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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

Ultrastructure of the soma of Retzius neurons in relation to somatic exocytosis. (A) Low magnification electron micrograph of a neuron that had been stimulated at 1 Hz, a frequency that fails to evoke somatic serotonin exocytosis. Vesicle clusters located around the nucleus (n) are indicated with arrows. Clusters in the region midway between the nucleus and the plasma membrane are indicated as vc. Vesicle clusters and multivesicular bodies (mvb) are absent in the peripheral cytoplasmic area next to the plasma membrane. Some multivesicular bodies are interspersed within the vesicle clusters. Golgi apparatuses (go) are also more concentrated around the nucleus. Mitochondria (m) are near vesicles and endoplasmic reticulum (er) are located near the plasma membrane (pm). The arrowhead points to microtubule bundle arriving at the plasma membrane. The neuron is surrounded by layers of the giant glial cell (g). Scale = 2 μm. (B) Electron micrograph of a neuron stimulated at 20 Hz. A majority of the vesicle clusters are apposed to the plasma membrane (arrowheads). Multivesicular bodies lie within peripheral vesicle clusters and mitochondria can be seen around them (small arrows). Note that even after stimulation, vesicle clusters remained near the nucleus (n). Scale = 2 μm. (C) Magnified electron micrograph of a peripherally located vesicle cluster in a neuron that had been stimulated at 1 Hz. Vesicle clusters are located away from the plasma membrane and the intermediate space contains endoplasmic reticulum. A bundle of microtubules (mt) extends from the vesicle cluster vesicles to a hemi-desmosome (arrow) apposed to an invagination of the plasma membrane (arrowheads). Glial cell fingers characteristic of these structures penetrate into the somatic invagination. Scale = 1 μm. (D) Magnified electron micrograph of a neuron stimulated at 20 Hz. A vesicle cluster is apposed to the plasma membrane near an invagination (arrowheads). Vesicle rows formed radial arrays with multivesicular bodies. Scale = 1 μm. (E) Graph of the percentage of vesicle clusters apposed to the plasma membrane in neurons stimulated at frequencies of 1 vs. 20 Hz. Individual clusters were identified and counted from digital images low magnification and scanned at high resolution such as those in (A) and (B). As seen in (C,D), this vesicle cluster counting could be done easily since the clusters are self-delimited and they only contain vesicles and multivesicular bodies.
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Figure 1: Ultrastructure of the soma of Retzius neurons in relation to somatic exocytosis. (A) Low magnification electron micrograph of a neuron that had been stimulated at 1 Hz, a frequency that fails to evoke somatic serotonin exocytosis. Vesicle clusters located around the nucleus (n) are indicated with arrows. Clusters in the region midway between the nucleus and the plasma membrane are indicated as vc. Vesicle clusters and multivesicular bodies (mvb) are absent in the peripheral cytoplasmic area next to the plasma membrane. Some multivesicular bodies are interspersed within the vesicle clusters. Golgi apparatuses (go) are also more concentrated around the nucleus. Mitochondria (m) are near vesicles and endoplasmic reticulum (er) are located near the plasma membrane (pm). The arrowhead points to microtubule bundle arriving at the plasma membrane. The neuron is surrounded by layers of the giant glial cell (g). Scale = 2 μm. (B) Electron micrograph of a neuron stimulated at 20 Hz. A majority of the vesicle clusters are apposed to the plasma membrane (arrowheads). Multivesicular bodies lie within peripheral vesicle clusters and mitochondria can be seen around them (small arrows). Note that even after stimulation, vesicle clusters remained near the nucleus (n). Scale = 2 μm. (C) Magnified electron micrograph of a peripherally located vesicle cluster in a neuron that had been stimulated at 1 Hz. Vesicle clusters are located away from the plasma membrane and the intermediate space contains endoplasmic reticulum. A bundle of microtubules (mt) extends from the vesicle cluster vesicles to a hemi-desmosome (arrow) apposed to an invagination of the plasma membrane (arrowheads). Glial cell fingers characteristic of these structures penetrate into the somatic invagination. Scale = 1 μm. (D) Magnified electron micrograph of a neuron stimulated at 20 Hz. A vesicle cluster is apposed to the plasma membrane near an invagination (arrowheads). Vesicle rows formed radial arrays with multivesicular bodies. Scale = 1 μm. (E) Graph of the percentage of vesicle clusters apposed to the plasma membrane in neurons stimulated at frequencies of 1 vs. 20 Hz. Individual clusters were identified and counted from digital images low magnification and scanned at high resolution such as those in (A) and (B). As seen in (C,D), this vesicle cluster counting could be done easily since the clusters are self-delimited and they only contain vesicles and multivesicular bodies.

Mentions: Ultra thin sections from five somata that had been stimulated in the ganglion with trains at 1 Hz, a frequency that does not evoke somatic exocytosis, contained dense core vesicle clusters distributed in two main areas (Figures 1 and 2). A perinuclear region contained vesicles together with Golgi apparatuses, mitochondria, and endoplasmic reticulum. Small clear vesicles were located next to Golgi apparatuses and some small vesicles displayed dense cores. At larger distances from Golgi, vesicles were large and had dense cores. Multivesicular bodies were mainly located adjacent to the perinuclear vesicle clusters and were characterized by their bag shapes and membrane multilayers filled with dense core and clear vesicles (Figure 1, see also below).


