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Ultrastructural studies of the effects of reserpine on mouse abdominal sympathetic paraganglia.

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Ultrastructural Studies of the Effects of Reserpine
on
Mouse Abdominal Sympathetic Paraganglia '
JOE A. MASCORRO
AND
ROBERT D. YATES2
Department of Anatomy, The University of Texas Medical Branch,
Galveston, Texas 77550
ABSTRACT
Male and female A/Jax mice 10-18 days of age were given one,
two, or three daily subcutaneous injections of Serpasil (Reserpine USP; 2.5 mg/
kg) and sacrificed 24 hours after the last injection. Abdominal extra-adrenal
tissue was processed for electron microscopy to determine the effects of this
catecholamine depleting drug on the dense cared cytoplasmic granules of the
parenchymal chief cells.
Electron microscopic investigations of sympathetic paraganglia from treated
animals revealed a marked decrease in granule opacity as compared to that seen
in cells from control animals. The cells with granules reduced in opacity following reserpine treatment could be consistently distinguished from those of nontreated animals which led us to assume that the drug depleted the amine content
from its storage site in the granule without completely destroying the granule
structure. These results further substantiate our earlier speculations that the
granules in abdominal paraganglion chief cells of the mouse contain catecholamines.
The term paraganglion is one used to
describe several widely scattered groups of
cells located in the retroperitoneum which
have certain similarities with adrenal
chromaffin cells. Paraganglion cells are
embryologically and morphologically similar to those of the adrenal medulla and are
composed of groups of cells usually surrounding capillaries. The paraganglia and
adrenal medulla are said to comprise the
chromaffin system with the paraganglia
constituting the extra-adrenal elements.
This extra-adrenal chromaffin tissue has
been the subject of recent investigations
with the light and electron microscopes.
The postnatal distribution and ultrastructural morphology of this tissue in the
guinea pig, mouse, rat and rabbit have
been well documented by several investigators (Coupland, '60; VirAgh and Kordnyi
Both, '67; Winckler, '69; Bock, '70; Coupland and Weakley, '70; Mascorro and
Yates, '70). Although paraganglia in mice
appear to degenerate and ultimately disappear soon after birth, these organs persist
throughout life in rabbits and guinea pigs
(Coupland, '56, '60). This has led some
investigators to speculate that the paraANAT. REC.,170: 269-280.
ganglia and the adrenal medulla serve as
storage sites for biogenic amines and that
both can release a potent pressor substance
when stimulated (West et al., '53; Lempinen, '64).
Cytoplasmic granules reported in extraadrenal chromaffin cells are similar in
morphology to those seen in adrenal medullary cells which are known to contain
catecholamines. Fluorometric and electron
microscopic studies have shown that paraganglion cell granules in rabbits contain
almost exclusively norepinephrine (Brundin, '65; Brundin and Nilsson, '65). These
findings have been substantiated by Battaglia ('68, '69) and Muratori et al., ('65)
who reported the presence of norepinephrine in abdominal aortic paraganglia of
the human fetus and neonatal cat using a
glutaraldehyde ammoniacal silver method
for the demonstration of biogenic amines.
A technique involving the use of glutaraldehyde has been described for differentiating norepinephrine from epinephrine
Received Oct. 27, '70. Accepted Nov. 24, '70.
lThi!s research supported by USPHS grants HE
12751, N S 05665 and 00690.
2 Recipient of Career Research Development Award
1-K3-GM 28064.
269
270
JOE A. MASCORRO AND ROBERT D. YATES
in the adrenal medulla (Coupland and
Hopwood, '66). In the process glutaraldehyde reacts vigorously with norepinephrine resulting in granules with a very dense
core surrounded by a smooth surfaced
membrane.
Experimental studies with the light
microscope have demonstrated an effect of
insulin as well as drugs such as reserpine,
aldomet and chlorpromazine on paraganglia. Yamaji ('65) and Tominaga ('67)
noted pronounced differences in catecholamine stores in rabbits following reserpine
treatment ( 5 mg/kg) and concluded that
this drug caused a decrease in the amine
content of the paraganglia.
