Endocrinology Vol. 140, No. 5 2326-2333
Copyright © 1999 by The Endocrine Society
Immunocytochemical Localization of Prolactin-Releasing Peptide in the Rat Brain
Minoru Maruyama,
Hirokazu Matsumoto,
Ken Fujiwara,
Chieko Kitada,
Shuji Hinuma,
Haruo Onda,
Masahiko Fujino and
Kinji Inoue
Department of Regulation Biology, Faculty of Science, Saitama
University (M.M., K.F., K.I.), 255 Shimo-ohkubo, Urawa 338-0825; and
Discovery Research Laboratories I, Pharmaceutical Discovery Research
Division, Takeda Chemical Industries Ltd. (H.M., C.K., S.H., H.O.,
M.F.), 10 Wadai, Tsukuba, Ibaraki 300-4293, Japan
Address all correspondence and requests for reprints to: Kinji Inoue, Ph.D., Department of Regulation Biology, Faculty of Science, Saitama University, 255 Shimo-ohkubo, Urawa 338-0825, Japan. E-mail:
kininoue{at}seitai.saitama-u.ac.jp
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Abstract
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A hypothalamic peptide that stimulates PRL release has recently been
found as a ligand of an orphan receptor and named PRL-releasing peptide
(PrRP). PrRP and its receptor were mainly detected in the hypothalamus
and pituitary gland, respectively. Its characteristics suggested PrRP
to be a novel hypophysiotropic peptide that stimulates the anterior
pituitary PRL cell; however, this remained to be confirmed
morphologically. We therefore performed an immunocytochemical study to
locate PrRP in the rat brain using the region-specific monoclonal
antibodies, P2L-1C and P2L-1T, which recognize the C-terminal and the
internal sequence of PrRP, respectively. Our results clearly show that
dense immunoreactive nerve fiber networks are present in the
paraventricular hypothalamic nucleus, supraoptic nucleus, paratenial
thalamic nucleus, basolateral amygdaloid nucleus, and bed nucleus of
the stria terminalis. A small number of PrRP nerve fibers was also
observed in the neural lobe of the hypophysis. However, no
immunopositive fiber was observed in the external region of the median
eminence, which is known to be the release site of the classical
hypophysiotropic hormones. Also, the distribution of PrRP was not
changed during the estrous cycle. We therefore concluded that PrRP
probably differs from classical hypothalamic releasing hormones. We
found the immunoreactive cell bodies to be mainly in the caudal portion
of the dorsomedial hypothalamic nucleus and solitary nucleus. A double
immunocytochemical procedure revealed that some PrRP-positive neurons
showed synaptic contact with oxytocin-positive cell bodies in the
paraventricular hypothalamic nucleus, which suggests that PrRP
regulates the function of oxytocin neurons. This is the first report to
demonstrate the localization of the novel hypothalamic peptide, PrRP,
and we therefore suggest that it takes part in a variety of brain
functions. However, it is not yet known how PrRP is transported to the
pituitary gland, which is the site that contains the greatest
concentration of receptors to this new peptide. Therefore, additional
work will be required to resolve this discrepancy between ligand and
receptor site location.
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Introduction
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THE DISCOVERY of the hypophysiotropic
peptides and their delivery to the pituitary via the hypophyseal portal
system was the most dramatic event in endocrinology during this
century. Regulation of most of these pituitary hormones has been
explained through the action of these hypothalamic hormones. PRL, which
is included as one of the major anterior pituitary hormones, is known
to have its secretion controlled by a variety of hypothalamic factors.
