Cycling of Dense Core Vesicles Involved in Somatic Exocytosis of Serotonin by Leech Neurons

Abstract

We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear region.

Patterns and distribution of presynaptic and postsynaptic elements within serial electron microscopic reconstructions of neuronal arbors from the medicinal leech Hirudo verbana

“…With our staining protocol, there appeared to be at least two basic types of these vesicles, intensely stained (maximum diameter 100 nm) and lightly stained (maximum diameter 170 nm). Some of the intensely stained dense‐core vesicles likely contained serotonin on the basis of the pattern of somata containing them in our juvenile ganglion volume (Lent et al, 1991), in accordance with prior evidence (Muller, ; Kuffler et al, 1987; Trueta et al, ). Intensely stained dense‐core vesicles are a hallmark of serotonergic vesicles and have been described in the crustacean stomatogastric ganglion (Friend, ; King, ) and mollusk (Cottrell and Osborne, ; Goldman et al, ).…”

“…Based on the locations of these neurons (Fig. C, blue neurons), the neurons containing the intensely staining vesicles corresponded to the six serotonergic neurons of midbody ganglia according to previous observations (Lent et al, 1991; Trueta et al, ). Many of the somata containing large low‐density vesicles did not match any previously identified neurons (Fig.…”

“…A few somata contain numerous dense-core vesicles, but most do not (Coggeshall and Fawcett, 1964). In at least two neurons, the large serotonergic Retzius cells, these vesicles can be released directly from the somata (Trueta et al, 2012). Dense-core vesicles are also observed in the processes of the nociceptive N cell that closely wrap around the somata and the primary neurite of sensory neurons in adjacent ganglia (French and Muller, 1986).…”

Biophysics of Active Vesicle Transport, an Intermediate Step That Couples Excitation and Exocytosis of Serotonin in the Neuronal Soma

“…Serotonin is stored in clusters of large (100 nm diameter) dense core vesicles [16]–[18] and electrical stimulation with trains of ten impulses at 20 Hz evokes exocytosis for the following 2–5 minutes, contrary to the effect of 1 Hz stimulation, which fails to evoke exocytosis [3]. Electron micrographs taken from neurons at rest or after 1 Hz stimulation contain clusters of dense core vesicles resting away from the plasma membrane, whereas after 20 Hz stimulation a large proportion of the vesicle clusters appear closely apposed to the plasma membrane [19], [20]. 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.…”

Serotonin release from the neuronal cell body and its long-lasting effects on the nervous system

“…Figure 1 shows micrographs obtained from the soma of neurons stimulated at 1 and 20 Hz. Micrographs obtained after stimulation at 1 Hz show clusters of vesicles resting at a distance from the plasma membrane, whereas micrographs of neurons stimulated at 20 Hz show about 50% of the vesicle clusters opposed to the plasma membrane [ 2 ]. Morphological evidence of exocytosis came from experiments stimulating exocytosis in the presence of peroxidase in the bathing solution.…”