Synaptic vesicle pool‐specific modification of neurotransmitter release by intravesicular free radical generation

Abstract

Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) - a haem-containing plant enzyme - or antibodies against synaptotagmin-1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H O ) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization-induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP-tagged syt1 construct and subsequent H O application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss-of-function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

Quintuple labeling in the electron microscope with genetically encoded enhanced horseradish peroxidase

“…eHRPsyt1 was targeted to the lumen of SVs and when a SV fused with the plasma membrane, extracellular hydrogen peroxide was cleaved and the radicals rendered the released SV dysfunctional. With this technique it was possible to dissect SV cycling in physiological experiments [ 28 ].…”

Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components

“…It is important to note, however, that some antibodies might affect protein distribution and trafficking in live cells. Thus, rabbit polyclonal antibodies against the luminal domain of synaptotagmin 1 have been suggested to alter synaptic function (Afuwape et al, 2017). At the same time, mouse antibodies against the same target, which are usually used for vesicle tracking experiments (Matteoli et al, 1992; Kraszewski et al, 1995; Sara et al, 2005; Fernández-Alfonso et al, 2006; Wienisch and Klingauf, 2006; Hua et al, 2010), have not been reported to have such an effect, and do not perturb vesicle trafficking even when used for several days (Truckenbrodt et al, 2018b).…”

GFP nanobodies reveal recently-exocytosed pHluorin molecules

“…However, this approach is not free of potential controversies. Synaptotagmin 1 antibodies have been in use for more than two decades 10,11 and they do not appear to impair synaptic function 8,12 , but a significant modulation of vesicle recycling via the antibodies has been recently noted 13 . Thus, the usage of such antibodies is likely to provide a realistic image of the vesicle protein organization after exocytosis, but may have some modulatory side effects.…”