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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

FM1-43 staining upon subsequent depolarizations. (A) Equatorial image of the soma of a cultured neuron that had been depolarized with 40 mM potassium in the presence of FM1-43 in the external solution. Ten minutes after depolarization the dye was washed out from the fluid and neurons displayed their characteristic spotted pattern. Labeled spots are marked with the small arrowheads and small gray arrow. The asterisk marks an unspecific spot that was used as a reference for the following images in the series. (B) The FM1-43 fluorescence was partially photobleached to allow the testing of whether spots that were originally stained became restained. (C) After a second depolarization in the presence of fresh external FM1-43, some of the spots were restained (large arrowheads), some were not restained but could still be detected in their previous position (gray small arrows), others disappeared completely (small arrowheads), and others were newly formed (white arrows). Scale bar = 20 μm. (D) Graph of the distribution of the total number and percentage of fluorescent spots in each category described above.
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Figure 3: FM1-43 staining upon subsequent depolarizations. (A) Equatorial image of the soma of a cultured neuron that had been depolarized with 40 mM potassium in the presence of FM1-43 in the external solution. Ten minutes after depolarization the dye was washed out from the fluid and neurons displayed their characteristic spotted pattern. Labeled spots are marked with the small arrowheads and small gray arrow. The asterisk marks an unspecific spot that was used as a reference for the following images in the series. (B) The FM1-43 fluorescence was partially photobleached to allow the testing of whether spots that were originally stained became restained. (C) After a second depolarization in the presence of fresh external FM1-43, some of the spots were restained (large arrowheads), some were not restained but could still be detected in their previous position (gray small arrows), others disappeared completely (small arrowheads), and others were newly formed (white arrows). Scale bar = 20 μm. (D) Graph of the distribution of the total number and percentage of fluorescent spots in each category described above.

Mentions: Electrical stimulation of cultured Retzius neurons with a 20-Hz train in the presence of FM1-43 in the external fluid produces a spotted fluorescent pattern, in which each fluorescent spot is due to exocytosis followed by endocytosis from a dense core vesicle cluster (Trueta et al., 2003). The fact that the FM1-43 fluorescence in Retzius neurons is diminished as a result of prolonged depolarization by high extracellular potassium solution has suggested that vesicles in the clusters may undergo a second round of vesicle fusion (Trueta et al., 2003). However, a plausible alternative is that the fluorescence is reduced as vesicles become transported back to more central parts of the neuronal soma. In addition, the finding that electrical stimulation at increasing frequencies increases the number of fluorescent spots suggests that new clusters of vesicles are transported to and fuse at different release sites (Trueta et al., 2003). These possibilities were explored in four neurons in which the fluorescent FM1-43 spots formed upon high potassium depolarization were partially bleached by continuous light exposure followed by additional high potassium depolarization in the presence of fresh FM1-43. When counting the number of fluorescent spots in the equatorial plane of the soma in the same focal plane of the neurons before and after the second depolarization, we found four complementary effects (Figure 3): (1) 41% of the spots became restained, suggesting either, the arrival of a second vesicle cluster, maybe from the perinuclear region of the cell, or a second round of fusion of the same vesicles. However the presence of two pools of vesicle clusters and their correlation with the double sigmoidal kinetics of the FM1-43 fluorescence (P. Noguez, C. Bustos, and F. F. De-Miguel, in preparation) support the idea of the arrival and fusion of a second vesicle cluster over the reuse of the same dense core vesicles. The following three evidences provide further support to this possibility. (2) 19% of the spots that were bleached were not restained in response to the second depolarization, although they stayed at the same position. (3) 22% percent of the prestained spots disappeared, suggesting their intracellular transport out of the focal plane. (4) New spots were formed in places that were previously not stained, accounting for 18% of the total final spots. This was consistent with the recruitment of new vesicle clusters that were fused at new release sites, as it happens with increases in the frequency of stimulation (see Figure 4 in Trueta et al., 2003) and suggest the slow vesicle recycling and the transport of fused vesicles back to internal cell sites.


