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Department of Physiology, School of Medicine, University of Occupational and Environmental Health (R.S., Y.U., M.N., Y.Y., I.S., H.Y.), Kitakyushu 807-8555; the Department of Foods and Human Nutrition, Faculty of Human Life Sciences, Notre Dame Seishin University (Y.Har., Y.Hat.), Okayama 700-8516; the First Department of Internal Medicine, Miyazaki Medical College (K.Ki.), Kihara Kiyotake, Miyazaki 889-1601; and the National Cardiovascular Center Research Institute (K.Ka.), Fujishirodai, Suita, Osaka 565-0873, Japan; and the Department of Physiology, University Medical School (J.A.R.), Edinburgh, United Kingdom EH8 9AG
Address all correspondence and requests for reprints to: Hiroshi Yamashita, M.D., Ph.D., Department of Physiology, University of Occupational and Environmental Health School of Medicine, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yama{at}med uoeh.-u.ac.jp.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Yokoi et al. demonstrated that intracerebroventricular (icv) administration of AM had no significant effect on the basal plasma arginine vasopressin (AVP) level (11). However, icv administration of AM attenuated the plasma AVP increase induced by hyperosmolality and hypovolemia (11). The hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) are well known to synthesize AVP and oxytocin (OXT) and then release them into the general circulation through terminal axons located in the posterior pituitary. In addition, AM-like immunoreactive (LI) cells exist in the PVN and SON in the rat (12) and in humans (13).
There has been no evidence about whether central AM may have some effects on OXT release. In the present study, first we examined the effects of icv administration of AM on the plasma oxytocin level, using RIA for OXT. Second, we examined the effects of icv administration of AM on c-Fos protein (Fos) and c-Fos messenger RNA (mRNA) in the PVN and SON, using immunohistochemistry and in situ hybridization histochemistry. Finally, we examined the effects of pretreatment with AM receptor antagonists on the expression of c-fos mRNA in the PVN and SON induced by central administration of AM. The expression of the c-fos gene has recently been widely used to detect neuronal activity in the CNS (14). The expression of Fos in the dorsal brain stem, including the area postrema (AP) was also examined using immunohistochemistry, as the AP is thought to be one of the primary sites in the CNS activated by AM (15, 16).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgical procedures
For icv administration of AM, AM antagonist, or vehicle, the
animals were anesthetized (sodium pentobarbital, 50 mg/kg BW, ip
injection) and then placed in a stereotaxic frame in a prone position.
A stainless steel guide cannula (550-µm od; 10.5-mm length) was
implanted stereotaxically, following coordinates given in the atlas of
Paxinos and Watson (17). These coordinates were 0.8 mm posterior to
bregma, 1.4 mm lateral to midline, and 2.0 mm below the surface of the
left cortex, such that the tip of the cannula was 1.5 mm above the left
cerebral ventricle. Two stainless steel anchoring screws were fixed on
the skull, and the cannula was secured in place by dental acrylic
cement. The animals were then returned to their cages and allowed to
recover for at least 5 days. The animals were handled and housed
individually before the start of the experiments.
Central administration of the AM, AM antagonist, or vehicle
For icv administration of AM, a stainless steel injector
(300-µm od) was introduced through the cannula to a depth of 1.5 mm
beyond the end of the guide. The total volume of solution injected into
the lateral ventricle was 10 µl. Rat AM and human
AM-(22–52)-NH2 were purchased from the Peptide Institute (Minoh, Japan). Both were dissolved in sterile 0.9%
saline solution.
Experimental procedure
In the first experiment, icv administration of AM (1, 10
µg/rat) or vehicle was performed (n = 5–8 in each group). Five,
10, and 15 min after the icv injection, the animals were decapitated.
Trunk blood was collected for measurement of plasma OXT level by
RIA.
In the second experiment, icv administration of AM (1, 5, and 10 µg/rat) or vehicle was performed (n = 2 or 3 in each group). Ninety minutes after icv injection, the animals were anesthetized deeply with an ip injection of sodium pentobarbital (75 mg/kg BW) and then used for immunohistochemistry for Fos, OXT, and AVP.
In the third experiment, icv administration of AM (1 and 10 µg/rat) or vehicle was also performed (n = 6 in each group). Thirty minutes after injection, the animals were decapitated, and the brains were removed and then placed on powdered dry ice for in situ hybridization histochemistry for c-fos mRNA. Other animals were decapitated 0, 30, 60, or 180 min after icv administration of AM (5 µg/rat; n = 6 in each group).