Factors inhibiting its secretion are dopamine and somatostatin (1),
whereas TRH, vasoactive intestinal polypeptide, serotonin (1), oxytocin
(2), substance P, neurotensin (3), arginine vasopressin (4), pituitary
adenylate cyclase-activating peptide, and galanin (5) all act to
stimulate its secretion. However, the above secretagogues are all
multifunctional peptides and are not specifically targeted to PRL
release. Therefore, the search for the true PRL-stimulating factor
continued (6, 7), but these efforts provided no good candidate for
authentic PRL cell-stimulating factor. Recently, a novel hypothalamic
peptide with potent PRL-releasing activity has been reported. It was
discovered to be a ligand of an orphan receptor (hGR3) and has received
the name PRL-releasing peptide (PrRP) (8). This peptide is known to
have two molecular forms, one with a 31-residue peptide (PrRP31) and
another with its C-terminal 20-residue peptide (PrRP20). Their amino
acid sequences were determined to be
SRAHQHSMEIRTPDINPAWYAGRGIRPVGRF-amide and TPDINPAWYAGRGIRPVGRF-amide,
respectively. These two peptides, PrRP31 and PrRP20, are known to
derived from a single precursor molecule, and their sequences are
highly conserved among several species, such as human, bovine, and rat.
This, then, suggests that PrRP plays some important role in these
animals. Interestingly, PrRP showed a stronger and more specific
PRL-releasing activity in vitro compared with that of the
the known PRL-releasing hypothalamic peptides. It has been further
observed that PrRP messenger RNA is mostly located in the medulla
oblongata, but a large amount of the peptide exists in the
hypothalamus. Additionally, a high concentration of PrRP receptor
messenger RNA has been detected in the pituitary gland. These
characteristics of PrRP indicate that this novel peptide is a good
candidate for the role of the true hypothalamic PRL-releasing factor.
If PrRP is a PRL-releasing hypophysiotropic factor similar to the
classical hypothalamic factors, it must be transported to the external
layer of the median eminence (ME), where it can be released into the
hypophyseal portal vessels. Immunocytochemistry is one of the best
methods for to demonstrate neural projection of PrRP in the
hypothalamus. We, therefore, raised specific monoclonal antibodies that
recognize the C-terminal and the internal sequences of PrRP and
performed immunocytochemistry. By use of these region-specific
monoclonal antibodies, we succeeded in localizing PrRP neurons in the
rat brain, but failed to detect PrRP neurons in the external
regions of ME. We report here that PrRP differs from the classical
hypothalamic releasing factors. However, its numerous and specific
neural localizations in the hypothalamus suggest that PrRPs play
important roles in neuroendocrinology.
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Materials and Methods
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Synthetic peptides
The peptides used in this study were synthesized by an automatic
peptide synthesizer (model 430A, PE Applied Biosystems,
Foster City, CA). These peptides included human (h), rat (r), and
bovine (b) PrRP31, PrRP20, and PrRP-related peptides with a C-terminal
amide group [hPrRP-(1–30)amide,
[Cys17]hPrRP-(17–31)amide and [Cys25]hPrRP
(12–25)amide], and peptides with a free carboxyl group in the
C-terminus[bPrRP-(1–31)OH and bPrRP-(1–31)Gly-Arg-OH] (8).
Antibodies
[Cys17]hPrRP-(17–31)amide and
[Cys25]hPrRP-(12–25)amide were used for immunogens.
Conjugation of 1.7 mmol of these immunogens with 30 nmol bovine
thyroglobulin previously maleimidated with
N-(
-maleimidobutyryloxy)succinimide was performed. These
immunogens (40 mg/mouse) together with complete or incomplete Freund’s
adjuvant were sc injected into BALB/c mice (female, 8 weeks old) at
3-week intervals. Four days after iv injecting each mouse with 200 mg
of the immunogen, spleen cells were separated and fused with mouse
myeloma cells (P3-X63Ag8-U1) as described previously (9). Monoclonal
antibodies, P2L-1T (IgG2a, 
) and P2L-1C (IgG2a, ) were selected
and purified from ascites fluid with a protein A-immobilized column
(IPA-300, Repligen, Cambridge, MA).
