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Cycling of dense core vesicles involved in somatic exocytosis of serotonin by leech neurons.

Trueta C, Kuffler DP, De-Miguel FF - Front Physiol (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto Nacional de Psiquiatría "Ramón de la Fuente Muñiz," México D. F., México.

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.

No MeSH data available.


Related in: MedlinePlus

Formation of multivesicular bodies after electrical stimulation. (A) Several multivesicular bodies near the plasma membrane with dense core vesicles around. The neuron was stimulated at 20 Hz and fixed 10 min later. Arrows point to vesicles associated with different multivesicular bodies, as they seem to be forming through the wrapping of endoplasmic reticulum. The arrowhead points to a multivesicular body containing different types of vesicles without a noticeable external layer of membranes. Clear vesicles (cv) and dense core vesicles (dcv) inside the multivesicular bodies can also be seen. (B) Multivesicular body surrounded by a radial array of dense core vesicles, endoplasmic reticulum (arrowheads), microtubules (mt), and mitochondria (m). Asterisk marks a bundle of microtubules attached to the plasma membrane through a hemi-desmosome. Stimulation was as in (A). (C) Formation of a multivesicular body (arrow) with microtubule coiling (arrowhead). (D) A multivesicular body after the neuron was stimulated in the presence of extracellular peroxidase, displaying gold marks, and containing clear and dense core vesicles (dcv). Arrowheads point to gold particles inside multivesicular bodies and small arrows point to gold particles on top of vesicles and membrane corpses, maybe produced upon endocytosis. Scale bar = 1 μm.
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Figure 6: Formation of multivesicular bodies after electrical stimulation. (A) Several multivesicular bodies near the plasma membrane with dense core vesicles around. The neuron was stimulated at 20 Hz and fixed 10 min later. Arrows point to vesicles associated with different multivesicular bodies, as they seem to be forming through the wrapping of endoplasmic reticulum. The arrowhead points to a multivesicular body containing different types of vesicles without a noticeable external layer of membranes. Clear vesicles (cv) and dense core vesicles (dcv) inside the multivesicular bodies can also be seen. (B) Multivesicular body surrounded by a radial array of dense core vesicles, endoplasmic reticulum (arrowheads), microtubules (mt), and mitochondria (m). Asterisk marks a bundle of microtubules attached to the plasma membrane through a hemi-desmosome. Stimulation was as in (A). (C) Formation of a multivesicular body (arrow) with microtubule coiling (arrowhead). (D) A multivesicular body after the neuron was stimulated in the presence of extracellular peroxidase, displaying gold marks, and containing clear and dense core vesicles (dcv). Arrowheads point to gold particles inside multivesicular bodies and small arrows point to gold particles on top of vesicles and membrane corpses, maybe produced upon endocytosis. Scale bar = 1 μm.

Mentions: The formation of multivesicular bodies has been studied in a variety of cell types (for review, see Piper and Katzmann, 2007). Our electron micrographs fit with the general scheme by indicating that multivesicular bodies form when membranous structures, including vesicles, are surrounded by microtubules and endoplasmic reticulum. In Figure 1 it was shown that after 20 Hz stimulation multivesicular bodies appear within the vesicle clusters near the plasma membrane. Figure 6 shows several examples of multivesicular bodies in the periphery of vesicle clusters from neurons fixed 10 min after electrical stimulation at 20 Hz. Individual or aligned dense core vesicles are associated to multivesicular bodies (see also Figure 1), at parts in which they still seem to be opened. Some multivesicular bodies contain dense core and clear vesicles (Figure 6A). As shown above (Figure 5), multivesicular bodies formed by neurons stimulated in the presence of external peroxidase and their surrounding vesicles also displayed electron dense gold particles. This also occurred in multivesicular bodies in the apparent process of formation shown in Figures 6C,D and is consistent with the early observations that multivesicular bodies are capable to accumulate peroxidase (Rosenbluth and Wissig, 1964; Holtzman and Peterson, 1969; Birks et al., 1972; Weldon, 1975).