In the final experiment, the effects of pretreatment with the AM receptor antagonist, human AM-(22–52)-NH2 (100 µg/5 µl saline, icv) on the induction of c-fos gene expression by icv administration of AM (1 µg/5 µl saline) were examined.
All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.
RIA for OXT
Plasma OXT levels were determined using a RIA with a specific
OXT antiserum and [125I]OXT tracer as previously
described (18), using antiserum provided by Prof. T. Higuchi. The
sensitivity of the assay was 3–5 pg/ml. The inter- and intraassay
variations were 12% and 15%, respectively.
Dual detection of Fos and OXT/AVP immunoreactivities
Deeply anesthetized animals were perfused transcardially with
0.1 M phosphate buffer (PB; pH 7.4) containing heparin
(1000 U/liter) followed by 4% paraformaldehyde and 0.2% picric acid
in 0.1 M PB. The brains were then removed and divided into
blocks, which included the hypothalamus and the brain stem. The blocks
were postfixed with 4% paraformaldehyde and 0.2% picric acid in 0.1
M PB for 48 h at 4 C. The tissues were then
cryoprotected in 20% sucrose in 0.1 M PB for 24 h at
4 C. Serial sections of either 30 µm for dual staining for Fos and
OXT/AVP or 40 µm for immunostaining for Fos were cut using a
microtome. The sections were rinsed twice with 0.1 M PBS
containing 0.3% Triton X-100 and then incubated in 0.1 M
PBS containing 0.3% Triton X-100 with 1% hydrogen peroxidase for 60
min. The sections were rinsed twice with 0.1 M PBS
containing 0.3% Triton X-100. The floating sections were incubated
with a primary Fos antibody (sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then diluted at 1:100 in 0.1
M PBS containing 0.3% Triton X-100 at 4 C for 3 days.
After washing for 20 min in 0.1 M PBS solution containing
0.3% Triton X-100, the sections were further incubated for 120 min
with a biotinylated secondary antibody solution (1:250) and finally
with an avidin-biotin peroxidase complex (Vectastain ABC
kit, Vector Laboratories, Inc., Burlingame, CA) for 120
min. The peroxidase in the sections was visualized with 0.02%
diaminobenzidine in a Tris buffer containing 0.05% hydrogen peroxidase
for 10–15 min. In the dual staining for OXT and AVP, they were
sequentially incubated in OXT antibody (INCSTAR Corp.,
Stillwater, MN; diluted 1:2000) or AVP antibody (INCSTAR Corp.; diluted 1:6000) for 5 days each at 4 C. The avidin-biotin
peroxidase complex was visualized with simainobenzidine using nickel
sulfate enhancement. Both OXT- and AVP-like immunoreactivity (LI) were
revealed to be a violet cytoplasmic and axonal precipitate. Fos-LI was
revealed as dark brown-labeled nuclei. Details of the
imunohistochemistry have been published previously (19, 20). The
sections were mounted onto slides coated with gelatin, air-dried,
dehydrated in 100% ethanol, cleared with xylene, and then finally
coverslipped. To count the double labeled cells, four serial sections,
including the PVN and SON, per animal were chosen and observed by
microscope.
In situ hybridization histochemistry for c-fos mRNA
In situ hybridization histochemistry was performed on
frozen 12-µm thick coronal brain sections cut by cryostat at -20 C,
thawed, and mounted onto gelatin/chrome alum-coated slides that had
been kept at -80 C until needed. The locations of the PVN and SON were
determined according to coordinates given by Paxinos and Watson (17).
Eight sites from four sections in the PVN and SON per rat were used to
measure the density of the autoradiographs. The slides were warmed to
room temperature and allowed to dry for 10 min, then fixed in 4%
formaldehyde in PBS for 5 min. They were then washed twice in PBS and
incubated in 0.9% NaCl containing 0.25% acetic anhydride (vol/vol)
and 0.1 M triethanolamine at room temperature for 10 min.
