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Max Planck Institute of Psychiatry (M.B.M., R.L., J.P., I.S., A.E.K., M.E.K., S.Z., F.H., W.W.), 80804 Munich, Germany; GSF Research Center, Institute for Mammalian Genetics (W.W.), 85764 Munich, Germany; and Department of Medical Biochemistry and Biophysics, Molecular Neurobiology Division, Karolinska Institute (A.E.K.), Stockholm, Sweden
Address all correspondence and requests for reprints to: Wolfgang Wurst, Ph.D., Max Planck Institute of Psychiatry, Molecular Neurogenetics, Kraepelinstrasse 2–10, 80804 Munich, Germany. E-mail: wurst{at}mpipsykl.mpg.de
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Introduction |
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Recently, mice deficient for the CRHR1 (Crhr1-/-) have been created (10, 11), and their phenotype confirms the obligatory role of CRHR1 in both the stress-associated response of the HPA system and anxiety; in particular, Crhr1-/- mutants display a severe impairment of stress-induced HPA system activation and marked glucocorticoid deficiency and exhibit significantly reduced anxiety-related behavior. However, despite the lack of functional CRHR1 on pituitary corticotropes, basal plasma ACTH concentrations in homozygous Crhr1-/- mutants are similar to those found in wild-type controls (10), suggesting that basal ACTH secretion is regulated or compensated for by signaling pathways other than CRH/CRHR1.
The neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) are mainly synthesized in the magnocellular neurons of the hypothalamic paraventricular (PVN) and supraoptic nuclei that project to the posterior pituitary. In addition, parvocellular neurons of the PVN coexpressing AVP and CRH coordinate HPA system activity and project to the external layer of the median eminence, where AVP and CRH are released into the portal blood (for review, see Refs. 12 and 13). Numerous investigations have consistently shown that AVP potently synergizes with CRH to stimulate pituitary ACTH release in vitro and in vivo (for review, see Ref. 12). A recent study provided indirect evidence that vasopressin might be activated to maintain HPA system activity in Crhr1-/- mutants (14). OXT, although less effectively than AVP, has also been shown to markedly potentiate CRH-induced ACTH release from pituitary corticotropes in rodents (15, 16).
Although the ACTH secretagogue potency of both AVP and OXT has been extensively described, little information is available about 1) whether one or both neuropeptides may compensate for disrupted CRH/CRHR1 signaling in mice congenitally deficient for the CRHR1, and 2) whether such a compensatory role of AVP and OXT, having been characterized mainly in the rat (17, 18, 19, 20), is also relevant in mice. Therefore, we investigated the roles of AVP and OXT in compensatorily maintaining HPA system activity in the absence of functional CRHR1, both under basal conditions and in response to ethologically relevant stressors.
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Materials and Methods |
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Homozygous and heterozygous transgenic mice deficient for the CRHR1 and wild-type controls were generated as originally described by Timpl et al. (10). The present experiments were performed with 8- to 12-week-old male mice, weighing 25–35 g. No significant difference in body weight was observed between homozygous Crhr1-/- mutants and heterozygous and age-matched wild-type animals. In addition, homozygous Crhr1-/- mutants do not show any significant difference in their total amount of water and food intake (21). Measurement of plasma sodium concentrations revealed no significant difference between male wild-type mice and homozygous Crhr1-/- mutants (wild-type mice, 157 ± 4.7 mmol/liter; homozygous Crhr1-/- mutants, 161 ± 5 mmol/liter; n = 8). The animals were housed four to six per cage in the breeding unit of the Max Planck Institute of Psychiatry under standard conditions with a 12-h light, 12-h dark cycle (lights on from 0600–1800 h; 22 ± 1 C; 40–60% humidity) and received standard pelleted food and water ad libitum.
Blood collection and stress experiments
Two weeks before the experiments, animals were separated and
housed singly to avoid uncontrolled stress reactions.
