IMMUNOHISTOCHEMICAL LOCALIZATION OF HYPOTHALAMIC ...
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864
THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY
Copyright #{174}1976 by The Histochemical Society, Inc.
Vol. 24, No. 7, pp. 864-871, 1976
Printed in U.S.A.
IMMUNOHISTOCHEMICAL LOCALIZATION OF HYPOTHALAMIC
HORMONES
GEORGES PELLETIER, RACHEL LECLERC AND DONALD DUBE�
Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier de l’Uniuersit#{235}Laval, Quebec Gi V 4G2, Canada
Using an immunoperoxidase technique at the light and electron microscope levels, thelocalization of somatostatin and luteinizing hormone-releasing hormone (LH-RH) has beenperformed in the brain, as well as the pancreas and stomach. The following generalizationsmay be drawn on the localization of LH-RH and somatostatin. (a) LH-RH and somatostatinare contained in axons and nerve endings of the median eminence and organum vasculosumof the lamina terminalis. (b) LH-RH and somatostatin are present in different nerve endings.(c) In the subcomn�issural organ, subfornical organ and area postrema, the two neurohor-mones are present in the cytoplasm of ependymal and subependymal cells. (d) In the
endocrine pancreas and gastric mucosa, somatostatin is localized within the secretorygranules of specific endocrine cells.
The activity of the anterior pituitary gland is
controlled by neurohormones produced by the
hypothalamus. During the last few years, two
hypothalamic releasing hormones and one re-
lease-inhibiting hormone have been character-
ized and synthesized (3, 4, 7, 8, 22, 30). While it
is well known that hypothalamic regulatory
hormones are concentrated in the median cmi-
nence (19), it has been recently reported that
thyrotropin-releasing hormone and somato-
statin are present in some extrahypothalamic
areas (5, 6). Moreover, a direct inhibitory effect
of somatostatin in the secretion of insulin (15),
glucagon (15) and gastnin (13) has also been
reported, thus suggesting that this neurohor-
mone could be produced in the pancreas and
gastrointestinal tract. By measuring contents of
very small pieces oftissue obtained by microdis-
section (5, 6) or purified nerve endings (33), it is
not possible to determine which cells and gran-
ules contain the hormone in the nervous tissue.
Only use of immunohistochemical techniques at
both light and electron microscope levels can
achieve a precise identification of the cellular
elements responsible for the production of the
hypothalamic hormones. In order to gain a
better understanding of the mechanisms of
packaging and secretion of luteinizing hormone-
releasing hormone (LH-RH) and somatostatin
in the brain and target tissues, detailed im-
munohistochemical studies were performed on
the localization of these peptides.
IMMUNOHISTOCHEMICAL METHODS
The development by Sternberger (32) of a sensitive
immunoperoxidase technique involving the use of a
penoxidase-antiperoxidase (PAP) complex has pen-mitted the localization of neurohormones. With thistechnique, which is 100-1000 times more sensitive
than immunofluorescence, it has been possible todemonstrate small polypeptides such as vasopnessin(20), which has only eight amino acids. At the lightmicroscope level, the localization studies were per-formed in thick (7-�.cm) and semithin (2-�.cm) sections.The antisera against LH-RH and somatostatin were
supplied by Dr. A. Arimura (New Orleans) and wereproduced in our laboratory (by J. C#{244}t#{233}).The anti-serum to vasopressin was supplied by Dr. J. Roth(Bethesda, Md.), whereas the antiserum to oxytocinwas prepared in our laboratory (by Dr. A. Belanger).
The PAP was a generous gift from Dr. L. A. Stem-berger (Edgewood Arsenal, Md.). All of the im-munohistochemical reactions were carefully con-
trolled by immunoabsorption of the antiserum withthe synthetic hormone. Detection of different hor-
mones in consecutive sections was also used as a
control of specificity.
LOCALIZATION OF LH-RH
Luteinizing hormone-releasing hormone (LH-
RH), a decapeptide, has been studied by many
groups who used either fluorescence or peroxi-
dase labeling methods. In many species,
LH-RH was consistently observed in the exter-
nal zone of the median eminence (1, 2, 17, 18,
23, 31). The reaction was generally more con-
centrated in the lateral portions of the median
eminence (Fig. 1A) and appeared along the
entire sweep of the external zone, although more
concentrated in the caudal median eminence.
By immunoelectron microscopic studies, we
have clearly established that LH-RH is present
exclusively in nerve endings of the external zone
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FIG. 1. A, immunohistochemical localization of LH-RH in a coronal section of the caudal portion of themedian eminence. The reaction product (arrow) is concentrated in the dorsal region of the external zone. x 190.B, localization of somatostatin in a section adjacent to that shown in A. The reaction (arrows) is present in theventral and lateral portion of the external zone, whereas the dorsal region is relatively free of reaction. x 190.
FIG. 2. Electron micrograph showing a section of the external zone of the median eminence. A nerve ending
containing granules positive for LH-RH (arrow) is present. Other nerve endings (NE) are negative. x32,000.