The sections were then dehydrated using a series of 70% (1 min), 80%
(1 min), 95% (2 min), and 100% (1 min) ethanol solutions in a
stepwise manner and delipidated in 100% chloroform for 5 min. The
slides were then partially rehydrated in first 100% (1 min) and then
95% (1 min) ethanol and allowed to dry briefly in air. Hybridization
was performed at 37 C overnight in 45 µl buffer solution consisting
of 50% formamide and 4 x SSC (1 x SSC = 150
mM NaCl and 15 mM sodium citrate), which
contains 500 µg/ml sheared salmon sperm DNA (Sigma Chemical Co., St. Louis, MO), 250 µg/ml baker’s yeast total RNA
(Boehringer Mannheim, Mannheim, Germany), 1 x
Denhardt’s solution, and 10% dextran sulfate (500,000 mol wt;
Sigma Chemical Co.). The hybridization was performed under
a Nescofilm (Bando Chemical IMD Ltd., Osaka, Japan) coverslip. A
35S 3'-end-labeled deoxyoligonucleotide complementary to
transcripts coding for c-fos (complementary to bases
138–185 of the rat c-fos gene) and OXT (complementary to
bases 912–941 of rat OXT nucleotides) was used. The specificity of the
probes has been described previously (21, 22). A total of 1 x
106 cpm/slide for c-fos transcripts was used. A
total of 3 x 105 cpm/slide for OXT transcripts was
used. After hybridization, the sections were washed for 1 h in
four separate 1 x SSC rinses at 55 C and for a further 1 h
in two changes of 1 x SSC at room temperature. All independent
experimental sections were treated simultaneously to minimize the
variable effects of hybridization and wash stringency. Hybridized
sections of the PVN and SON were apposed using autoradiography film
(Hyperfilm, Amersham, Aylesbury, UK) for 7–14 days for
c-fos transcripts and 8 h for OXT transcripts. The
resulting images were analyzed by computerized densitometry using an
MCID imaging analyzer (Imaging Research, Inc., Ontario,
Canada). The mean optical density of autoradiographs was measured by
comparing it with simultaneously exposed 14C microscale
samples (Amersham). Slides hybridized with the
c-fos probe were dipped in a nuclear emulsion (K-5, Ilford,
Cheshire, UK) and further exposed for 28 days.
Statistical analysis
Data for plasma oxytocin levels are expressed as the mean
± SEM. Each group within an experiment was compared with
the control group. The data were analyzed using a one-way fractional
ANOVA followed by a Bonferroni-type adjustment for multiple comparison.
The statistical significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the changes in plasma OXT level
after central administration of AM or vehicle.
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Figure 2C showed that parvocellular cells predominantly expressed
Fos-LI, and few magnocellular cells expressed Fos-LI in the PVN. The
Fos-LI in the magnocellular cells was particularly within the
ventromedial zone, where there are OXT cells that are likely to be
projecting to the medulla. A few Fos-LI cells were observed in the PVN
and SON after icv administration of vehicle (saline; Fig. 2, A and D).
The pattern of distribution of the Fos-LI in the PVN and SON after
administration of 1 µg AM/rat icv was not different from that after
administration of 10 µg/rat (Fig. 2, B and C, E, and F). The 5
µg/rat dose also caused a similar induction of Fos-LI in the PVN and
SON. Fos-LI cells were seen throughout the ependymal cells of the
cerebral ventricles (Fig. 2, A, B, and C). In the brain stem, Fos-LI
cells were observed in the AP and the nucleus of the tractus solitarius
(NTS) after icv administration of AM (10 µg/rat; Fig. 3, C and F). Fos-LI cells in the AP after
AM administration were distributed in the peripheral regions of the
rostral and ventral AP. Although Fos-LI cells were observed in the AP
and NTS after the administration of 1 and 5 µg AM/rat, these were
fewer than after 10 µg/rat (Fig. 3).
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, B and E). The
numerous Fos-LI cells in the parvocellular parts of the PVN were
reactive for neither OXT nor AVP (Fig. 4, C and F). In the SON, OXT-LI
cells predominated in the nuclear Fos-LI region compared with AVP-LI
cells (Fig. 5).
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Effects of icv administration of AM on c-fos gene induction in the
hypothalamus
In situ hybridization histochemistry revealed
that c-fos gene expression in the PVN and SON increased in a
dose-related manner 30 min after icv administration of AM (1 and 10
µg/rat; Fig. 6A). Thirty minutes after
administration of AM (1 µg/rat), c-fos gene expression
increased significantly in the PVN (3995 ± 873.8 vs.