Since it is known that reserpine will
cause catecholamine depletion from the
adrenal medullary cell granules in the
hamster, rat and mouse (Camanni and
Molinatti, '58; E r h k o and Hopsu, '58),
accompanied by changes in granule ultrastructure (Yates, '63), the present research
was undertaken to determine if similar fine
structural changes could be observed in
the cells of tissue classified as sympathetic
par aganglia.
washed in 0.1 M phosphate buffer with
10% sucrose for a minimum of two hours,
post-fixed for one hour in 1% phosphate
buffered osmium tetroxide, rapidly dehydrated in a graded series of ethyl alcohols
and embedded in Epon 812.
Sections approximately 1 thick were
examined with the light microscope and
used to localize the paraganglia within the
tissue slices. After appropriate trimming
of the blocks containing chromaffin cells,
sections 900-1500 A in thickness were cut
with a Sorvall MT2-B Ultramicrotome,
picked up on 200 mesh grids, stained with
lead citrate and viewed in an RCA EMU3G microscope. Initial micrographs were
made at magnifications of 2,650 to 8,440
diameters and subsequently enlarged
photographically to the desired size.
RESULTS
Non-treated animals. The fine structural features and cell types of abdominal
sympathetic paraganglia have been reported previously (Mascorro and Yates,
'70). The parenchymal cells of the sympathetic paraganglia are of two types;
chief and supporting. The chief cells are
MATERIALS AND METHODS
irregular in shape, contain round or oval
Twelve AIJax mice 10-18 days of age nuclei with evenly dispersed chromatin
were divided into non-treated ( 3 animals) and eccentrically located nucleolar maand treated (9 animals) groups. Those terial, small dark, elongated or round mitofrom the latter group received daily sub- chondria and membranes of the Golgi
cutaneous injections of Serpasil (Reserpine complex and granular endoplasmic reticuUSP; 2.5 mg/kg body weight) for one, lum. Perhaps the most prominent cytotwo or three days and were sacrificed 24 plasmic structures are the numerous memhours after the last injection. Young ani- brane bound granules which appear less
mals used during this work showed an concentrated in juxtanuclear areas largely
erratic tolerance to reserpine and usually occupied by the endoplasmic reticulum,
expired prior to the third injection. Only Golgi membranes and mitochondria. These
those animals which survived were used granules are characterized by an oval,
round or irregular mass of dark material
for these studies.
All animals were anesthetized with surrounded by, but usually separated from,
Sodium Amobarbital (12.5 mg/kg; IP) a smooth-surf aced limiting membrane. The
and prepared for light and electron mi- central dark core at times does adhere to
croscopy by perfusion with 2% glutaral- the inner portion of the limiting memdehyde buffered to pH 7.4 with 0.1 M phos- brane giving the complete granule strucphate. Following the perfusion procedure, ture an overall heterogeneous appearance.
a retroperitoneal tissue block which in- Other components observed within paracluded the renal vessels, inferior vena ganglion chief cells are multivesicular
cava and abdominal sympathetic ganglia bodies, centrioles and lysosomes. Glvcogen
was excised, divided into three or four particles are a constant feature of these
slices cut transversely across the aorta cells and appear distributed at random
and further fixed in the perfusion fluid for through the cytoplasm. Membranes of the
two hours. The tissues were thoroughly granular endoplasmic reticulum occur
RESERPINE EFFECTS
sparsely and contain ribosomes along their
surf aces (figs. 1 , 2 ) . Typical nerve endingparenchymal cell contacts have not been
noted in non-treated or reserpinized
material.
The supporting cells are somewhat irregular in shape and partly or completely
surround individual or clusters of chief
cells (fig. 2). The fine structural features
are the same as those described by Mascorro and Yates ('70).
Paraganglion cells are consistently observed lying close to or surrounded by capillaries which possess thin and occasionally fenestrated endothelium (fig. 1). The
entire paraganglion cell, or group of cells,
is surrounded by collagen fibers and other
connective tissue elements. The bloodparenchymal cell barrier varies from place
to place within the paraganglion. The
endothelial cells are separated from the
parenchymal cells by two basal laminae
between which collagen fibers may or may
not be present. The adjacent parenchymal
cells may be either chief or supporting.
Treated alzimals. Electron microscopic
studies of sympathetic paraganglion chief
cells of animals given one, two or three
subcutaneous injections of reserpine revealed a progressive reduction in the central core opacity of the granules (figs. 3,
4,5). However, not all the granules within
a given cell exhibited the same reduction
in density but the majority of those in animals treated for two or three days were
much less dense than in the cells of control
tissues. In the treated and control animals
the granules were distributed throughout
the cytoplasm. On occasion the granule
membrane appeared to coalesce with the
cell membrane but such relationships were
not observed in greater numbers in the
cells from the treated animals. No alterations in the fine structure of the supporting cells were noted after reserpine
injections.