Basal hormone levels. To determine the basal morning plasma levels of AVP, OXT, ACTH, CORT, and sodium, mice (n = 6–9/genotype) were left undisturbed throughout the night before the experiment. Blood sampling was performed in the early morning (0700–0800 h) by rapid retroorbital bleeding, with time from first handling of the animal to completion of bleed not exceeding 45 sec. Blood was sampled into prechilled EDTA tubes.
Poststress levels of hormones.
Forced swim test.
Animals were subjected to the forced swim
stress as a predominantly physical stressor. On the day of testing,
between 0800–1200 h, each mouse (n = 6–8/genotype) was placed
for 5 min in a glass beaker filled with tap water (21 C; diameter, 12.5
cm; height, 14 cm). Subsequently, the animals were returned to their
homecages, and blood collection was performed by retroorbital bleeding
5 min after the end of stress exposure.
Social defeat.
Male, singly housed mice served as resident
stimulus animals for the experimental subjects. In preparation of the
social defeat stress procedure, male resident mice were evaluated for
their display of aggressive behavior by placing a group-housed intruder
male into the resident’s homecage. Usually, within three tests the
resident reliably attacked the intruder within less than 2 min (adapted
with modifications from Ref. 22). Social defeat stress
consisted of introducing a naive, singly housed, experimental mouse
(intruder; male homozygous
Crhr1-/-, heterozygous
mutants, and wild-type mice; n = 6–8/group) into the resident’s
homecage. Immediately after being attacked by the resident for the
first time, the intruder was separated from the resident by wire mesh
within the resident’s homecage. The resident continued to attack and
threaten the intruder while the latter was protected from physical
injury but was exposed to auditory, visual, and olfactory stimulation
for 5 min. Subsequently, the experimental mouse was returned to the
homecage, and blood collection was performed by retroorbital bleeding 5
min after the end of the stressor.
Administration of an AVP receptor (V1)
antagonist
After 1 week of daily handling, male homozygous and heterozygous
Crhr1-/-
mutants and wild-type littermates (8–12 weeks; n = 6/group) were
sc injected with either vehicle or 5 µg of a selective
V1 receptor antagonist
(dP[Tyr(Me)2]AVP, provided by Dr. M. Manning,
Toledo, OH) (23, 24) dissolved in 50 µl 0.9% saline
(injection time, 0800–0830 h). One hour after the injection, the
animals were killed by rapid decapitation, and trunk blood was
collected for determination of plasma ACTH levels.
Measurement of plasma AVP after corticosterone (CORT)
treatment
Male homozygous
Crhr1-/-
mutants and wild-type littermates (8–12 weeks; n = 8/group) were
housed singly and received standard pelleted food and water ad
libitum.
Oral administration of CORT was performed by adding water-soluble CORT 21-sulfate (Sigma, Deisenhofen, Germany; 13.5 mg/liter) to the drinking water. After the animals had been given CORT 21-sulfate continuously for 2 weeks, they were left undisturbed throughout the night before the experiment. Blood collection was performed between 0700–0800 h by rapid retroorbital bleeding, and blood samples for measurements of plasma AVP and CORT concentrations were collected.
Treatment of blood samples and hormone analysis
Blood samples were collected in prechilled tubes containing EDTA
and a protease inhibitor (10 µl aprotinin; Trasylol, Bayer Corp., Leverkusen, Germany) and centrifuged (10 min, 3500
rpm, 4 C). Plasma samples were stored at -80 and -20 C until assay.
Plasma ACTH (50 µl) and CORT (10 µl) levels were measured using
commercially available kits (Biochem, Freiburg, Germany) according to
the respective protocols. AVP and OXT contents were measured in
lyophilized plasma samples after extraction by highly sensitive and
selective RIAs [detection limit, 0.1 pg/sample; cross-reactivity of
the antisera with other related peptides, including AVP (for the
anti-OXT antiserum) and OXT (in case of the anti-AVP antiserum,
respectively), <0.7%] (25). Synthetic AVP and OXT
(Ferring Pharmaceuticals Ltd., Malmo, Sweden) were used as
standards, and iodinated nonapeptide (SA, 2,200 Ci/mmol; NEN Life Science Products, Boston, MA) was used as a tracer. Rabbit
antibodies raised in our laboratory were used at a dilution of
1:350,000.