Cycling of dense core vesicles involved in somatic exocytosis of serotonin by leech neurons.

Trueta C, Kuffler DP, De-Miguel FF - Front Physiol (2012)

Formation of multivesicular bodies after electrical stimulation. (A) Several multivesicular bodies near the plasma membrane with dense core vesicles around. The neuron was stimulated at 20 Hz and fixed 10 min later. Arrows point to vesicles associated with different multivesicular bodies, as they seem to be forming through the wrapping of endoplasmic reticulum. The arrowhead points to a multivesicular body containing different types of vesicles without a noticeable external layer of membranes. Clear vesicles (cv) and dense core vesicles (dcv) inside the multivesicular bodies can also be seen. (B) Multivesicular body surrounded by a radial array of dense core vesicles, endoplasmic reticulum (arrowheads), microtubules (mt), and mitochondria (m). Asterisk marks a bundle of microtubules attached to the plasma membrane through a hemi-desmosome. Stimulation was as in (A). (C) Formation of a multivesicular body (arrow) with microtubule coiling (arrowhead). (D) A multivesicular body after the neuron was stimulated in the presence of extracellular peroxidase, displaying gold marks, and containing clear and dense core vesicles (dcv). Arrowheads point to gold particles inside multivesicular bodies and small arrows point to gold particles on top of vesicles and membrane corpses, maybe produced upon endocytosis. Scale bar = 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Formation of multivesicular bodies after electrical stimulation. (A) Several multivesicular bodies near the plasma membrane with dense core vesicles around. The neuron was stimulated at 20 Hz and fixed 10 min later. Arrows point to vesicles associated with different multivesicular bodies, as they seem to be forming through the wrapping of endoplasmic reticulum. The arrowhead points to a multivesicular body containing different types of vesicles without a noticeable external layer of membranes. Clear vesicles (cv) and dense core vesicles (dcv) inside the multivesicular bodies can also be seen. (B) Multivesicular body surrounded by a radial array of dense core vesicles, endoplasmic reticulum (arrowheads), microtubules (mt), and mitochondria (m). Asterisk marks a bundle of microtubules attached to the plasma membrane through a hemi-desmosome. Stimulation was as in (A). (C) Formation of a multivesicular body (arrow) with microtubule coiling (arrowhead). (D) A multivesicular body after the neuron was stimulated in the presence of extracellular peroxidase, displaying gold marks, and containing clear and dense core vesicles (dcv). Arrowheads point to gold particles inside multivesicular bodies and small arrows point to gold particles on top of vesicles and membrane corpses, maybe produced upon endocytosis. Scale bar = 1 μm.
Mentions: The formation of multivesicular bodies has been studied in a variety of cell types (for review, see Piper and Katzmann, 2007). Our electron micrographs fit with the general scheme by indicating that multivesicular bodies form when membranous structures, including vesicles, are surrounded by microtubules and endoplasmic reticulum. In Figure 1 it was shown that after 20 Hz stimulation multivesicular bodies appear within the vesicle clusters near the plasma membrane. Figure 6 shows several examples of multivesicular bodies in the periphery of vesicle clusters from neurons fixed 10 min after electrical stimulation at 20 Hz. Individual or aligned dense core vesicles are associated to multivesicular bodies (see also Figure 1), at parts in which they still seem to be opened. Some multivesicular bodies contain dense core and clear vesicles (Figure 6A). As shown above (Figure 5), multivesicular bodies formed by neurons stimulated in the presence of external peroxidase and their surrounding vesicles also displayed electron dense gold particles. This also occurred in multivesicular bodies in the apparent process of formation shown in Figures 6C,D and is consistent with the early observations that multivesicular bodies are capable to accumulate peroxidase (Rosenbluth and Wissig, 1964; Holtzman and Peterson, 1969; Birks et al., 1972; Weldon, 1975).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto Nacional de Psiquiatría "Ramón de la Fuente Muñiz," México D. F., México.

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.

No MeSH data available.


Related in: MedlinePlus