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Ultrastructural characteristics of glomus cells in the external carotid artery during larval development and metamorphosis in bullfrogs Rana catesbeiana.

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THE ANATOMICAL RECORD 233:461-466 (1992)
Ultrastructural Characteristics of Glomus Cells in the External
Carotid Artery During Larval Development and Metamorphosis in
Bullfrogs, R a m catesbeiana
Department of Anatomy, Yokohama City University School of Medicine,
Yokohama 236, Japan
Electron microscopic observations of the external carotid artery
in the larvae of the bullfrog, Rana catesbeiana, showed that glomus cells are
present in the subendothelial stroma of the septum between the expanded region
of the external carotid artery and the carotid arch. There were some differences in
the ultrastructure of the glomus cells a t each stage of larval development. At the
early stages (stages I, 111, V, XI, most glomus cells were isolated and free from the
covering of a supporting cell. The cytoplasm of the glomus cells contained fewer
dense-cored vesicles. No synaptic junctions were observed. At the middle stages
(stages XV, XX, XXI), some glomus cells showed a tendency to form small clusters.
Between adjacent cells in a cluster, gap junctions were often observed. The number
of dense-cored vesicles increased remarkably. Intimate apposition of the glomus
and smooth muscle cells (g-s connection) was also observed. Nerve terminals containing clear vesicles were observed in synaptic contact with glomus cells at this
At the metamorphic climax (stages XXII-XXV), in addition to g-s connections,
the glomus cells made intimate apposition to the cells around the glomus cells. The
afferent synapses described in other amphibians were not encountered in this
These findings suggest that the glomus cells at the early stages of development
are nonfunctional, the vascular regulation via the g-s connection starts at the
middle stages, and the chemoreception starts after metamorphosis.
0 1992 Wiley-Liss, Inc.
In adult amphibians, a pair of carotid labyrinths is
situated at the point where the common carotid artery
bifurcates into the internal and external carotid arteries (Adams, 1958).The inside of the carotid labyrinth is
a complicated maze of vasculature (Kobayashi and Murakami, 1975; Toews et al., 1982; Kusakabe, 1990a). In
the intervascular stroma, there are glomus cells which
are characterized by a large number of dense-cored vesicles in their cytoplasm (Rogers, 1963; Ishii and
Oosaki, 1969; Kobayashi, 1971; Poullet-Krieger, 1973;
Ishii and Kusakabe, 1982; Kusakabe, 1990b, 1991a,b).
Arterial chemoreceptor, vascular regulatory, and endocrine functions have been proposed on the basis of the
ultrastructural features of the glomus cells. The
chemoreceptor and vascular regulatory functions have
been confirmed physiologically and pharmacologically
(Ishii et al., 1966; Kasakabe et al., 1987). Thus it appears that the amphibian carotid labyrinth is involved
in these multiple roles.
Recently the ontogenesis of the carotid labyrinth in
the bullfrog during larval development and metamorphosis was studied using vascular corrosion casting
and scanning electron microscopy (Kusakabe, 1991b).
The morphogenesis of the carotid labyrinth starts a t
the point where the carotid arch descends to the internal gills by making contact with the slightly expanded
external carotid artery. Through the early and middle
phases of the larval development, this contact region
does not show any maze-like structures. At stage XXII,
a primitive maze-like structure first appears, and this
expansion is completely surrounded by a simple vascular maze at stage XXIV. These findings suggested that
the incomplete carotid labyrinth immediately before
the completion of metamorphosis has a vascular regulatory function. Although this method is suitable for
the analysis of the three-dimensional fine vascular arrangement of the complicated vasculature, no histological information on the structure of the vascular wall
has been provided. The functions of the carotid labyrinth described above have been usually discussed in
relation to the ultrastructural characteristics of the
glomus cells in the vascular wall, but it is unclear
whether the glomus cells exist in the larval stages or
During the course of the ultrastructural observations
of the vascular expansion in the bullfrog larvae, I found
glomus cells in the septum between the external ca-
Received September 28, 1991; accepted December 30, 1991
1-3: Photomicrograph of a semithin cross section of the expan ed region of the external carotid artery at stage I11 (Fig. l),stage
XXII (Fig. 21, and stage XXV (Fig. 3).A slender septum with a few
perforations (arrows) lies between the carotid arch (ca) and the exter-
nal carotid artery (eca). At stage XXV, the vascular maze (mz) is
clearly seen. cch, central chamber. Toluidine blue stain. Bar = 100
rotid artery and the carotid arch. In the present study,
I describe the ultrastructural characteristics of the glomus cells in larval stages, and discuss their possible
function in relation to the morphogenesis of the carotid
acetate and lead citrate. The specimens were examined
with a transmission electron microscope.