AVP and OXT in situ hybridization histochemistry
Animals (n = 5) were killed with an overdose of
halothane. Thereafter, brains were quickly removed. Tissue was frozen
on dry ice and stored at -80 C. For subsequent in situ
hybridization experiments, brains (n = 5/genotype) were sectioned
in a cryostat. All brains were cut in five parallel series (20 µm,
coronal sections, spanning the region of the PVN from bregma -0.70 mm
to bregma -1.22 mm), with every fifth section being thaw-mounted on
the same glass slide. Sections were stored at -20 C until use.
The following oligonucleotide DNA probes were used for in situ hybridization: AVP (48-mer), 5'-GCA GAA GGC CCC GGC CGG CCC GTC CAG CTG CGT GGC GTT GCT CCG GTC-3' (directed against the last 16 amino acids of the glycoprotein that are not shared with oxytocin) (26); and oxytocin (48-mer), 5'-CTC GGA GAA GGC AGA CTC AGG GTC GCA GGC GGG GTC GGT GCG GCA GCC (directed against nucleotides 890–937 of the rat oxytocin sequence) (26). The specificity of these oligonucleotides has been described in detail previously (27).
All in situ hybridization experiments were carried out as
previously described in detail (28), and for each
oligonucleotide probe all sections were run in the same experiment
under identical conditions. Briefly, the synthesized
oligonucleotides were labeled at the 3'-end with
[
-35S]deoxy-ATP (NEN Life Science Products-DuPont, Boston, MA) using terminal dexoynucleotidyl
transferase (Roche, Mannheim, Germany). Radiolabeled probe
(106 cpm/200 µl/slide) was diluted into
hybridization buffer consisting of 1 x Denhardt’s solution, 0.25
mg/ml yeast transfer RNA (Sigma), 0.5 mg/ml salmon sperm
DNA (Sigma), 10% dextran sulfate, 10
mM dithiothreitol, and 50% formamide; applied to
the slides; and incubated for 20 h at 42 C. After hybridization,
the slides were washed in 1 x SSC (55 C) four times for 15 min
each time, dehydrated in ethanol, and air-dried. Finally, the slides
were dipped in Kodak NTB2 emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 in distilled water, exposed for
5 weeks at 4 C, and then developed in Kodak D19 solution.
The developed slides were lightly counterstained with cresyl
violet and examined using a Leica Corp. microscope
(Bensheim, Germany) with both bright- and darkfield
condensers.
Immunohistochemistry
Animals (n = 4/genotype) were deeply anesthetized with
phenobarbital and transcardially perfused with phosphate-buffered 4%
paraformaldehyde. Brains were removed from the skull, postfixed for
6 h in 4% paraformaldehyde, and then transferred to 15% sucrose
in PBS (pH 7.4). Serial 30-µm coronal frozen sections were cut in a
cryostat into PBS and processed as free floating sections. For each
animal, all sections spanning the region of the median eminence
(between bregma -1.58 mm and bregma -2.30 mm) were analyzed to allow
for exact comparison of the different genotypes. All of the following
steps were interposed by copious washes in PBS, and all reagents for
immunohistochemistry were diluted in PBS with 1% BSA unless otherwise
specified. After blocking endogenous peroxidase in absolute methanol
with 0.01% hydrogen peroxide for 15 min, preincubation with 5% normal
goat serum for 2 h was performed. The sections were then incubated
with the primary antibody diluted 1:10,000 (polyclonal rabbit anti-AVP
antibody (IHC 8103, Peninsula Laboratories, Inc., Belmont,
CA) at 4 C. The specificity of this antibody has been tested by the
manufacturer. The sections were then incubated with a biotinylated goat
antirabbit secondary antibody diluted 1:300 for 45 min at room
temperature (Vector Laboratories, Inc., Burlingame,
CA), followed by incubation with avidin-biotinylated horseradish
peroxidase complex (ABC Elite Universal kit, Vector Laboratories, Inc.) for 45 min. at room temperature (dilution, 1:300). Finally,
the sections were developed in a substrate solution of 0.05%
diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in
0.05 M Tris-HCl (pH 7.6), washed, mounted on glass slides,
air-dried, and lightly counterstained with hematoxylin. Appropriate
negative controls were performed by omission of the primary
antibody.
