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, the localization of somatostatin and luteinizing hormone-releasing hormone (LH-RH) has been performed in the brain, as well as the pancreas and stomach. The following generalizations may be drawn on the localization of LH-RH and somatostatin. (a) LH-RH and somatostatin are contained in axons and nerve endings of the median eminence and organum vasculosum of the lamina terminalis. (b) LH-RH and somatostatin are present in different nerve endings. (c) In the subcomnissural 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 secretory granules 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 this technique, which is 100-1000 times more sensitive than immunofluorescence, it has been possible to demonstrate small polypeptides such as vasopnessin (20), which has only eight amino acids. At the light microscope 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 were produced 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 oxytocin was 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 with the 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 by guest on October 5, 2016 jhc.sagepub.com Downloaded from
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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
by guest on October 5, 2016jhc.sagepub.comDownloaded from
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.
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(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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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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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
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