In the sections stained with toluidine blue, through
the early and middle phases of the larval development
(Stages I-XXI) a slender septem, 30-50 pm in width,
Thirty-five larvae of bullfrogs, Rana catesbeiana, with some small perforations was seen between the
were used in this study. Tadpoles were kept in glass carotid arch and the expansion of the external carotid
tanks at 23-25°C. Stages during larval development artery (Fig. 1).At stages XXII and XXIII, the vascular
were referred to those in the normal development for wall near both bases of the septum began to branch off
Rana pipiens larvae described by Taylor and Kollros and lengthen to make a primitive maze-like structure
(1946). Larvae were sacrificed at the early larval phase (Fig. 2). At stages XXIV and XXV, a primitive maze
(stages I, 111, V, X), middle phase (stages XV, XX, XXI), enclosed the expansion, and the vascular maze took the
and metamorphic climax (stages XXII-XXV). At least form of the carotid labyrinth, although it was not so
three larvae of each stage were examined. After anes- complicated as in adults (Fig. 3). Light microscopically
thetization by immersion in a 1%aqueous solution of the following cells were distinguished in the septum
tricaine-methanesulfate (MS-222) for a few minutes, and the intervascular stroma: endothelial cells, perithe thoracic cavity was opened to expose the region of cytes, smooth muscle cells, fibroblasts, melanophores,
the vascular expansion. Through a thin nylon tube in- and oval cells which seemed to be glomus cells.
serted into the aortic trunk, both sides of the vascular
Electron microscopy confirmed the presence of gloexpansions were washed with heparinized (1IUlml) 0.1 mus cells at different stages of differentiation. At the
M cacodylate buffer, pH 7.3, and then perfused with early stages of the larval development (stages I, 111, V,
2.5% glutaraldehyde in the same buffer at a pressure of XI, most glomus cells were oval in shape and separated.
60 cm H,O for 5 minutes. Then the expansions of both They were free from the covering of supporting cell
sides were removed from the body and immersed in the cytoplasm, and the basal lamina closely followed the
same fixative for a n additional 3 hours at 4°C. After contour of their plasmalemma (Fig. 4). They had a
washing with buffer solution, the specimens were post- large nucleus with relatively low electron density. The
fixed in 1%osmium tetroxide buffered with 0.1 M ca- cytoplasm contained fewer granules, 50-60 nm in dicodylate for 1 hour a t 4°C. Following dehydration in ameter. Exocytosis of these was not observed a t the cell
ascending concentrations of ethanol, the specimens surface. The Golgi apparatus was poorly developed. At
were embedded in Epon-Araldite mixture. Sections of 1 stage 111, although a few nerve fibers were close to the
pm were stained with toluidine blue to identify the glomus cells (Fig. 5), no synaptic vesicles were found.
glomus cells by light microscopy. Ultrathin sections
Midway through the larval development (stages XV,
were serially cut, and stained with saturated uranyl XX, XXI), some glomus cells showed a tendency to form
small clusters (Fig. 6). A supporting cell which possessed a large nucleus with dense chromatin was
closely connected with the cluster, and its thin cytoplasm partially enveloped the cluster. Between adjacent glomus cells in a cluster, gap junctions were often
observed (Fig. 7). The number of dense-cored vesicles,
60-80 nm in diameter, increased remarkably, and the
coated pits indicating the final stage of exocytosis were
sometimes seen. A well-developed Golgi complex was
found in the perinuclear region. Many oval mitochondria, numerous free ribsomes, a relatively small
amount of rough endoplasmic reticulum, some lipid
droplets, and a few bundles of thin filaments were dispersed throughout the cytoplasm.
At stage XV, there were some glomus cells which
made close contact with the vascular smooth muscle
cells (g-s connection). In the g-s connection, the distance between the plasma membrane of the glomus cell
and that of the smooth muscle cell was about 10-20
nm. The basal lamina followed the contour of both glomus cells and smooth muscle cells. Synaptic junctions
with typical membrane thickenings were first observed
between some of the glomus cells and the nerve endings at stage XV (Fig. 8). These synapses were morphologically efferent because they were characterized
by the accumulation of many clear snyaptic vesicles,
40-60 nm in diameter.