Optical densitometry
Representative areas of the hypothalamic PVN were scanned
by a digital camera under dark-light conditions. Care was taken to scan
all images under identical light conditions. Quantitative analysis of
messenger RNA (mRNA) expression was performed blind to the animal’s
genotype as previously described (28), using the
Macintosh-based public domain image analysis program NIH image, version
1.6.1 (developed at the NIH and available on the internet at
http://rsb.info.nih.gov./nih-image). At least three parallel tissue
sections per animal and region were analyzed, and the mean values for
each animal and region were calculated.
AVP mRNA. Levels of mRNA expression (optical density) were determined by measuring the mean gray value on inverted (in situ hybridization signal, black), automatically thresholded images in the PVN, analyzing the left and right hemispheres separately. The region of interest was outlined according to adjacent sections that had been stained with cresyl violet to determine orientation and the precise anatomical localization and borders of the nuclei. As the three-dimensional structure of these hypothalamic nuclei varies from rostral to caudal, at least three parallel sections per animal and region were analyzed, spanning the section containing the highest signal intensity.
OXT mRNA. As neurons expressing OXT mRNA are scattered throughout the PVN, quantitative analysis of OXT mRNA expression was performed as 1) an automatic count of labeled objects (=cells) on thresholded and binary images, and 2) an automatic measurement of the optical density over each object (cell) automatically outlined, representing the density of silver grains per labeled cell. The means for both the number of labeled cells and the optical density per cell were calculated for each animal.
Statistical analysis
Results are presented as the mean ± SEM.
Statistical analysis was performed with a software package (GB Stat,
version 6.5 PPC, Dynamic Microsystems, Inc., Silver Spring, MD).
Statistical significance of differences between groups was determined
by ANOVA (one-way ANOVA, factor genotype, or two-way ANOVA, factors
genotype and treatment, where appropriate), followed by
post-hoc Newman-Keuls test. A significant difference in
plasma sodium concentration was determined by Mann-Whitney U test.
P < 0.05 was considered statistically significant.
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Results |
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).
Post-hoc analysis revealed significantly higher basal
morning plasma AVP levels in
Crhr1-/-
mice compared with wild-type mice (P < 0.01) and
heterozygous mutants (P < 0.01). After forced swim
stress or social defeat, plasma AVP in homozygous
Crhr1-/-mice
fell to levels indistinguishable from those in heterozygous mutants or
wild-type controls. Significant differences in poststress plasma AVP
levels compared with basal morning plasma AVP concentrations were
detected in homozygous
Crhr1-/-
mice only (forced swim stress, P < 0.01; social
defeat, P < 0.01).
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). Basal morning plasma
oxytocin levels did not differ significantly among the three genotypes.
In all three groups and in both stress paradigms, poststress plasma OXT
levels were significantly increased compared with the respective basal
morning levels (forced swim: wild-type, P < 0.05;
heterozygous mutants, P < 0.01; homozygous
Crhr1-/-
mutants, P < 0.01; social defeat: wild-type,
P < 0.01; heterozygous mutants, P <
0.01; homozygous
Crhr1-/-
mutants, P < 0.01). In wild-type mice only, plasma OXT
levels after social defeat were significantly higher than the
respective plasma concentrations after forced swim stress
(P < 0.01).