865
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866 PELLETIER, LECLERC, DUBE
of the median eminence (24). As evidenced in
Figure 2, the reaction is localized in the secre-
tory granules of the positive nerve endings. The
diameter of the LH-RH-containing granules
ranges from 75 to 95 nm. Absolutely no reaction
was observed in the tanycytes of the median
eminence.
Most investigators have been unable to show
immunostained cell bodies in the hypothalamus
(1, 17, 18, 31). In guinea pigs previously injected
with colchicine, Barry et al. (2), using immuno-
fluorescence, observed a few LH-RH positive
cell bodies scattered in a large area extending
from the preoptic area to the caudal part of the
tuber. Zimmerman et al. (36) described the
presence of LH-RH in the penikaryon of a few
neurons of the arcuate nucleus in the mouse.
However, the picture seems to indicate that
most of the staining is really around the neurons
instead of being inside the perikarya. Using
serial paraffin sections, we have been unable to
find any immunostained cell bodies in the rat
brain (23).
At the light microscope level, LH-RH was
localized around the organum vasculosum of
the lamina tenminalis (OVLT) of the mouse
(36), guinea pig (9) and rat (Fig. 3A). The
reaction was usually very strong and located in
close proximity to the capillaries of the onganum
vasculosum. With the electron microscope,
LH-RH was cleanly observed in the secretory
granules of a few nerve endings located in
contact with the basement membrane of the
fenestrated capillaries. These observations sug-
gest that LH-RH is secreted from this organ into
the general circulation. As in the median emi-
nence, all of the secretory granules (of a diame-
ter of 70-100 nm) were labeled in a positive axon
(23).
The other peniventnicular organs were also
studied for LH-RH detection. It was found that
the subfonnical organ, the subcommissural
organ (Fig. 4A) and the area postrema showed a
positive reaction. A common pattern for the
distribution of LH-RH was observed in these
organs: a strong reaction was localized in the
ependymal and subependymal layers and also
at the vicinity of blood vessels. At the ultra-
structural level, a diffuse reaction was observed
in the cytoplasm of the ependymal and subep-
endymal cells (Fig. 5). This reaction was not
clearly associated with any onganelles. Some
cell processes were also found to be labeled
mainly at the proximity of the capillaries.
LOCALIZATION OF SOMATOSTATIN IN THE BRAIN
By immunopenoxidase, somatostatin, the
most recently characterized hypothalamic hor-
mone, has been localized in the external zone of
the median eminence of both rat and guinea pig
(9, 16, 25, 26). The immunostaining was gener-
ally more intense in the lateral parts of the
median eminence, although the somatostatin
fibers are generally medial to LH-RH fibers
(Fig. 1B). Using an immunofluonescence tech-
nique, Hockfelt et a!. (14) have localized soma-
tostatin in the external zone of the median
eminence and the ventnomedial nucleus of the
guinea pig brain. With the aid of ultrastructural
localization, we have localized somatostatin
within secretory granules in about 30% of the
nerve endings in the external zone (25). These
granules were slightly larger than those contain-
ing LH-RH (90-110 nm versus 75-95 nm).
These results cleanly indicate that somatostatin
and LH-RH fibers, although belonging to the
tuberoinfundibular system, are separate sub-
systems.
Using serial paraffin, it was demonstrated
that somatostatin was located in the OVLT, the
subcommissural organ, the subfornical organ
and the pineal gland (10, 23, 26). In the OVLT,
immunostaining was found close to the capil-
laries, whereas ependymal cells remained un-
stained. As demonstrated on serial sections, the
localization of somatostatin was completely
different from that of LH-RH (Fig. 3B). With
high resolution immunohistochemistry, soma-
tostatin was detected in the secretory granules
(of a diameter of 90-120 nm) of many nerve
endings. In the subfornical organ, subcommis-
sunal organ and area postrema, the reaction was
FIG. 3. A, coronal section through the organum vasculosum of the lamina terminalis. LH-RH (arrows) islocalized in proximity to the capillaries (C) of the organum vasculosum. x440. B, detection of somatostatin of asection adjacent to that shown in A. The distribution of reaction product (arrows) dlose to the capillaries (C) isdifferent from that observed for LH-RH. x 440.
FIG. 4. Localization of LH-RH (A) and somatostatin (B) in two consecutive sections of the subcommissuralorgan. The distribution of the two neurohormones, which are mainly concentrated in the ependymal layers(arrows), is very similar. x540.
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LOCALIZATION OF HYPOTHALAMIC HORMONES867
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FIG. 5. Electron micrograph showing the localization of LH-RH in the subcommissural organ. A diffusepositive reaction is observed in the cytoplasm of many ependymal cells (E). V, third ventricle. x 16,000.
Fic. 6. Immunohistochemical localization of somatostatin in the gastric mucosa. Accumulation of PAPmolecules can be detected over the secretory granules (arrows) and the cytoplasm of an endocrine cell. Anotherendocrine cell (EC) contains granules which are completely negative. x 17,000.
868
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LOCALIZATION OF HYPOTHALAMIC HORMONES 869
similar to that observed for LH-RH (Fig. 4B).
The accumulation of PAP molecules was pres-
ent over the cytoplasm of ependymal and sub-
ependymal cells. In the pineal gland, a diffuse
reaction was found in the cytoplasm of many
cells surrounding the capillaries.