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

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

FM1-43 staining upon subsequent depolarizations. (A) Equatorial image of the soma of a cultured neuron that had been depolarized with 40 mM potassium in the presence of FM1-43 in the external solution. Ten minutes after depolarization the dye was washed out from the fluid and neurons displayed their characteristic spotted pattern. Labeled spots are marked with the small arrowheads and small gray arrow. The asterisk marks an unspecific spot that was used as a reference for the following images in the series. (B) The FM1-43 fluorescence was partially photobleached to allow the testing of whether spots that were originally stained became restained. (C) After a second depolarization in the presence of fresh external FM1-43, some of the spots were restained (large arrowheads), some were not restained but could still be detected in their previous position (gray small arrows), others disappeared completely (small arrowheads), and others were newly formed (white arrows). Scale bar = 20 μm. (D) Graph of the distribution of the total number and percentage of fluorescent spots in each category described above.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: FM1-43 staining upon subsequent depolarizations. (A) Equatorial image of the soma of a cultured neuron that had been depolarized with 40 mM potassium in the presence of FM1-43 in the external solution. Ten minutes after depolarization the dye was washed out from the fluid and neurons displayed their characteristic spotted pattern. Labeled spots are marked with the small arrowheads and small gray arrow. The asterisk marks an unspecific spot that was used as a reference for the following images in the series. (B) The FM1-43 fluorescence was partially photobleached to allow the testing of whether spots that were originally stained became restained. (C) After a second depolarization in the presence of fresh external FM1-43, some of the spots were restained (large arrowheads), some were not restained but could still be detected in their previous position (gray small arrows), others disappeared completely (small arrowheads), and others were newly formed (white arrows). Scale bar = 20 μm. (D) Graph of the distribution of the total number and percentage of fluorescent spots in each category described above.
Mentions: Electrical stimulation of cultured Retzius neurons with a 20-Hz train in the presence of FM1-43 in the external fluid produces a spotted fluorescent pattern, in which each fluorescent spot is due to exocytosis followed by endocytosis from a dense core vesicle cluster (Trueta et al., 2003). The fact that the FM1-43 fluorescence in Retzius neurons is diminished as a result of prolonged depolarization by high extracellular potassium solution has suggested that vesicles in the clusters may undergo a second round of vesicle fusion (Trueta et al., 2003). However, a plausible alternative is that the fluorescence is reduced as vesicles become transported back to more central parts of the neuronal soma. In addition, the finding that electrical stimulation at increasing frequencies increases the number of fluorescent spots suggests that new clusters of vesicles are transported to and fuse at different release sites (Trueta et al., 2003). These possibilities were explored in four neurons in which the fluorescent FM1-43 spots formed upon high potassium depolarization were partially bleached by continuous light exposure followed by additional high potassium depolarization in the presence of fresh FM1-43. When counting the number of fluorescent spots in the equatorial plane of the soma in the same focal plane of the neurons before and after the second depolarization, we found four complementary effects (Figure 3): (1) 41% of the spots became restained, suggesting either, the arrival of a second vesicle cluster, maybe from the perinuclear region of the cell, or a second round of fusion of the same vesicles. However the presence of two pools of vesicle clusters and their correlation with the double sigmoidal kinetics of the FM1-43 fluorescence (P. Noguez, C. Bustos, and F. F. De-Miguel, in preparation) support the idea of the arrival and fusion of a second vesicle cluster over the reuse of the same dense core vesicles. The following three evidences provide further support to this possibility. (2) 19% of the spots that were bleached were not restained in response to the second depolarization, although they stayed at the same position. (3) 22% percent of the prestained spots disappeared, suggesting their intracellular transport out of the focal plane. (4) New spots were formed in places that were previously not stained, accounting for 18% of the total final spots. This was consistent with the recruitment of new vesicle clusters that were fused at new release sites, as it happens with increases in the frequency of stimulation (see Figure 4 in Trueta et al., 2003) and suggest the slow vesicle recycling and the transport of fused vesicles back to internal cell sites.

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