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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

Migration of fluorescent FM1-43 spots. Images taken from the site of contact with the dish bottom (left) and at 5 and 20 μm depth. Each area was imaged at 12, 36, and 48 h after depolarization of cultured neurons in bathing solution containing 40 mM potassium and FM1-43. Note the accumulation of spots to more internal regions of the soma at the 20-μm focal plane. Scale bar = 20 μm.
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Figure 7: Migration of fluorescent FM1-43 spots. Images taken from the site of contact with the dish bottom (left) and at 5 and 20 μm depth. Each area was imaged at 12, 36, and 48 h after depolarization of cultured neurons in bathing solution containing 40 mM potassium and FM1-43. Note the accumulation of spots to more internal regions of the soma at the 20-μm focal plane. Scale bar = 20 μm.

Mentions: The destiny of the material contained inside multivesicular bodies remains a matter of debate (for review, see Von Bartheld and Altick, 2011), however, that multivesicular bodies in our electromicrographs appear in the perinuclear region suggest they have been transported after their peripheral formation. To explore if after electrical stimulation vesicles and or multivesicular bodies become transported toward the plasma region, FM1-43 fluorescent spots of four neurons that had been stimulated at 20 Hz were imaged in the same focal planes 12, 36, and 48 h after stimulation (Figure 7). At the end of the experiments the neurons were known to be healthy because they had good resting potentials and the capacity to produce normal action potentials. As shown in Figure 7, at all times tested, fluorescence remained restricted to small spots, and the label gradually appeared in more central parts of the soma, while there was a decrease in the number of peripheral spots. The transport pathway (as exemplified by the sequence of images made at a depth of 20 μm from the plane of contact of the cell with the culture dish), produced a large concentration of spots at the center of the soma. This suggests that the mobilization of the spots did not occur randomly but followed well-organized patterns.


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

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

Migration of fluorescent FM1-43 spots. Images taken from the site of contact with the dish bottom (left) and at 5 and 20 μm depth. Each area was imaged at 12, 36, and 48 h after depolarization of cultured neurons in bathing solution containing 40 mM potassium and FM1-43. Note the accumulation of spots to more internal regions of the soma at the 20-μm focal plane. Scale bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Migration of fluorescent FM1-43 spots. Images taken from the site of contact with the dish bottom (left) and at 5 and 20 μm depth. Each area was imaged at 12, 36, and 48 h after depolarization of cultured neurons in bathing solution containing 40 mM potassium and FM1-43. Note the accumulation of spots to more internal regions of the soma at the 20-μm focal plane. Scale bar = 20 μm.
Mentions: The destiny of the material contained inside multivesicular bodies remains a matter of debate (for review, see Von Bartheld and Altick, 2011), however, that multivesicular bodies in our electromicrographs appear in the perinuclear region suggest they have been transported after their peripheral formation. To explore if after electrical stimulation vesicles and or multivesicular bodies become transported toward the plasma region, FM1-43 fluorescent spots of four neurons that had been stimulated at 20 Hz were imaged in the same focal planes 12, 36, and 48 h after stimulation (Figure 7). At the end of the experiments the neurons were known to be healthy because they had good resting potentials and the capacity to produce normal action potentials. As shown in Figure 7, at all times tested, fluorescence remained restricted to small spots, and the label gradually appeared in more central parts of the soma, while there was a decrease in the number of peripheral spots. The transport pathway (as exemplified by the sequence of images made at a depth of 20 μm from the plane of contact of the cell with the culture dish), produced a large concentration of spots at the center of the soma. This suggests that the mobilization of the spots did not occur randomly but followed well-organized patterns.

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