Abstract
Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52–951 vesicles/cluster were transported along 0.60–6.18 µm distances at average 11–95 nms−1 velocities. The ATP cost per vesicle fused (0.4–72.0), calculated from the ratio of the ΔGprocess/ΔGATP, depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6–2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a quantitative method for analyzing similar phenomena in other cell types.
Highlights
Serotonin and other molecules in the nervous system act as conventional transmitters when released from presynaptic endings or as modulators when released extrasynaptically from the soma, dendrites and axon varicosities [1,2]
In addition to these previous observations, we report here that after 20 Hz stimulation, the vesicle clusters that had arrived at the plasma membrane were near microtubules and mitochondria (Fig. 2A, C; Fig. 3), suggesting that they had been transported together with their energetic machinery
The kinetics of exocytosis and a mathematical model we predict that the kinetics of exocytosis in individual active zones depends on the number of vesicles in the cluster, their initial distance from the plasma membrane and their transport velocity
Summary
Serotonin and other molecules in the nervous system act as conventional transmitters when released from presynaptic endings or as modulators when released extrasynaptically from the soma, dendrites and axon varicosities [1,2]. Since somatic secretion in Retzius neurons and in other neuron types depends on transmembrane calcium entry followed by calcium release from intracellular stores [19,21], a plausible hypothesis is that increases of free cytoplasmic calcium trigger the transport of vesicles towards the plasma membrane through the activation of cytoskeletal-based molecular motors. This may explain, at least in part, the minute scale duration of exocytosis, which is 1–2 orders of magnitude longer than the duration of depolarization [3]
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