Cells from animals receiving two reserpine injections contained granules with
reduced opacity within their central core.
Although the response to the drug was
more obvious in material from animals
receiving more than one injection, not all
granules showed a uniform effect in relation to reduction of core material as some
could still be seen to contain more density
- PARAGANGLIA
271
than others. In material from animals
given daily injections of reserpine for three
days and sacrificed 24 hours later, the
cyto~dasmwas noticeably lacking in granules in comparison with cells of control
anirrials. Since the degree of depletion increased as injections continued, it is probable that further treatment would eventually deplete all granules.
DISCUSSION
Ex tra-adrenal chromaffin tissue usually
classified as chromaffin paraganglia has
been likened in structure and function to
the well studied chromaffin cells of the
adrenal medulla. Coupland and Weakley
('70) clearly demonstrated ultrastructural
similarities between these two cell types
during their extensive studies dealing with
developing chromaffin tissue in the adrenal
medulla and extra-adrenal sites in rabbits.
Fine structural studies of paraganglia have
demonstrated the presence of electron
opaque cytoplasmic granules resembling
the catecholamine containing ones in the
adrenal medulla (Brundin and Nilsson,
'65; YirAgh and Korknyi Both, '67; Bock,
'70; Mascorro and Yates, '70; Yates and
Mascorro, '70).
Fluorometric studies by Brundin ('65)
showed that paraganglionic tissue in fetal
rabbits contained almost exclusively noradrenaline and Nakata ('64), employing
Hillarp and Hokfelt's ('55) potassium
iodate technique for the demonstration of
biogenic amines, demonstrated a positive
reaction for this amine in rabbits, cats
and dogs. Radioautographic and cytochemical studies performed by Chen and
Yates ('70) on parasympathetic (vagal)
paraganglia of the Syrian hamster prcjved
positive for the presence of unsubstituted
catecholamines. At the electron microscope level, Battaglia ('68, '69) has employed the glutaraldehyde-ammoniacal silver method to demonstrate biogenic amines
in the aortic abdominal bodies of the
human fetus.
Coupland and Hopwood ('66) have described a highly reliable technique for differentiating between epinephrine and norepinephrine containing cells in the adrenal
medullae of various animals at the electron
microscope level. The mechanism of the
differential reaction involves the action of
2 72
JOE A. MASCORRO AND ROBERT D. YATES
glutaraldehyde as a primary fixative. This
chemical has a solubilizing effect on epinephrine, but reacts very vigorously with
norepinephrine resulting in granules which
exhibit a dense core surrounded by a variable space and limited by a smooth surfaced membrane, On the basis of this
technique the paraganglion cell granules
observed during the present study are of
the norepinephrine type.
At the light microscope level Japanese
investigators have observed a pronounced
loss of extra-adrenal catecholamine stores
in rabbits and dogs following the administration of reserpine and chlorpromazine
(Yamaji, '65; Tominaga, '67). The most
conspicuous effect noted in reserpinized
material during the present research was a
reduction in opacity of the granule core
indicating a diffusion of amine from the
granule. While not all granules exhibited
uniform effects of the drug, particularly
after one injection, all paraganglion cells
of animals subjected to reserpine clearly
possessed at least some granules which differed markedly in appearance from their
normal counterparts.
Yates ('63) demonstrated a marked reduction in granules accompanied by an
increase in empty vesicles and vacuoles in
hamster adrenal medullary cells following
three reserpine injections. The empty
vesicular elements presumably represent
former catecholamine storage sites depleted of their hormone content by the
drug treatment, possibly indicating a disruption in the process of formation or
packaging of arnine concurrent with reserpine treatment. A total loss of core material from granules in the paraganglion
chief cells of animals subjected to three
reserpine injections was never observed,
only a reduction in core capacity. Duncan
and Yates ('67) observed a persistence of
dense granules in cat carotid bodies from
animals subjected to massive doses of
reserpine while similar granules disappeared from sympathetic nerve endings
and nearly all adreno-medullary cells.
They speculated that the binding between
catecholamines and other components of
the storage vesicles may be more resistant
to reserpine than similar but different complexes in the medulla and nerve endings.
The electron opacity remaining in para-
ganglion chief cell granules following reserpine treatment could represent products
of catecholamine binding material: or
perhaps the granule populations from different cells or animals represent differing
tolerances to reserpine treatment.