The stress-induced increase in plasma ACTH and CORT levels is
severely impaired in homozygous Crhr1-/-mutants: differential effects of emotional vs. combined physical and
emotional stressor on ACTH release
Plasma ACTH levels. Two-way ANOVA revealed a significant
effect of both genotype (P = 0.01) and treatment condition
(P < 0.0001), with a significant interaction between
both factors (P < 0.0001; Fig. 1). Under basal
conditions, no significant differences in plasma ACTH levels were
observed. Both forced swim stress and social defeat induced a
significant increase in plasma ACTH in wild-type mice compared with
basal levels (forced swim, P < 0.01; social defeat,
P < 0.01). In heterozygous mutants and homozygous
Crhr1-/-
mutants, a significant increase in plasma ACTH was observed after
social defeat only (heterozygous mutants, P < 0.01;
homozygous
Crhr1-/-
mutants, P < 0.05).
Plasma CORT levels. Two-way ANOVA revealed a significant
effect of both genotype (P < 0.0001) and treatment
condition (P < 0.0001) as well as a significant
interaction of both factors (P < 0.0001). No
significant differences between the groups could be detected in basal
morning plasma CORT levels; however, basal morning levels of CORT in
homozygous
Crhr1-/-
mutants were consistently below the assay detection limit. Both
heterozygous mutants and wild-type mice showed a significant increase
in plasma CORT levels after forced swim stress (heterozygous mutants,
P < 0.01; wild-type mice, P < 0.01;
Fig. 1) and social defeat stress (heterozygous mutants,
P < 0.01; wild-type mice, P < 0.01;
Fig. 1). In homozygous
Crhr1-/-
mutants, in contrast, plasma CORT concentrations did not increase
significantly after any of the stress conditions. No differential
effects of the different stressors on plasma CORT levels were observed
in either wild-type or heterozygous mice.
Plasma ACTH concentrations decrease after administration of a
V1 receptor antagonist in homozygous
Crhr1-/- mutants
Administration of a selective V1 receptor
antagonist under resting conditions significantly decreased plasma ACTH
levels in homozygous
Crhr1-/- mice
compared with those in both vehicle-treated mutants (P
< 0.01) and antagonist-treated wild-type mice (P <
0.01; Fig. 2). There was no statistical
difference between the two vehicle-treated groups. The
V1 receptor antagonist did not exert any
significant effect on plasma ACTH levels in the wild-type controls.
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and 3).
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) compared with wild-type
littermates. No obvious difference was found between heterozygous
mutants and wild-type controls (data not shown). In homozygous Crhr1-/- mice, the zona externa displayed many prominent and strongly immunoreactive axons, whereas in wild-type mice, only weakly immunoreactive structures were present in the zona externa. No obvious difference in the number of AVP-immunoreactive neurons in the PVN could be detected between the different genotypes, nor was there any clear difference in the distribution pattern or projection sites of paraventricular neurons in homozygous Crhr1-/- mutants (data not shown).
Administration of CORT normalizes basal plasma AVP levels in
homozygous Crhr1-/- mutants
After continuous oral treatment with CORT 21-sulfate for
2 weeks, basal plasma CORT levels were indistinguishable between
homozygous
Crhr1-/-
mutants and wild-type controls (wild-type mice, 22.3 ± 6.3 ng/ml;
homozygous
Crhr1-/-
mutants, 18.7 ± 4.1 ng/ml; Fig. 4).
Concomitantly, basal plasma AVP concentrations decreased to levels
indistinguishable from those in control mice (wild-type mice, 2.5
± 1.3 pg/µl; homozygous
Crhr1-/-
mutants, 2.1 ± 0.6 pg/µl; Fig. 4).
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Selective activation of the vasopressinergic system maintains basal
HPA system activity in the absence of CRHR1
Since the discovery of CRH by Vale et al.