At the metamorphic climax (stages XXII-XXV), the
intimate apposition of the glomus cells and the neighboring endothelial cells, pericytes, and the melanophores was frequently observed (Figs. 9,101 in addition
to the g-s connection. These cellular connections with
the glomus cells were not found a t the earlier stages,
with the exception of the supporting cells and the
smooth muscle cells. The frequency of g-s connections
was higher than in the previous phase, and a t some of
the g-s connections, desmosome-like membrane thickening and aggregation of dense-cored vesicles could be
seen on the glomus cell side (Fig. 11).At these contact
regions and on their circumference, all glomus cells
had no covering of supporting cells. At the contact with
melanophores, the processes of the glomus cell often
enclosed a part of the melanophore with many melanosomes, and exocytosis of melanosomes was also found
near the junction (Fig. 9). Few gap junctions were
found. At these stages, many ovoid, elliptical, or slender dense-cored vesicles were mingled with round vesicles in the cytoplasm of some glomus cells (Fig. 9).
These ultrastructural studies in the expanded region
of the external carotid artery in the bullfrog larvae
have shown that glomus cells can be found even as
early as the initial stages of the development. However, as would be expected, these cells differentiate in
cellular features, attaining the adult form by the middle phase of development. The glomus cells in the early
stages were in an immature state, and no synaptic
junctions were observed. It therefore appears that, at
the initial stages of development, the glomus cells are
Some glomus cells in the middle phases of the larval
development (stages XV, XX, XXI) showed ultrastructural similarity to those previously reported in some
adult amphibians (Rogers, 1963; Ishii and Oosaki,
1969; Kobayashi, 1971; Poullet-Krieger, 1973; Ishii
and Kusakabe, 1982; Kusakabe, 1990b, 1991a, 1992).
The features observed in the maturation process of the
glomus cell include cellular contacts with the smooth
muscle cells (g-s connection), efferent synapses, and the
appearance of exocytotic vesicles. This suggests that
the g-s connection is involved in some functions. Ishii
and Kusakabe (1982) demonstrated g-s connections
and exocytotic catecholamine-containing granules in
the glomus cells of the Xenopus carotid labyrinth. Furthermore, Kusakabe et al. (1987) confirmed physiologically that the vascular tone of the labyrinth is modulated by the catecholamines which are released a t the
g-s connections. That is, the vascular regulation via the
g-s connection serves as a supplementary system to
control by the sympathetic nervous system. Through
the middle larval phase, there is no maze-like structure, but there are some small channels which connect
the carotid arch and the expansion of the external carotid artery. The g-s connections found in this phase
may take part in the regulation of the diameter of
small channels, or may be only a preliminary arrangement for the next stages.
Thereafter the number of the g-s connections increases as the maze-like structure grows more complicated. It can be certain that the g-s connections observed during the metamorphic climax contribute to
the vascular regulation by the same mechanism as described above. In addition, the membrane thickening
and the aggregation of dense-cored vesicles a t the g-s
connections in this phase indicate a clear functional
relation between the glomus cells and the smooth muscle cells as suggested in the pulmonary artery of the
tortoise (Kusakabe et al., 1988).
The present study showed that no afferent synapses
could be found throughout the larval stages. In juvenile
bullfrogs immediately after metamorphosis, however,
both efferent and afferent synapses are noted on the
glomus cells (Kusakabe, 1992), as reported in many
species of animals: mouse (Kobayashi, 19711, rat (McDonald and Mitchell, 1975; Kondo, 19761, rabbit (Matsumoto et al., 1980), turtle (Kusakabe et al., 19881,
toad (Ishii and Oosaki, 1969), clawed toad (Ishii and
Kusakabe, 1982), and newt (Kusakabe, 1990b). Some
synapses are seen lying side by side. Recently, Kariya
et al. (1990) reported in the glomus cells of the rabbit
fetus that efferent synapses are first noted on the 25th
day of gestation and afferent ones on the 30th day of
gestation. In the arterial chemoreceptor organs, efferent innervation to the glomus cells may appear in advance of afferent innervation.
By stage XXI, the forelegs appear on both sides, and
the larvae often go ashore. At this point or a little later,
the respiratory system begins to change gradually from
branchial to pulmonary or cutaneous. In the present
study no afferent synapses were found in any larval
stage, indicating that the glomus cells are not involved
in chemoreception as receptor cells. Distinct afferent
synapses, which are characterized by membrane thickenings with the aggregation of dense-cored vesicles on
the glomus cell side, have been observed in juvenile
bullfrogs as mentioned above (Kusakabe, 1992). It appears that the glomus cells begin t o contribute to the
chemoreception immediately after metamorphosis.
