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

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.

Keywords: dense core vesicle; endocytosis; extrasynaptic exocytosis; leech; serotonin; somatic exocytosis; vesicle cycle; volume transmission.

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.

Figure 2

Ultrastructural relationships of perinuclear vesicles with the nucleus (n), Golgi (go), and vesicle clusters (vc). (A) Low magnification image of the soma of a neuron that had been stimulated with 20 Hz trains in a bathing solution in which magnesium was substituted for calcium. Under these conditions, electrical activity fails to evoke somatic exocytosis and vesicle mobilization, and as can be seen, the areas immediate to the plasma membrane (pm) are devoid of vesicle clusters. Scale = 2.0 μm. (B) Golgi apparatus in the perinuclear area surrounded by clear and dense core vesicles. On the right, there are rows of small vesicles (arrows), some of which are clear while others have electron dense material (small arrowheads). More distantly there are small vesicles with dense cores continued by vesicles with large dense cores (large arrowheads). A fragment of a multivesicular body (mvb) can also be seen. On the top left electron dense vesicles align with the layers of the Golgi apparatus (large arrowhead). (C) Higher magnification image of vesicles adjacent to the nucleus, in the vicinity of mitochondria (m) and Golgi. (D) Clear and dense core vesicles are adjacent to the nuclear membrane (nm). A filament projecting from the vesicle toward the nuclear membrane is marked by the arrow and amplified in the inset. Scale bars for (B–D) = 500 nm.

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.

Figure 4

Dense core vesicle fusion after electrical stimulation (arrow). To the right of the fusing vesicle are small clear vesicles and cisternae (arrowheads) atypical of somatic clusters before electrical stimulation. This suggests vesicle formation as part of the endocytic pathway. Glial cell fingers (g) can be seen outside the Retzius cell. Scale bar = 250 nm.

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.

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.

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.

Figure 8

Perinuclear multivesicular bodies. (A) Multivesicular bodies (mvb) coexisting with Golgi apparatus (go). The multivesicular body on the right top contains mostly clear vesicles and is in association with endoplasmic reticulum (er) and mitochondria (m). The one in the right bottom has a membrane discontinuity (arrowhead). On the left side of the image, small vesicles near the Golgi endings (asterisks) display electrodense material inside. (B) Contents of the multivesicular bodies are in association with the nuclear membrane and near Golgi apparatus (n = nucleus). The arrowhead points to structures possibly binding the multivesicular body fragment with the nuclear membrane. Scale bars = 500 nm.