1012 ± 170.4 arbitrary units) and SON (2203 ± 166.1
vs. 639.7 ± 35.0 arbitrary units; n = 6 in each
case; P < 0.01; Fig. 6A). Thirty minutes after icv
administration of AM (10 µg/rat), c-fos gene expression
had also increased significantly in the PVN (12740 ± 2575
vs. 1012 ± 170.4 arbitrary units) and SON (5794
± 746.0 vs. 639.7 ± 35.0 arbitrary units; n = 6
in each case; P < 0.01; Fig. 6A). The icv
administration of AM at 0, 30, 60, and 180 min (5 µg/rat) induced
expression of the c-fos gene to the greatest degree at 30
min in the PVN (5339 ± 757.4 arbitrary units) and SON (2427
± 68.7 arbitrary units; Figs. 6B and 7). The expression of the
c-fos gene in the PVN began to decrease by 60 min and
eventually reached basal levels 180 min after icv administration of AM
(5 µg/rat). The expression of the c-fos gene in the SON
also decreased after 30 min, but remained significantly above basal
levels even 60 and 180 min after icv administration of AM (5 µg/rat).
The expression of the c-fos gene in the PVN and SON at
0, 30, 60, and 180 min after icv administration of vehicle did not
change significantly (Fig. 6B). Strong expression of the
c-fos gene was seen throughout the ependymal cells of the
cerebral ventricle 30 min after icv administration of vehicle (Fig. 7, F and N).
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Effects of icv pretreatment with an AM antagonist on AM-induced
c-fos gene expression
Thirty minutes after icv administration of AM (1 µg/5 µl), the
expression of the c-fos gene had significantly increased in
the PVN (18,680 ± 868.5 vs. 5981 ± 364.0
arbitrary units in vehicle controls) and SON (10,600 ± 460.0
vs. 3948 ± 233.6 arbitrary units in controls; n =
6 in each case; P < 0.01; Fig. 8). Thirty minutes after icv
administration of the AM receptor antagonist, human
AM-(22–52)-NH2 (100 µg/5 µl), and AM (1 µg/5 µl),
the expression of the c-fos gene had increased in the PVN
(10,440 ± 813.1 arbitrary units) and SON (5,808 ± 203.8
arbitrary units; n = 6 in each case; P < 0.01).
However, the induction of c-fos gene expression as caused by
icv administration of AM was significantly reduced by pretreatment with
the AM receptor antagonist, human AM-(22–52)-NH2 (Figs. 8 and 9; P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding from dual immunohistochemical staining for Fos and OXT/AVP that Fos was predominantly in OXT-LI cells in the PVN and SON indicates that central AM preferentially activates OXT-producing magnocellular neurosecretory cells in the PVN and SON. We did not detect a significant change in the expression of the OXT gene after AM injection. It may be difficult to determine whether transcription of the OXT gene is rapidly activated by icv administration of AM because of the large cytoplasmic pool of OXT mRNA.
Physiological stimuli such as hyperosmolality and hypovolemia stimulate the release of both AVP and OXT (23). However, systemic cholecystokinin is known to stimulate the release of OXT, but not AVP, in rats (24). It has been demonstrated in previous studies that peripheral administration of cholecystokinin activates OXT-secreting cells in the PVN and SON via noradrenergic neurons in the caudal NTS in the region of the brain stem receiving visceral vagal afferents (25, 26). Verbalis et al. reported that central OXT may inhibit salt appetite and feeding behavior (27). They suggested that release of OXT into the systemic circulation may correlate with the activation of central OXTergic pathways. The dual immunostaining study indicates that activation of both centrally OXT projecting neurons and magnocellular OXT neurons projecting to the posterior pituitary may be elicited by central administration of AM. Central administration of AM also inhibited salt appetite, which had been induced by peripheral administration of polyethylene glycol (10), and feeding induced by fasting (28). It is thus hypothesized that the inhibitory effects of central AM on salt appetite and feeding may involve central OXTergic pathways. It has been further shown that icv administration of AM inhibited water drinking, which had been induced by icv administration of angiotensin II, dehydration, and hyperosmotic challenge (9). However, it is unclear whether a central OXTergic pathway is associated with the inhibitory effects of AM on water-drinking behavior.
In the present study only a few AVP-LI cells in the PVN and SON exhibited nuclear Fos-LI after icv administration of AM. Recent studies have demonstrated that icv administration of AM had no significant effect on the basal level of plasma AVP in the rat (11) or sheep (29) and, further, that it inhibited the release of AVP that had been evoked by hypertonic and hypovolemic stimulation in the rat (11). Thus, central AM may not stimulate the release of AVP under basal conditions.
