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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

Endocytosis of peroxidase following electrical stimulation. (A) Vesicle cluster in a neuron fixed during stimulation in the presence of external peroxidase. Large multivesicular bodies (mvb) are also present. Peroxidase was detected by a polyclonal antibody coupled to 10 nm gold particles. The black electron dense spots are associated with vesicles in the clusters located near the plasma membrane (pm), and labeling was considerably lower outside of the cluster. However, multivesicular bodies (arrows) and membranes of endoplasmic reticulum (arrowheads) also displayed gold particles. Scale = 1.0 μm. (B) Higher magnification of a vesicle cluster with electron dense spots accumulated over the vesicles. Scale = 200 nm. (C) Cluster with dense core vesicles and multivesicular bodies bearing gold particles. Scale = 1 μm. (D) Higher magnification of a multivesicular body that accumulated gold particles. Scale = 500 nm.
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Figure 5: Endocytosis of peroxidase following electrical stimulation. (A) Vesicle cluster in a neuron fixed during stimulation in the presence of external peroxidase. Large multivesicular bodies (mvb) are also present. Peroxidase was detected by a polyclonal antibody coupled to 10 nm gold particles. The black electron dense spots are associated with vesicles in the clusters located near the plasma membrane (pm), and labeling was considerably lower outside of the cluster. However, multivesicular bodies (arrows) and membranes of endoplasmic reticulum (arrowheads) also displayed gold particles. Scale = 1.0 μm. (B) Higher magnification of a vesicle cluster with electron dense spots accumulated over the vesicles. Scale = 200 nm. (C) Cluster with dense core vesicles and multivesicular bodies bearing gold particles. Scale = 1 μm. (D) Higher magnification of a multivesicular body that accumulated gold particles. Scale = 500 nm.

Mentions: As shown in Figure 4, neurons fixed during 20 Hz stimulation had some dense core vesicles with omega-shaped structures, suggesting exo/endocytosis. In addition, adjacent to the release sites we found clear vesicles and cisternae, thus suggesting that these structures were formed also as result of endocytosis. To explore if dense core vesicles also underwent endocytosis, the extracellular fluid was added with peroxidase before stimulation and peroxidase was detected by colloidal gold-coupled antibodies. As shown in Figure 5, multiple dense core vesicles became labeled, confirming endocytosis of full dense core vesicles and also supporting that large numbers of vesicles in the cluster become fused upon electrical stimulation.


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

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

Endocytosis of peroxidase following electrical stimulation. (A) Vesicle cluster in a neuron fixed during stimulation in the presence of external peroxidase. Large multivesicular bodies (mvb) are also present. Peroxidase was detected by a polyclonal antibody coupled to 10 nm gold particles. The black electron dense spots are associated with vesicles in the clusters located near the plasma membrane (pm), and labeling was considerably lower outside of the cluster. However, multivesicular bodies (arrows) and membranes of endoplasmic reticulum (arrowheads) also displayed gold particles. Scale = 1.0 μm. (B) Higher magnification of a vesicle cluster with electron dense spots accumulated over the vesicles. Scale = 200 nm. (C) Cluster with dense core vesicles and multivesicular bodies bearing gold particles. Scale = 1 μm. (D) Higher magnification of a multivesicular body that accumulated gold particles. Scale = 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Endocytosis of peroxidase following electrical stimulation. (A) Vesicle cluster in a neuron fixed during stimulation in the presence of external peroxidase. Large multivesicular bodies (mvb) are also present. Peroxidase was detected by a polyclonal antibody coupled to 10 nm gold particles. The black electron dense spots are associated with vesicles in the clusters located near the plasma membrane (pm), and labeling was considerably lower outside of the cluster. However, multivesicular bodies (arrows) and membranes of endoplasmic reticulum (arrowheads) also displayed gold particles. Scale = 1.0 μm. (B) Higher magnification of a vesicle cluster with electron dense spots accumulated over the vesicles. Scale = 200 nm. (C) Cluster with dense core vesicles and multivesicular bodies bearing gold particles. Scale = 1 μm. (D) Higher magnification of a multivesicular body that accumulated gold particles. Scale = 500 nm.
Mentions: As shown in Figure 4, neurons fixed during 20 Hz stimulation had some dense core vesicles with omega-shaped structures, suggesting exo/endocytosis. In addition, adjacent to the release sites we found clear vesicles and cisternae, thus suggesting that these structures were formed also as result of endocytosis. To explore if dense core vesicles also underwent endocytosis, the extracellular fluid was added with peroxidase before stimulation and peroxidase was detected by colloidal gold-coupled antibodies. As shown in Figure 5, multiple dense core vesicles became labeled, confirming endocytosis of full dense core vesicles and also supporting that large numbers of vesicles in the cluster become fused upon electrical stimulation.

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