Competitive enzyme immunoassay (EIA)
The reactivity of antibodies was investigated with competitive
EIAs using [Cys17]hPrRP-(17–31)amide or
[Cys25]hPrRP-(12–25)amide labeled with horseradish
peroxidase (HRP). Conjugation of these peptides with HRP was performed
as described previously (9). In the EIA, 100 µl culture medium of
hybridomas, properly diluted with buffer C [0.02 M phosphate buffer
(pH 7.0) containing 0.4 M NaCl, 2 mM EDTA, and
1% BSA] and 50 µl standard PrRP or PrRP-related peptides in buffer
C were added to a goat antimouse Ig (IgG fraction, Cappel Laboratories,
Durham, NC; coated microtest plate, 96 wells, Nunc, Naperville,
IL) and incubated at room temperature for 6 h. Subsequently, 50
µl HRP-labeled PrRP-(17–31) or PrRP-(12–25) diluted 300 times with
buffer C were added to the plate and incubated at 4 C for
16 h. The plate was washed with PBS, and the bound enzyme activity
was measured with a TMB microwell peroxidase system (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Animals and immunocytochemistry
Adult female rats (F344 strain) were housed individually in
cages under controlled conditions (23.0 ± 0.5 C; lights on,
0900–2100 h) and provided with standard rat chow and water ad
libitum. All procedures were performed in accordance with
institutional guidelines for animal care at Saitama University.
For immunocytochemistry, the animals were deeply anesthetized with
sodium pentobarbital (50 mg/kg, ip) and perfused first with saline and
then with 5% acrolein in a 0.07 M phosphate buffer
(pH 7.4) as previously reported (10). The brains were quickly removed
and immersed for more than 24 h in PBS containing 30% sucrose.
Frozen serial frontal and sagittal sections (60 µm thick) of the
brain were made with a cryomicrotome. A sensitive method of
immunocytochemistry that employs a free floating technique was used
as described previously (11, 12). Briefly, the cryosections were washed
with PBS and immersed sequentially in the following solutions: 1) 0.5%
sodium metaperiodate in PBS for 20 min, 2) 1% sodium borohydride in
PBS for 20 min, 3) 1% normal horse serum and 0.4% Triton X-100 in PBS
(TNBS) for 1 h, 4) 40 µg/ml P2L-1C in TNBS or 80 µg/ml P2L-1T
in TNBS for 24 h, 5) biotin-conjugated donkey antimouse IgG
(Chemicon International, Temecula, CA) diluted 1:300 in TNBS for
2 h, 6) avidin-biotinylated HRP-complex (ABC, Vector Laboratories, Inc., Burlingame, CA) for 30 min, and 7) 0.02%
3,3-diaminobenzidine-tetrachloride mixed with 0.06% hydrogen peroxide
in 0.05 M Tris-HCl, pH 7.6, for 4–6 min. After processing,
the sections were dehydrated, mounted, and examined by light
microscopy. Immunocytochemical controls were carried out as follows.
Sections were incubated for 24 h with monoclonal antibodies, which
were preabsorbed with 1 mg/ml synthetic PrRP31 for 1 h instead of
the nonabsorbed monoclonal antibodies.
Double immunostaining
After staining the PrRP neurons with diaminobenzidine, the free
floating sections were first washed with 0.1 M glycine-HCl
buffer (pH 2.2) for 2 h and then incubated for 24 h with
rabbit antiserum against vasopressin (HAC-HM07–02RBP90, provided by
Dr. Wakabayashi, Gunma University; 1:2000) or oxytocin
(Calbiochem, La Jolla, CA; 1:100). The sections were
treated with biotin-conjugated goat antirabbit IgG (ABC kit,
Vector Laboratories, Inc.) diluted 1:300 in TNBS for
2 h and further incubated with avidin-biotinylated HRP complex
(ABC, Vector Laboratories, Inc.) for 30 min. Finally, the
sections were visualized with 0.02% 4-chloro-1-naphthol mixed with
0.005% hydrogen peroxide in 0.05 M Tris-HCl (pH 7.6) for
10–15 min. After processing, the sections were mounted and examined by
light microscopy.
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Results
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Character and specificity of monoclonal antibodies
The reactivities of two monoclonal antibodies with PrRP and
related peptides were analyzed by competitive EIAs using HRP-labeled
PrRP-(12–25) or PrRP-(17–31) (Fig. 1
).
