Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

8-POS Board 8 EXTRASYNAPTIC EXOCYTOSIS FROM THE AXON OF IDENTIFIED SEROTONERGIC NEURONS Montserrat G Cercós 1 ; Citlali Trueta 1 ; 1 Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Neurophysiology, Mexico City, Distrito Federal, Mexico Serotonin exocytosis is activated by single impulses from presynaptic terminals and by high- frequency trains of impulses from extrasynaptic sites at the neuronal soma. Vesicle-containing sites at the axons of serotonergic neurons suggest extrasynaptic secretion, but axonal extrasynaptic exocytosis has not been functionally demonstrated and its regulation mechanisms are unknown. Here we show axonal extrasynaptic exocytosis and analyze its firing-frequency and calcium-source dependence. Exocytosis and intracellular calcium were analyzed from the fluorescence of FM styryl dyes and Fluo-4, respectively, in identified leech serotonergic neurons. Ten impulses at 1 or 20 Hz produced one or two axonal exocytosis phases, respectively, suggesting different vesicle pools arriving from increasing distances to the membrane, which were corroborated by electron microscopy. Two calcium transients underlie exocytosis upon stimulation at both frequencies: a fast transient synchronized with electrical activity, which activates exocytosis from the closest vesicle pool and determines the mobilization of distant pools, and a slower and longer submembrane transient, produced by a positive feedback mechanism activated by released serotonin, which allows exocytosis of all mobilized vesicles. The fast calcium transient in response to 20-Hz stimulation was twice larger than that upon 1-Hz stimulation, and in contrast to the later, propagated through the axoplasm. Depletion of intracellular calcium stores revealed that this propagation requires calcium-induced calcium release (CICR), and is necessary for mobilizing distant vesicles towards the membrane. Blockade of L-type calcium channels, generally associated with extrasynaptic exocytosis, did not impair axonal exocytosis or the propagation of the fast calcium transient upon 20-Hz stimulation, suggesting that calcium entry through non-L synaptic channels activates CICR. Axonal extrasynaptic exocytosis has intermediate mechanisms between synaptic and somatic exocytosis and expands the possibilities of single neurons to produce different effects in neural circuits.

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