However, the ultrastructural findings are only indirect
Fig. 4. An electron micrograph showing a n immature glomus cell a t
stage V. The glomus cell cytoplasm contained fewer dense-cored vesicles than in later stages (Fig. 6). E, endothelial cell; L, lumen of the
external carotid artery; Sm, smooth muscle cell. Bar = 1 pm.
Fig. 5. A small nerve fiber (arrow) adjacent to the glomus cell at
stage 111. Bar = 1 pm.
Fig. 6. An electron micrograph of a cluster of three glomus cells
(1-3) and a supporting cell (S)at stage XX. The density of dense-cored
vesicles is higher than at earlier stages of development (Fig. 4). Bar =
1 pm.
Flg. 7. A gap junction (arrow) between two adjacent glomus cells at
stage XV. F, filament bundle. Bar = 0.2 pm.
Fig. 8. Efferent synaptic junctions between a glomus cell and a
nerve ending (Ne) at stage XV. Many clear vesicles are aggregated at
the synaptic region (arrows). Bar = 0.2 pm.
Figs. 9-1 1. The close apposition of glomus cells and three types of
neighboring cells observed at metamorphic climax.
Fig. 9. A cellular connection between a glomus cell and two processes of a melanophore at stage XXIV. The arrow indicates the exocytosis of a melanosome. Bar = 0.5 pm.
evidence for chemoreception. Electrophysiological analysis, as we have provided in other cases (Ishii et al.,
1985a,b; Kusakabe et al., 1988) will also be necessary.
A special feature of the glomus cells in the metamorphic climax was the frequent intimate apposition of the
glomus cells to endothelial cells, pericytes, and melanophores in the vascular wall, in addition to the g-s connection. Such appositions have been noted in the newt
carotid labyrinth, between glomus cells and endothe-
Fig. 10. A cellular connection between a glomus cell and a pericyte
(P) a t stage XXIV. E, endothelium; L, lumen of sinusoid. Bar = 0.5
Fig. 11. A g-s connection with membrane specialization (arrow) at
stage XXV. Some dense-cored vesicles are aggregated near this region. Sm, smooth muscle cell. Bar = 0.2 pm.
lial cells, and between glomus cells and pericytes
(Kusakabe, 1990b). It has been reported that endothelial cells have the filament bundles in their cytoplasm
(Rohlich and Olah, 1967; Chen and Weiss, 1972; Yohro
and Burnstock, 1973; De Bruyn and Cho, 1974), and
they are stained by fluorescent antibodies to actin, aactinin, and myosin (Wong et al., 1983). On the other
hand, although the contractility of the pericytes is
questionable, there is some ultrastructural evidence
that the pericytes transform into smooth muscle cells
(Movat and Fernando, 1964; Rhodin, 1968). If the exocytosis of the granules occurs in these connections,
there is a possibility that these connections also participate in the vascular regulation as well as the g-s
In the present work, intimate apposition of glomus
cells to melanophores was observed for the first time in
amphibians. In amphibians, polymorphic melanophores occur frequently in the adventitia of the vascular system as well as in the epidermis. Therefore, they
often appear in the intervascular stroma of the carotid
labyrinth. Recently, electron microscopic observation
of the newt carotid labyrinth revealed the occurrence of
glomus cells with clusters of melanosomes (MG cells),
which has not been reported in the carotid labyrinth so
far (Kusakabe, 1991a). Regarding the mechanism of
melanosome transfer, Kusakabe (1991a) suggested
that the melanosomes detached from the melanophores
are taken into a glomus cell as a group by phagocytosis.
The occurrence of this type of connection strongly supports this suggestion.
In conclusion, the regulation of vascular tone via the
g-s connection starts in correlation with the appearance of the vascular maze. I suggest that the vascular
regulatory function of the carotid labyrinth begins a t
an early stage of the metamorphic climax, and that the
chemoreceptor function begins immediately after
I wish to thank Dr. R.C. Goris of Yokohama City
University School of Medicine for his help in preparing
the manuscript.
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development, artery, characteristics, carotid, external, catesbeiana, cells, rana, ultrastructure, metamorphosis, bullfrog, larvae, glomus
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