P2L-1T and P2L-1C reacted with rPrRP31 and rPrRP20 as well as both
hPrRPs. P2L-1T reacted with bPrRP-(1–31)OH and bPrRP-(1–31)Gly-Arg-OH
as well as bPrRP31, indicating that it recognized not only mature PrRP
but also the prepro form of PrRP. P2L-1C reacted with PrRP31 and
PrRP20, but not with hPrRP-(1–30)amide, bPrRP-(1–31)OH, or
bPrRP-(1–31)Gly-Arg-OH, indicating that it recognized only mature PrRP
especially the C-terminal portion of PrRP containing
Phe31-amide. As expected, the P2L-1T antibody stained both
nerve fibers and cell bodies, while P2L-1C stained nerve fibers alone.
However, the PrRP nerve fibers were more strongly stained by P2L-1C
than by P2L-1T. Therefore, we used the P2L-1T antibody to detect cell
bodies and P2L-1C to detect nerve fibers. To test for
immunocytochemical specificity, we performed absorption controls as
described in Materials and Methods. The specificity was
verified by the complete abolishment of immunostaining in the
absorption control (data not shown).
Localization of PrRP neurons
As shown in Fig. 2, many PrRP fibers
were observed in the bed nucleus of the stria terminalis, and they were
most dense in the ventral portion of anterior commissure. Additionally,
many PrRP-immunoreactive fibers were observed to surround the third
ventricle, especially in the paraventricular hypothalamic nucleus (Fig. 3A), the paratenial thalamic nucleus
(Fig. 3B), and the supraoptic nucleus (Fig. 3C). Some of the PrRP
fibers surrounded magnocellular cell bodies. Other PrRP-immunolabeled
fibers were present at the basolateral amygdaloid nucleus (Fig. 3D),
the retrochiasmatic area, and the superior cerebellar peduncle (data
not shown). PrRP nerve fibers were also observed in the medulla
oblongata. As shown in Fig. 6D, clear PrRP-immunoreactive fibers were
found in the solitary nucleus (Fig. 6D) and in the spinocerebellar
tract (data not shown).
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Figure 2. Photomicrograph showing P2L-1C-immunoreactive
fibers in the bed nucleus of the stria terminalis (frontal view).
Magnification, x100. The dense PrRP fiber network is located in the
ventral portion of the anterior commissure. BST, Bed nucleus of the
stria terminalis; CA, anterior commissure.
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Figure 3. Frontal view of P2L-1C-immunoreactive neurons in
the paraventricular hypothalamic nucleus (A), the paratenial
thalamic nucleus (B), the supraoptic nucleus (C), and the basolateral
amygdaloid nucleus (D). Magnification, x100. ABL, Basolateral
amygdaloid nucleus; CO, optic chiasm; PT, paratenial thalamic
nucleus; PVH, paraventricular hypothalamic nucleus; SO, supraoptic
nucleus; V, ventricle.
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Figure 6. Photomicrographs showing P2L-1T-immunoreactive
cell bodies in the DM (A), the spinocerebellar tract (B), and solitary
nucleus (C). The immunoreactive cells and fibers in the solitary
nucleus stained by P2L-1C are shown in D. Magnification: A, x40; B–D,
x100. All micrographs in this figure were obtained from frontal
sections (A–D). P2L-1T-immunoreactive cell bodies are found in the DM,
solitary nucleus, and spinocerebellar tract. Note the monoclonal
antibody P2L-1T stained many PrRP cell bodies in both the DM and the
solitary nucleus. In contrast to P2L-1T, the P2L-1C antibody stained
many fiber networks in the solitary nucleus, but only slightly stained
the cell bodies. Arrowheads indicate
P2L-1C-immunoreactive cell bodies. C, Central canal; SOL, solitary
nucleus; TSC, spinocerebellar tract; V, ventricle.
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One of the most important results of this investigation was the
demonstration of the distribution of PrRP neurons around the ME.
Although only a small number of PrRP nerve fibers were observed in the
internal region of the ME, pituitary stalk (Fig. 4, A–C), and posterior pituitary gland
(Fig. 4D), no immunopositive neuron was detected in the external zones
of the ME (Fig. 4, A–C). The distribution of the PrRP neurons can be
more clearly followed by combining sagittal and frontal sections. As
shown in Fig. 5, A and B, the
immunoreactive fiber network extended from the solitary nucleus to the
superior cerebellar peduncle, then to the third ventricle.