In a previous report (Mascorro and
Yates, '70) we described the fine structure
of abdominal paraganglia in the mouse
and showed these to be composed of cells
containing many catecholamine type
granules compactly grouped around capillaries exhibiting fenestrated endothelium.
These observations, along with our current
results involving the use of reserpine, lead
us to believe that abdominal paraganglia
are probably endocrine type structures secreting catecholamines. However, paraganglion cells closely approximating capillaries
and containing similar granules have been
located within sympathetic ganglia and
evidence exists that they function as connector or interneurons interposed between
pre- and postganglionic elements. In such
a position they could modify neuronal activity by means of synapses or catecholamine release (Siegrist et al., '68; Williams,
'67a,b; Matthews and Raisman, '69). The
paraganglia studied during this work have
been localized in the retroperitoneum
around the level of the left renal vein.
In this position these extra-adrenal chromaffin bodies are not immediately apposing nor are they located within a ganglion.
Nerve endings similar to those observed
on carotid body and parasympathetic paraganglion chief cells have not been seen
adjacent to the parenchymal cells in this
study. One could speculate that paraganglion function is related to anatomical
position. Thus, some paraganglia represent
chromaffin tissue closely apposing or lying
within sympathetic ganglia and functioning as interneurons. On the other hand,
paraganglia in the retroperitoneum situated
some distance from a ganglion, as in the
present case, may be accessory chromaffin
organs subserving the adrenal medulla in
a pure endocrine capacity by releasing
their amine content. Experiments are in
progress to further verify these results.
LITERATURE CITED
Battaglia, G. 1968 Sulla presenza di amine
biogene nei paragangli carotidei e aortico-ab-
RESERPINE EFFECTS - PARAGANGLIA
dominali del feto umano. Boll. SOC.Ital. Biol.
Sper., 44: 1664-1665.
1969 Ultrastructural observations on
the biogenic amines i n the carotid and aorticabdominal bodies of the human fetus. Z. Zellforsch., 99: 529-537.
Bijck, P. 1970 Die Feinstruktur des paraganglionaren Gewebes im Plexus suprarenalis des
Meerschweinchens. Z. Zellforsch., 1 0 5 : 389404.
Brundin, T. 1965 Catecholamines in the preaortal paraganglia of fetal rabbits. Acta
Physiol. Scan., 64: 287-288.
Brundin, T., and S. E. G . Nilsson 1965 Osmiophilic granules in preaortal paraganglia from
newborn rabbits. Acta Physiol. Scand., 65:
287-288.
Camanni, F., and G. M. Molinatti 1958 Selective depletion of noradrenaline in the adrenals
of the hamster produced by reserpine. Acta
Endocrin., 29: 369-374.
Chen, I-Li, and R. D. Yates 1970 Ultrastructural studies of vagal paraganglia in Syrian
hamsters. Z. Zellforsch., 108: 309-323.
Couuland. R. E. 1956 The development and
fate of 'chromaffin tissue in the rabbit. J. Anat.,
90: 527-537.
1960 The post-natal distribution of the
abdominal chromaffin tissue in the guinea
pig, mouse and white rat. J. Anat., 94: 244-256.
Coupland, R. E., and D. Hopwood 1966 Mechanism of a histochemical reaction differentiating
between adrenaline- and noradrenaline-storing
cells i n the electron microscope. Nature, 209:
590-591.
Coupland, R. E., and B. S. Weakley 1970 Electron microscopic observations on the adrenal
medulla and extra-adrenal chromaffin tissue of
the postnatal rabbit. J. Anat., 106: 213-231.
Duncan, D., and R. D. Yates 1967 Ultrastructure of the carotid body of the cat as revealed
by various fixatives and the use of reserpine.
Anat. Rec., 157: 667-681.
Eranko, O., and V. Hopsu 1958 Effect of reserpine on the histochemistry and content of adrenaline and noradrenaline in the adrenal medulla of the rat and the mouse. Endocrin., 62:
15-23.
Hillarp, N.-A., and B. H6kfelt 1955 Histochemical demonstration of noradrenaline and adrenaline i n the adrenal medulla. J. Histochem.
Cytochem., 3: 1-5.
Lempinen, M. 1964 Extra-adrenal chromaffin
tissue of the rat and the effect of cortical hor-
273
mones on it. Acta Physiol. Scan., 62, Supp. 231:
1-91.