(29), it was rapidly established that AVP potently
synergizes with CRH to stimulate pituitary ACTH release; when CRH and
AVP are given together, hormone output is well above the added effects
of the two peptides alone in both rodents and humans (30, 31). This CRH/AVP synergism is known to be functionally relevant
under both physiological (19) and pathophysiological
conditions, such as stress (17, 20) or glucocorticoid
deficiency (18, 32). AVP is predominantly synthesized in
magno- and parvocellular neurons of the hypothalamic supraoptic nucleus
and PVN. Distinct neuronal entities and anatomical subnuclei have been
studied in detail in the rat hypothalamus (33); however,
only sparse information is available for the mouse to date. In
addition, considerable species differences in hypothalamic AVP
expression have been described in rodents (34). Compared
with the rat, the mouse vasopressinergic system has been found to be
strikingly more profuse (35), and AVP/CRH colocalization
studies revealed that all parvocellular CRH-positive nerve endings are
also immunoreactive for AVP (34). Evidence that AVP
derived not only from the parvocellular part of the PVN, but also from
the magnocellular neurosecretory system released en passant
from axons within the internal zone of the median eminence influences
pituitary ACTH release has become considerably strong
(12). Recently, Turnbull and colleagues provided indirect
evidence that vasopressin might be compensatorily activated to maintain
HPA system activity in
Crhr1-/-
mutants (14); mice were injected with a polyclonal
antiserum against AVP before measurements of plasma ACTH. However,
because of the structural homology between the two neuropeptides, AVP
and OXT, a polyclonal antiserum raised against AVP is likely to
cross-react with OXT. Therefore, this experiment cannot sort out
whether only AVP or both neuropeptides might be compensatorily
activated in
Crhr1-/-
mutants.
Several studies have shown that immunohistochemical detection of AVP in the median eminence may be considered a reliable indicator of hypothalamic vasopressinergic activity, allowing for a distinction between predominantly parvocellular (axons passing through the external zone of the median eminence) and magnocellular (axons passing through the internal zone of the median eminence) contributions to the hypothalamic vasopressinergic system (36, 37). The strong increase in AVP-like immunoreactivity in homozygous Crhr1-/- mutants in both the external and internal zones of the median eminence is in complete accordance with our observation that AVP mRNA is significantly increased in the PVN. No significant difference in plasma sodium concentration could be observed between homozygous Crhr1-/- mice and wild-type littermates. It is, therefore, most likely that in Crhr1-/- mutants chronically exposed to increased plasma vasopressin levels, a marked desensitization of renal vasopressin V2 receptors occurs to maintain water and electrolyte homeostasis.
To test the hypothesis that the observed activation of the vasopressinergic system is functionally relevant, we injected the mice with a selective V1 receptor antagonist. In Crhr1-/- mutants, the plasma ACTH concentration decreased significantly after administration of the V1 receptor antagonist, providing evidence that activation of the vasopressinergic system is critically involved in maintaining basal pituitary ACTH release in the absence of functional CRHR1. However, it is likely that additional factors besides AVP might be activated to maintain HPA system activity in Crhr1-/- mutants, and studies to identify other potential ACTH secretagogues in Crhr1-/- mutants are currently underway.
In the present investigation, stress-induced plasma AVP levels were lower than baseline values, confirming previous results in rats, in which AVP release into the blood has been described to be unaffected or even suppressed in response to emotional stressors (38, 39).
Glucocorticoid deficiency is the major driving force behind
activation of the vasopressinergic system in
Crhr1-/- mutants
It is well known that AVP expression is markedly increased under
conditions of glucocorticoid deficiency, such as adrenalectomy
(18, 32, 40). Accordingly, very low plasma CORT levels in
Crhr1-/-
mutants (10), leading to reduced negative feedback on the
activity of neurosecretory neurons, may be considered an additional and
potent stimulus for increased hypothalamic AVP expression. To
investigate whether glucocorticoid deficiency causally contributes to
hypothalamic vasopressinergic stimulation in homozygous
Crhr1-/-
mutants, we treated homozygous
Crhr1-/-
mutants with CORT 21-sulfate. Mutant mice reached basal plasma CORT
levels indistinguishable from those of wild-type littermates after
continuous administration of CORT 21-sulfate added to the drinking
water (13.5 mg/liter). Concomitantly, basal plasma AVP concentrations
decreased to wild-type levels in CORT-treated homozygous
Crhr1-/-
mutants. We can therefore conclude that glucocorticoid deficiency is
the major driving force behind the selective activation of the
hypothalamic vasopressinergic system.