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Figure 4. Photomicrographs of PrRP neurons adjacent to the
median eminence and posterior pituitary gland. A, Frontal view of the
median eminence. Magnification, x150. B, Sagittal view of the median
eminence. Magnification, x30. C, High power enlargement of B.
Magnification, x150. D, Sagittal view of the posterior pituitary.
Magnification, x150. Note that almost no immunoreactive PrRP neurons
are found in the external zone of the ME (A–C), but a small number of
PrRP neurons are present in the internal zones of the ME, pituitary
stalk, and posterior pituitary. Arrowheads indicate PrRP
fibers. IL, Intermediate lobe; OT, optic tract; PL, posterior lobe; V,
ventricle.
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Figure 5. Photomicrographs showing sagittal views of
P2L-1C-immunoreactive fibers. In A, PrRP fibers are seen to extend from
the superior cerebellar peduncle to the third ventricle. Magnification,
x100. In B, PrRP fibers extend from the solitary nucleus to the
superior cerebellar peduncle. Magnification, x100. In C, the PrRP
fiber network is also seen in the bed nucleus of the stria terminalis.
Magnification, x40. Here the dense PrRP fiber network is distributed
in the ventral portion of anterior commissure to form a ring-like
structure. 3V, Third ventricle; BST, bed nucleus of the stria
terminalis; BC, superior cerebellar peduncle; CA, anterior commissure;
SOL, solitary nucleus.
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The dense PrRP nerve fibers located in the bed nucleus of the stria
terminalis are shown in frontal view in Fig. 2
and also can be seen in
the sagittal view (Fig. 5C). As shown in Fig. 5C, a dense nerve fiber
network surrounds part of the hypothalamus like a circle. It is
important to note that the distribution of PrRP in all of the brain
structures in the rat is not affected or changed by the estrous cycle
(data not shown).
Localization of PrRP-positive cell bodies
Many immunopositive cell bodies were observed at the most caudal
portion of the dorsomedial hypothalamic nucleus (DM; Fig. 6A). A few of such cell bodies were also
observed in the spinocerebellar tract (Fig. 6B). In addition, many
strongly immunopositive cell bodies were observed in the solitary
nucleus (Fig. 6C). The cell bodies of the solitary nucleus were also
stained by the P2L-1C antibody, but were very weakly stained compared
with the P2L-1T antibody (Fig. 6C), as shown in Fig. 6D.
Double immunocytochemistry of oxytocin and vasopressin
Double staining for PrRP and oxytocin or for PrRP and vasopressin
neurons was performed with their specific antibodies. The results
clearly showed that some oxytocin cell bodies were in contact with
PrRP neurons (Fig. 7A). As shown in Fig. 7B, the PrRP nerve terminals were enlarged at the contact site. This
suggests that they were in synaptic contact. The vasopressin neurons,
however, did not make contact with PrRP cells (data not shown).
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Figure 7. Photomicrographs showing the double immunostaining
used to locate PrRP and oxytocin in the paraventricular hypothalamic
nucleus using P2L-1C and oxytocin antibodies. The
oxytocin-immunolabeled cell bodies (OX) are surrounded by
P2L-1C-immunoreactive fibers (A). Note the oxytocin-positive cell body
showing synaptic contact with PrRP-immunopositive fibers (B).
Magnification, x200. PrRP neurons and OX cell bodies show brownand blue staining, respectively.
Arrowheads indicate the synaptic contact.
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The locations of the PrRP neurons and their cell bodies in the rat
brain are schematically represented in Figs. 8 and 9.
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Figure 8. Schematic representation of the PrRP neurons
distribution in the rat brain. Large dots indicate the
PrRP-positive cell bodies, medium dots show the dense
fiber network, and small dots show the rough fiber
network. The brain sections are arranged from rostral to caudal on the
frontal plane. ABL, Basolateral amygdaloid nucleus; BST, bed nucleus of
the stria terminalis; BC, superior cerebellar peduncle; CA, anterior
commissure; CLA, claustrum; CO, optic chiasm; LHA, lateral hypothalamic
area; OT, optic tract; POA, lateral preoptic area; PT, paratenial
thalamic nucleus; PVH, paraventricular hypothalamic nucleus; SO,
supraoptic nucleus; SOL, solitary nucleus; TSC, spinocerebellar
tract.