The present study showed that expression of the c-fos gene in the PVN and SON was increased transiently after icv administration of AM in a dose-related manner. However, it remains unclear whether centrally administered AM induces the expression of the c-fos gene and Fos in the PVN and SON directly or indirectly. The distribution of Fos-LI cells in the PVN and SON 90 min after the icv administration of AM coincided with that in c-fos mRNA-expressing cells. As we did not perform a time-course study of the expression of Fos-LI and c-fos mRNA in the PVN and SON after icv administration of AM, we could not exclude the possibility that there might be a discrepancy between the expression of Fos-LI and c-fos mRNA in the PVN and SON after central administration of AM. The rapid and transient increase in plasma OXT levels is quite dramatic. The elevation of plasma OXT reached a peak 10 min after the icv administration of AM and returned to normal 5 min later. On the other hand, the marked increase in c-fos mRNA in the PVN and SON was observed 30 min after icv administration of AM. The number of Fos-LI cells in the PVN and SON was also increased 90 min after icv administration of AM. The effects of the centrally administrated AM on plasma OXT is much faster than the induction of c-fos mRNA and Fos in the PVN and SON when plasma OXT levels returned to normal. Therefore, it should be determined whether both events might be significantly related or independent of each other. At the least, induction of Fos and c-fos mRNA in the PVN and SON should reflect the neural activation either directly or indirectly. Given that previous studies have demonstrated that AM-LI cells exist in the PVN and SON in the rat (12) as well as in humans (13), the possibility exists, then, that the neuronal activity of neurons in the PVN and SON is modulated by the release of AM in an autocrine and/or paracrine manner.
Central administration of AM causes vasopressor and sympathetic responses in anesthetized rats (7) and conscious rats (8). As the PVN is known to be an integrative site for the coordination of neuroendocrine and autonomic functions (30, 31), centrally administered AM may activate the PVN neurons that project to the regions of the brain stem and the spinal cord related to the sympathetic outflow. In the present study we demonstrated that icv AM induced the expression of Fos and the c-fos gene in the subdivision of the PVN that may project to the brain stem and spinal cord. Central administration of AM causes hemodynamic changes that may be responsible for the expression of the c-fos gene within autonomic-related nuclei in the PVN. Central administration of AM provokes an alteration in blood pressure, which consequently activates the regions involved in the central cardiovascular system. The high doses of AM (5 and 10 µg/rat) used in the present study caused strong Fos expression in the PVN; this induction of Fos and c-fos gene expression after administration of icv administration of AM (5 and 10 µg/rat) may depend on hemodynamic changes. However, the lowest dose of AM (1 µg/rat) used here caused Fos expression in the PVN and SON. As this dose of centrally administered AM (1 µg/rat) does not cause any corresponding change in blood pressure (9, 11), this induction of Fos and c-fos gene expression after icv administration of AM (1 µg/rat) may be independent of changes in blood pressure.
The presence of Fos-LI after icv administration of AM was also observed in the parvocellular parts of the PVN. These parts of the PVN comprise cells containing hypophysiotropic factors, such as CRH- and TRH-producing cells. As the possibility that AM may play an important role in the regulation of the hypothalamo-pituitary-adrenal axis has been suggested (32, 33), the central effects of AM may include actions on the hypothalamo-adenohypophysial system as well as on the hypothalamo-neurohypophysial system.
In the present study it was demonstrated that pretreatment with a selective AM receptor antagonist partially blocked AM-induced c-fos mRNA induction in the PVN and SON. Centrally administered AM-induced suppression of feeding and vasopressor and sympathetic responses are partially blocked by pretreatment with a calcitonin gene-related peptide (CGRP) antagonist (8, 28). The possibility exists that AM-induced c-fos mRNA induction in the PVN and SON may be mediated not only by AM-selective receptors but also by CGRP receptors. A recent study demonstrated that a combination of calcitonin receptor-like receptor and receptor activity-modifying proteins, which are members of a new family of single transmembrane domain proteins, play an important role in AM and CGRP receptors (34). However, the regional expression of calcitonin receptor-like receptor and receptor activity-modifying proteins and their distribution in the CNS have not been identified.
In conclusion, centrally administered AM may activate OXTergic cells in the PVN and SON in part via an AM-selective receptor.
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Acknowledgments |
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Footnotes |
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Received June 18, 1998.
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