Abstract
The soma of many neurons releases large amounts of transmitter molecules through an exocytosis process that continues for hundreds of seconds after the end of the triggering stimulus. Transmitters released in this way modulate the activity of neurons, glia and blood vessels over vast volumes of the nervous system. Here we studied how somatic exocytosis is maintained for such long periods in the absence of electrical stimulation and transmembrane Ca2+ entry. Somatic exocytosis of serotonin from dense core vesicles could be triggered by a train of 10 action potentials at 20 Hz in Retzius neurons of the leech. However, the same number of action potentials produced at 1 Hz failed to evoke any exocytosis. The 20-Hz train evoked exocytosis through a sequence of intracellular Ca2+ transients, with each transient having a different origin, timing and intracellular distribution. Upon electrical stimulation, transmembrane Ca2+ entry through L-type channels activated Ca2+-induced Ca2+ release. A resulting fast Ca2+ transient evoked an early exocytosis of serotonin from sparse vesicles resting close to the plasma membrane. This Ca2+ transient also triggered the transport of distant clusters of vesicles toward the plasma membrane. Upon exocytosis, the released serotonin activated autoreceptors coupled to phospholipase C, which in turn produced an intracellular Ca2+ increase in the submembrane shell. This localized Ca2+ increase evoked new exocytosis as the vesicles in the clusters arrived gradually at the plasma membrane. In this way, the extracellular serotonin elevated the intracellular Ca2+ and this Ca2+ evoked more exocytosis. The resulting positive feedback loop maintained exocytosis for the following hundreds of seconds until the last vesicles in the clusters fused. Since somatic exocytosis displays similar kinetics in neurons releasing different types of transmitters, the data presented here contributes to understand the cellular basis of paracrine neurotransmission.
Highlights
In addition to the canonical release of transmitters from synapses, many neuron types release transmitters or peptides by exocytosis from their soma (For review see Trueta and De-Miguel, 2012)
We have previously shown (De-Miguel et al, 2012) that the sigmoidal shape of the fluorescence kinetics has the following components: its latency is determined by the resting distance from the vesicle clusters to the plasma membrane and by the transport velocity of the vesicle cluster; the slope of the fluorescence increase expresses the rate of exocytosis; the plateau is reached upon the end of exocytosis, and the maximum fluorescence level is proportional to the cumulative number of vesicles that fused
We have shown that a brief stimulation with 10 impulses at 20 Hz produces a fast Ca2+ transient that triggers exocytosis and determines its magnitude
Summary
In addition to the canonical release of transmitters from synapses, many neuron types release transmitters or peptides by exocytosis from their soma (For review see Trueta and De-Miguel, 2012). Since somatic exocytosis depends on transmembrane Ca2+ entry (Sun and Poo, 1987; Chen et al, 1996; Jaffe et al, 1998; Puopolo et al, 2001; Trueta et al, 2003; Soldo et al, 2004; Huang et al, 2007; Kaushalya et al, 2008; Hirasawa et al, 2009), one wonders how it is maintained for such long periods after electrical stimulation and transmembrane Ca2+ entry have ended To study this problem we took advantage of the serotonergic Retzius neurons of the leech, in which most fine mechanisms of serotonin (5-HT) exocytosis from the synapses and soma were first elucidated (For review see Nicholls and Kuffler, 1990; DeMiguel and Trueta, 2005).
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