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Figure 9. Schematic representation showing the distribution
of PrRP neurons in a sagittal section in the rat brain. Large
dots indicate the cell bodies containing PrRP, medium
dots show the dense fiber network, and small
dots show the rough fiber network. BST, Bed nucleus of the
stria terminalis; BC, superior cerebellar peduncle; CA, anterior
commissure; OT, optic tract; PT, paratenial thalamic nucleus; PVH,
paraventricular hypothalamic nucleus; SO, supraoptic nucleus; SOL,
solitary nucleus.
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Neural projections of PrRP fibers
The neural projections of PrRP were followed through
frontal and sagittal sections. PrRP neurons originated from solitary
nucleus ascended to the superior cerebellar peduncle where they then
extended ventrally, as shown Fig. 5, A and B. These PrRP fibers
originating in the solitary nucleus were then joined with fibers
originating from the DM at the dorsocaudal portion of hypothalamus.
Together they extended to the paraventricular hypothalamic nucleus, the
bed nucleus of the stria terminalis, and the paratenial thalamic
nucleus. The PrRP fibers in the stria terminalis then extended to the
amygdala. However, a small number of PrRP neurons in the ME and
posterior pituitary did not follow these tracts.
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Discussion
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The recently discovered PrRP is reported to be a ligand of an
orphan receptor (hGR3) (8). It is also known that this new peptide
(PrRP) has a potent PRL-releasing activity in vitro, and its
receptors are principally located in the pituitary gland. Additionally,
we know that the largest amount of this peptide is located in the
hypothalamus. These topological characteristics of PrRP suggest that
PrRP stimulates the anterior pituitary gland in the same manner as the
known classical hypophysiotropic factors, but if this novel PrRP is
indeed a true hypophysiotropic factor, its neurons must terminate in
the external region of median eminence, where they can release the
peptide into the hypophyseal portal system. In our present study,
however, no PrRP neurons were seen to project into the external region
of the median eminence. This fact, then, leads us to conclude that PrRP
differs from the classical hypophysiotropic factors. This conclusion
requires one to address the discrepancy presented by the observation
that the ligand site and the receptor site are different. It is
therefore necessary to attempt to uncover how the ligand might be
transported to its receptor sites in the anterior pituitary gland. The
presence of PrRP neurons in the posterior pituitary gland lends support
to the possibility that the PrRP might be transported to the anterior
pituitary through a short hypophyseal portal system that may exist
between the anterior and posterior pituitaries (13, 14, 15). However, the
following possibilities also cannot be dismissed. 1) PrRP is not
present in a high enough concentration to be detectable by the
immunocytochemical technique used in this study and thus presents a
negative result in the ME. 2) PrRP exists in the other tissues and
stimulates the anterior pituitary cells through an unknown mechanism.
In any case, further work will be required to resolve the paradox of
receptor and ligand localization.
In this study we are the first to demonstrate that PrRP-immunoreactive
fibers are localized in the paraventricular hypothalamic nucleus,
supraoptic nucleus, paratenial thalamic nucleus, basolateral amygdaloid
nucleus, and bed nucleus of stria terminalis. These fibers are
especially prominent in the bed nucleus of the stria terminalis. It has
been previously reported that the bed nucleus of the stria terminalis
shows sexual dimorphism (16); this is a region close to the preoptic
area where GnRH neurons reside (17). These data then give rise to the
possibility that PrRP is related to sexual activity. To be sure, more
data must be gathered before a definitive statement about this can be
made.
The paraventricular hypothalamic nucleus is another area where dense
PrRP neurons are localized. It is interesting that some immunolabeled
fibers were seen to be in contact with magnocellular neurons.
Therefore, we examined the possible association of PrRP with oxytocin
neurons or with vasopressin neurons, which are well known major
cellular element in the paraventricular hypothalamic nucleus (18). Our
double immunocytochemistry clearly demonstrated that
PrRP-immunolabeled fibers make contact with oxytocin-positive cell
bodies. This, then, suggests that PrRP regulates the function of
oxytocin neurons.