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

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

Ultrastructure of the soma of Retzius neurons in relation to somatic exocytosis. (A) Low magnification electron micrograph of a neuron that had been stimulated at 1 Hz, a frequency that fails to evoke somatic serotonin exocytosis. Vesicle clusters located around the nucleus (n) are indicated with arrows. Clusters in the region midway between the nucleus and the plasma membrane are indicated as vc. Vesicle clusters and multivesicular bodies (mvb) are absent in the peripheral cytoplasmic area next to the plasma membrane. Some multivesicular bodies are interspersed within the vesicle clusters. Golgi apparatuses (go) are also more concentrated around the nucleus. Mitochondria (m) are near vesicles and endoplasmic reticulum (er) are located near the plasma membrane (pm). The arrowhead points to microtubule bundle arriving at the plasma membrane. The neuron is surrounded by layers of the giant glial cell (g). Scale = 2 μm. (B) Electron micrograph of a neuron stimulated at 20 Hz. A majority of the vesicle clusters are apposed to the plasma membrane (arrowheads). Multivesicular bodies lie within peripheral vesicle clusters and mitochondria can be seen around them (small arrows). Note that even after stimulation, vesicle clusters remained near the nucleus (n). Scale = 2 μm. (C) Magnified electron micrograph of a peripherally located vesicle cluster in a neuron that had been stimulated at 1 Hz. Vesicle clusters are located away from the plasma membrane and the intermediate space contains endoplasmic reticulum. A bundle of microtubules (mt) extends from the vesicle cluster vesicles to a hemi-desmosome (arrow) apposed to an invagination of the plasma membrane (arrowheads). Glial cell fingers characteristic of these structures penetrate into the somatic invagination. Scale = 1 μm. (D) Magnified electron micrograph of a neuron stimulated at 20 Hz. A vesicle cluster is apposed to the plasma membrane near an invagination (arrowheads). Vesicle rows formed radial arrays with multivesicular bodies. Scale = 1 μm. (E) Graph of the percentage of vesicle clusters apposed to the plasma membrane in neurons stimulated at frequencies of 1 vs. 20 Hz. Individual clusters were identified and counted from digital images low magnification and scanned at high resolution such as those in (A) and (B). As seen in (C,D), this vesicle cluster counting could be done easily since the clusters are self-delimited and they only contain vesicles and multivesicular bodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368391&req=5

Figure 1: Ultrastructure of the soma of Retzius neurons in relation to somatic exocytosis. (A) Low magnification electron micrograph of a neuron that had been stimulated at 1 Hz, a frequency that fails to evoke somatic serotonin exocytosis. Vesicle clusters located around the nucleus (n) are indicated with arrows. Clusters in the region midway between the nucleus and the plasma membrane are indicated as vc. Vesicle clusters and multivesicular bodies (mvb) are absent in the peripheral cytoplasmic area next to the plasma membrane. Some multivesicular bodies are interspersed within the vesicle clusters. Golgi apparatuses (go) are also more concentrated around the nucleus. Mitochondria (m) are near vesicles and endoplasmic reticulum (er) are located near the plasma membrane (pm). The arrowhead points to microtubule bundle arriving at the plasma membrane. The neuron is surrounded by layers of the giant glial cell (g). Scale = 2 μm. (B) Electron micrograph of a neuron stimulated at 20 Hz. A majority of the vesicle clusters are apposed to the plasma membrane (arrowheads). Multivesicular bodies lie within peripheral vesicle clusters and mitochondria can be seen around them (small arrows). Note that even after stimulation, vesicle clusters remained near the nucleus (n). Scale = 2 μm. (C) Magnified electron micrograph of a peripherally located vesicle cluster in a neuron that had been stimulated at 1 Hz. Vesicle clusters are located away from the plasma membrane and the intermediate space contains endoplasmic reticulum. A bundle of microtubules (mt) extends from the vesicle cluster vesicles to a hemi-desmosome (arrow) apposed to an invagination of the plasma membrane (arrowheads). Glial cell fingers characteristic of these structures penetrate into the somatic invagination. Scale = 1 μm. (D) Magnified electron micrograph of a neuron stimulated at 20 Hz. A vesicle cluster is apposed to the plasma membrane near an invagination (arrowheads). Vesicle rows formed radial arrays with multivesicular bodies. Scale = 1 μm. (E) Graph of the percentage of vesicle clusters apposed to the plasma membrane in neurons stimulated at frequencies of 1 vs. 20 Hz. Individual clusters were identified and counted from digital images low magnification and scanned at high resolution such as those in (A) and (B). As seen in (C,D), this vesicle cluster counting could be done easily since the clusters are self-delimited and they only contain vesicles and multivesicular bodies.
Mentions: Ultra thin sections from five somata that had been stimulated in the ganglion with trains at 1 Hz, a frequency that does not evoke somatic exocytosis, contained dense core vesicle clusters distributed in two main areas (Figures 1 and 2). A perinuclear region contained vesicles together with Golgi apparatuses, mitochondria, and endoplasmic reticulum. Small clear vesicles were located next to Golgi apparatuses and some small vesicles displayed dense cores. At larger distances from Golgi, vesicles were large and had dense cores. Multivesicular bodies were mainly located adjacent to the perinuclear vesicle clusters and were characterized by their bag shapes and membrane multilayers filled with dense core and clear vesicles (Figure 1, see also below).

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