Masccrro, J. A., and R. D. Yates 1970 Microscopic observations on abdominal sympathetic
paraganglia. Texas Reports on Biol. and Med.,
28: 59-68.
Matthews, M., and G. Raisinan 1969 The ultrastructure and somatic efferent synapses of small
granule-containing cells in the superior cervical
ganglion. J. Anat., 105: 255-282.
Muratori, G., G. Battaglia and G. Modonesi 1965
Dimostrazione istochimica della noradrenalina
nel cromaffine cervico-toracico e aortico-addondnale degli amnioti 11) Osservazioni a1
microscopio elettronico. Boll. SOC. Ital. Biol.
Spei:., 41: 1185-1186.
Nakata, Y. 1964 Histochemical studies on catechol.amine with reference to the paraganglia.
Acta Neuroveg., 26: 75-92.
Siegrist, G., M. Dolivo, Y. Dunant, C. ForoglouKerameus, Fr. de Ribaupierre and Ch. Rouiller
1968 Ultrastructure and function of the chromafen cells in the superior cervical ganylion of
the rat. J. Ultrastruct. Res., 25: 381407.
Toniinaga, S. 1967 Histochemical studies on
catecholamines (with special reference to the
paraganglion). Arch. Jap. Chir., 36: 430442.
Viraglx, Sz., and A. Korknyi Both 1967 The
fine structure of abdominal paraganglia in the
newborn mouse. Acta Biol. Acad. Sci. Hung.,
18: 161-179.
West, G. D., D. M. Shepherd, R. B. Hunter and
A. G . MacGregor 1953 The function of the
organs of Zuckerkandl. Clinical Sci., 1 2 : 317325.
Willia:ms, T. H. 1967a The question of the
intraganglionic (connector) neuron of the autonomic nervous system. J. Anat., 101: 603-604.
____ 1976b Electron microscopic evidence
for a n autonomic interneuron. Nature, 214:
309-310.
Winckler, J. 1969 Zur Lage unal Funktion der
extramedullaren Chromaffinen Zellen. Z. Zellforsch., 96: 490-494.
Yamaji, S. 1965 Histochemical studies on catecholamines i n paraganglia and paraganglioma.
Arch. Jap. Chir., 34: 1197-1212.
Yates, R. D. 1963 A n electron microscopic
study of the effects of reserpine o n adrenomedullary cells of the Syrian hamster. Anat.
Rec., 146: 2 9 4 5 .
Yates, R. D., and J. Mascorro 1970 Electron
microscopic studies on sympathetic paraganglia.
Ana.t. Rec., 166: 400 (Abstract).
PLATE 1
EXPLANATION OF FIGURE
1
2 74
Survey micrograph of a part of a sympathetic paraganglion illustrafing the parenchymal cells and a capillary ( C ) . Note the opaque granules i n the cytoplasm of the chief cells (Ch). Supporting cells ( S )
are also shown. X 9,262.
-
RESERPINE EFFECTS
PARAGANGLIA
Joe A. Mascorro and Robert D. Yates
PLATE 1
275
PLATE 2
EXPLANATION OF FIGURES
276
2
Electron micrograph illustrating the chief cells of a sympathetic
paraganglion. Note the opacity of the granules. x 11,788.
3
Micrograph of chief cells from a n animal which had received one
injection and was sacrificed 24 hours later. Note that the cytoplasmic
granules (circle) are decreased in opacity. >: 8,420.
Inset A: granules from a chief cell of a n untreated animal. Note the
opacity of the granules. X 17,730.
Inset B: granules from a chief cell of a n animal which had received
two injections of reserpine and was sacrificed 24 hours later. Note
the reduction in granule central core opacity. x 17,730.
RESERPINE EFFECTS - PARAGANGLIA
Joe A. Mascorro and Robert D. Yates
PLATE 2
277
PLATE 3
EXPLANATION OF FIGURES
276
4
Electron micrograph of a paraganglion from a n animal which had
received two injections of reserpine. Note that the granules exhibit
reduced central core density (circles). x 10,109.
5
Micrograph of a paraganglion cell from a n animal which had received
three injections of reserpine. The density of the granules within the
circles is considerably reduced in comparison with those of the control
animals. x 14,184.
RESERPINE EFFECTS - PARAGANGLIA
Joe A. Mascorro and Robert D. Y a k s
PLATE 3
279
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