In the OVLT, the presence of somatostatin in
nerve endings suggests that this organ, besides
its possible role in the control of gonadotropins
secretion, may also play a role in the control of
growth hormone secretion. In the other peniven-
triculan organs, the presence of LH-RH and
somatostatin in the same cells, which are not
secreting neurons, may simply represent an
uptake of the neurohonmone.
LOCALIZATION OF SOMATOSTATIN IN PANCREAS AND
STOMACH
In the pancreas of many species, a cell type
present in Langerhans islets has been shown to
contain somatostatin (10, 12, 27, 29). Absolutely
no immunostaining was found in the exocnine
cells or nerve endings. In the rat pancreas, the
positive reaction was mainly observed in the
secretory granules of a few cells located at the
periphery of the Langerhans islets (12, 27). The
diameter of the somatostatin granules was
about 175-210 nm. The a and � cells were
always unstained by the antisomatostatin se-
rum. This somatostatin cell seems to come-
spond to the D cell type (23, 29). These results
suggest that somatostatin, which inhibits the
release of both insulin and glucagon by the
endocrine pancreas, may have some physiologic
importance in the regulation of the secretion of
these two hormones.
Somatostatin cells were also found in the
gastrointestinal tract (23, 29). They are particu-
larly abundant in the antral mucosa in the
neighborhood of gastnin-secreting cells. These
positive cells were generally located in the basal
region of the villi. At the ultrastructural level,
the accumulation of PAP molecules was local-
ized over the secretory granules (150-250 nm in
diameter) with some degree of diffusion in the
cytoplasm of a cell type which has not been
found in contact with the pyloric humen (Fig.
6). On the basis of histochemical and ultra-
structural characteristics, the somatostatin cell
appears to correspond very well to the intestinal
D cell (23, 29).
NEUROPHYSIN IN THE EXTERNAL ZONE OF THE
MEDIAN EMINENCE
Neurophysin and vasopnessin have rarely
been observed in the external zone of the rat
median eminence (20, 34). Since at the ultra-
structural level they are localized in granules
(150-200 nm in diameter) similar to those
observed in axons ofthe internal zone (20), they
are thought to belong to the hypothalamo-
neurohypophyseal system. Very recently, an
increase in the content of neumophysin in the ex-
tennal zone of the rat median eminence after
adrenalectomy has been noted (34, 35). Since
the origin of this neurophysin, possibly as-
sociated with corticotropin-neleasing hormone,
was not known, an ultrastructural study was
performed to identify cleanly the structures con-
taming neurophysin and vasopressin in the
adrenalectomized rat. It was found that both
neumophysin and vasopnessin are present in
small granules (about 80-100 nm in diameter)
in the external zone of the adrenalectomized
animals (Fig. 7A and B). These positive nerve
endings are very different from the axons of
the internal zone of the median eminence,
which are also positive for neurophysin and
vasopressin and contain granules about 150 nm
in diameter (28). Thus it appears that there are
two neurophysin-vasopressin systems. The first
one is a constituent of the hypothalamo-
neurohypophyseal tract, whereas the second
one probably originates from a parvicellulan
system and is dramatically influenced by
adrenalectomy. The origin and role of this
second neurophysin-vasopressin system remain
to be established.
SUMMARY AND CONCLUSION
With the help of a sensitive immunohisto-
chemical technique, many important conclu-
sions have recently been obtained in the field of
neu.rosecnetion. LH-RH and somatostatin have
been found within secretory granules of nerve
endings located near the portal capillaries in the
median eminence. These findings confirm the
earlier theory that regulatory hormones are
produced by neurons and stoned in axon endings
before being released into the capillaries of the
pituitary portal plexus. In the OVLT, LH-RH
and somatostatin have also been localized in
nerve endings near the fenestrated capillaries of
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870 PELLETIER, LECLERC, DUB�
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Ftc. 7. Sections of the external zone of the median eminence of adrenalectomized rats treated for
neurophysin (A) and vasopressin (B) detection. Both neurophysin and vasopressin have been localized in nerveendings (arrows) containing granules of about 80-100 nm. x 17,500.
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the onganum vasculosum. These findings, simi-
lam to that observed in the median eminence,
suggest that OVLT could be involved in the
secretion of these hypothalamic hormones. Fur-
then studies are needed to evaluate the impor-
tance of this organ.
In the endocrine pancreas and the gastroin-
testinal mucosa, somatostatin has been found
in specific secretory cells. These somatostatin-
producing cells have always been found in close
proximity to endocrine cells (pancreatic a and ficells and G cells) of which they inhibit the
secretion. The wide distribution of somatostatin
cells in the gastrointestinal tract suggest that
they can influence the secretion of other hor-
mones.
Finally, we have shown that neurophysin and
vasopressin are also constituent of a parvicellu-
lan system. Since this system seems to be
related with adrenocorticotrophic secretion, it is
possible that a corticotropin-releasing hormone,
immunologically related to vasopressin, had
been detected by immunocytochemistry.
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LOCALIZATION OF HYPOTHALAMIC HORMONES 871
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