains spontaneous release independent of dynamin activation. This result lends additional support to the notion that synapses harbor distinct vesicle populations with divergent release properties that maintain independent forms of neurotransmission.

The same synaptic vesicles drive active and spontaneous release

“…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.…”

“…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 .…”

“…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 ).…”

“…The systematic investigation of all possible loading/unloading paradigms (active-active, activespontaneous, spontaneous-active and spontaneous-spontaneous) had never been applied in past studies addressing the issue [6][7][8][9][10] . We tested four different synaptic preparations: the neuromuscular junctions (NMJs) of mouse, frog and Drosophila third instar larvae, cultured hippocampal neurons.…”

“…However, as most of the previous work [6][7][8][9][10] dealt with hippocampal cultures, we employed several additional experimental approaches in this model system. We first designed an assay in which the synaptic vesicle marker synaptotagmin was identified by biotinylated antibodies during one round of loading (actively for 30 seconds at 20 Hz, or spontaneously for 15 minutes).…”