An interesting point is that it is well established that not only PRL
release but also oxytocin release increase in response to nipple
stimulation, a phenomenon considered to be related to the milk let-down
reflex (19, 20). In the same vein, it is also noteworthy that the TRH,
vasoactive intestinal peptide, dopamine, and somatostatin neurons,
which are known as the stimulatory or inhibitory neurons of PRL
release, are also localized in the paraventricular hypothalamic nucleus
(21, 22). This close proximity of PrRP fibers to TRH fibers is
especially pronounced in the bed nucleus of the stria terminalis (10).
However, we need more extensive morphological and physiological
information to resolve the relationship between PrRP neurons and these
peptidergic neurons.
By use of the P2L-1T monoclonal antibody, we have demonstrated
PrRP-immunoreactive cell bodies in the DM, solitary nucleus, and
spinocerebellar tract. We found that especially the DM and solitary
nucleus contained numerous immunopositive cell bodies. As previously
reported, the cell bodies of many peptidergic neurons, such as
epinephrine, norepinephrine, -aminobutyric acid, somatostatin,
dynorphin, and pancreatic polypeptide Y exist in the solitary nucleus
(23), and it is well known that TRH, serotonin, neuropeptide Y, and
dopamine are present in the DM (24). However, the relationship between
PrRP neurons and these peptidergic neurons in the solitary nucleus and
DM remains to be elucidated.
On the other hand, the projections of PrRP-immunopositive fibers into
the amygdala, paraventricular hypothalamic nucleus, and bed nucleus of
the stria terminalis revealed in this paper are in good agreement with
the neural projection sites from the solitary nucleus (23). This, then,
supports the finding that the PrRP neurons in these areas originate in
the solitary nucleus.
The localization of many cell bodies in the DM is an interesting
result. The DM is known to be related to food intake, and DM-lesioned
rats show weight loss (24). Although we do not have any definitive
data, we cannot rule out the possibility that PrRP neurons in the DM
are related to weight regulation. In any case, PrRP can now be included
as a new member of the DM and solitary nucleus, and we believe that the
further study of PrRP will undoubtedly help clarify the function of
these nuclei.
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Acknowledgments
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The authors are grateful to Dr. K. Wakabayashi, Gunma University
of Gunma prefecture Japan, for the generous gift of antiserum to
vasopressin. We also thank Dr. E. F. Couch, Texas Christian
University (Fort Worth, TX), for his kind review of the English.
Received August 18, 1998.
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References
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|---|
-
Lamberts SWJ, Macleod RM 1990 Regulation of
prolactin secretion at the level of the lactotroph. Physiol Rev 70:279–318[Free Full Text]
-
Samson WK, Lumpkin MD, McCann SM 1986 Evidence for
a physiological role for oxytocin in the control of prolactin
secretion. Endocrinology 119:554–560[Abstract]
-
Rivier C, Brown M, Vale W 1977 Effect of
neurotensin, substance P, and morphine sulfate on the secretion of
prolactin and growth hormone in the rat. Endocrinology 100:751–754[Abstract]
-
Erfurth EM, Hender P, Lundin S, Ekman R 1996 Release of prolactin as well as adrenocorticotropin after
administration of arginine-vasopressin to healthy men. Horm Metab Res 28:599–602[Medline]
-
Hammond PJ, Smith DM, Akinsanya KO, Mufti WA, Wynick D,
Bloom SR 1996 Signalling pathways mediating secretory and
mitogenic responses to galanin and pituitary adenylate
cyclase-activating polypeptide in the 235–1 clonal rat lactotroph cell
line. J Neuroendocrinol 8:457–464[CrossRef][Medline]
-
Shin SH, Papas S, Obonsawin MC 1987 Current status
of the rat prolactin releasing factor. Can J Physiol Pharmacol 65:2036–2043[Medline]
-
Dannies PS 1997 A new releasing factor? With
biotechnology and a little bit of luck. Endocrinology 138:5085–5086[Free Full Text]
-
Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M,
Fukusumi S, Kitada C, Masuo Y, Asano T, Matsumoto H, Sekiguchi M,
Kurokawa T, Nishimura O, Onda H, Fujino M 1998 A
prolactin-releasing peptide in the brain. Nature 393:272–276[CrossRef][Medline]
-
Suzuki N, Matsumoto H, Kitada C, Masaki T, Fujino
M 1989 A sensitive sandwich-enzyme immunoassay for human
endothelin. J Immunol Methods 118:245–250[CrossRef][Medline]
-
Lechan RM, Jackson IMD 1982 Immunohistochemical
localization of thyrotropin-releasing hormone in the rat hypothalamus
and pituitary. Endocrinology 111:55–65[Abstract]
-
Ishikawa K, Taniguchi Y, Inoue K, Kurosumi K, Suzuki
M 1988 Immunocytochemical delineation of thyrotrophic area: origin
of thyrotropin-releasing hormone in the median eminence.
Neuroendocrinology 47:384–388[Medline]
-
Ishikawa K, Inoue K, Tosaka H, Shimada O, Suzuki M 1984 Immunohistochemical characterization of thyrotropin-releasing
hormone-containing neurons in rat septum. Neuroendocrinology 39:448–452[CrossRef][Medline]
-
Negm IM 1971 The blood supply of the mouse
hypophysis cerebri. Acta Anat 80:377–387[Medline]
-
Murakami T 1975 Pliable methacrylate casts of blood
vessels: use in a scanning electron microscope study of the
microcirculation in rat hypophysis. Arch Histol Jpn 38:151–168[Medline]
-
Gross PM, Joneja MG, Pang JJ, Polischuk TM, Shaver SW,
Wainman DS 1993 Topography of short portal vessels in the rat
pituitary gland: a scanning electron-microscopic and morphometric study
of corrosion cast replicas. Cell Tissue Res 272:79–88[CrossRef][Medline]
-
Segovia S, Guillamon A 1993 Sexual dimorphism in
the vomeronasal pathway and sex differences in reproductive behaviors.
Brain Res Rev 18:51–74[CrossRef][Medline]
-
Gibson MJ 1986 Role of the preoptic area in the
neuroendocrine regulation of reproduction: an analysis of functional
preoptic homografts. Ann NY Acad Sci 474:53–63[CrossRef][Medline]
-
Swanson LW, Sawchenko PE 1980 Paraventricular
nucleus: a site for the integration of neuroendocrine and autonomic
mechanisms. Neuroendocrinology 31:410–417[Medline]
-
Reichlin S 1989 Neuroendocrinology of the pituitary
gland. Toxicol Pathol 17:250–255[Medline]
-
Dawood MY, Khan-Dawood FS, Wahi RS, Fuchs F 1981 Oxytocin release and plasma anterior pituitary and gonadal hormones in
women during lactation. J Clin Endocrinol Metab 52:678–683[Abstract]
-
Kiss JZ 1988 Dynamism of chemoarchitecture in the
hypothalamic paraventricular nucleus. Brain Res Bull 20:699–708[CrossRef][Medline]
-
Swanson LW, Sawchenko PE, Berod A, Hartman BK, Helle KB,
Vanorden DE 1981 An immunohistochemical study of the organization
of catecholaminergic cells and terminal fields in the
paraventricular and supraoptic nuclei of the hypothalamus. J Comp
Neurol 196:271–285[CrossRef][Medline]
-
Ionescu DA, Lugoji G, Radula D 1986 The nucleus of
the solitary tract: a review of its anatomy and functions, with
emphasis on its role in a putative central-control of brain-capillaries
permeability. Neurol Psychiatry (Bucur) 24:69–85
-
Bernardis LL, Bellinger LL 1987 The dorsomedial
hypothalamic nucleus revisited: 1986 update. Brain Res 434:321–381[Medline]
Top
Abstract
Introduction
), hPrRP31 (
), hPrRP20(
), bPrRP31 (
),
bPrRP-(1–31)OH (
), bPrRP-(1–31)Gly-Arg-OH (
), and
hPrRP-(1–30)amide (
) by means of competitive EIAs using HRP-labeled
PrRP-(17–31) or PrRP-(12–25).