The oxytocinergic pathway is not activated in
Crhr1-/- mutants
OXT, although less effectively than AVP, has also been shown to
potentiate CRH-induced ACTH release from pituitary corticotropes
(15, 16) and was therefore considered a second candidate
to maintain HPA system activity in homozygous
Crhr1-/-
mutants. In contrast to AVP, basal as well as increased poststress
plasma OXT levels were indistinguishable among wild-type mice and
heterozygous and homozygous mutants. Accordingly, no differences in
either the number of OXT mRNA-expressing cells in the PVN or the mean
optical density per cell could be observed among the genotypes. Our
data therefore suggest that despite their close structural, functional,
and anatomical relationship, AVP and OXT are distinctly regulated and
independently involved in different mechanisms of HPA system regulation
and activity.
The stress-induced increase in plasma ACTH and CORT levels is
severely impaired in homozygous Crhr1-/-mutants: differential effects of social defeat vs. forced swim stress
on ACTH release
Forced swim stress, a combined physical and emotional
stressor, was previously shown not to elicit a significant increase in
plasma ACTH and CORT levels in homozygous
Crhr1-/-
mutants (10). However, it is well known that different
types of stressors (e.g. predominantly physical, emotional,
and combined physical-emotional stressors) are accompanied by specific
patterns of neuroendocrine activation. Most recently, a widespread
distribution of CRHR1 in the mouse brain and, in particular, in
autonomic pathways of the central nervous system has been described
(41). CRHR1 deficiency may therefore lead to significant
effects not only on the neuroendocrine system, but also on the
sympathetic nervous system and autonomic regulation. Social defeat by a
male conspecific can be considered one of the most severe stressors
among a number of laboratory stressful stimuli in terms of
neuroendocrine activation with respect to both the magnitude and the
quality of the stress response (for review, see Ref. 42).
Moreover, the stress response by social defeat in particular is
characterized by a strong sympathetic dominance, with both plasma
epinephrine and norepinephrine being significantly increased
(43). Therefore, we subjected
Crhr1-/-
mutants to social defeat stress to examine whether this severe
emotional stressor might lead, in contrast to the forced swim stress
paradigm, to a significant activation of the HPA system. Indeed, social
defeat induced a significant increase in plasma ACTH in homozygous
Crhr1-/-
mutants, whereas plasma ACTH levels after forced swim stress remained
indistinguishable from basal levels, herewith confirming our previous
findings (10). The differential effects of these two
stressors on pituitary ACTH release in homozygous
Crhr1-/-
mutants might be explained by the strong sympathetic activation
accompanying the social defeat stress paradigm. Adrenergic receptors
have been localized on pituitary corticotrophs (44), and a
stimulatory effect of central catecholamines on pituitary ACTH release
has been shown both in vitro and in vivo
(45 ; for review, see Ref. 46).
Taken together, we could demonstrate a selective activation of the hypothalamic vasopressinergic, but not the oxytocinergic, system and its critical involvement in the maintenance of basal HPA system activity in homozygous Crhr1-/- mutants. Heterozygous mutants, in contrast, are normal (that is, indistinguishable from their wild-type littermates) with respect to plasma AVP levels, hypothalamic AVP expression, and lack of responsiveness to the administration of a V1 receptor antagonist. Our data add to the complexity of HPA system regulation and stress hormone homeostasis. In addition, these findings are of particular importance for the question of potential side-effects of a long-term administration of selective CRHR1 receptor antagonists, the potential antidepressant and anxiolytic properties of which are currently under investigation in clinical trials (3, 47).
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Acknowledgments |
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Footnotes |
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Received May 2, 2000.
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A. J. Douglas, P. J. Brunton, O. J. Bosch, J. A. Russell, and I. D. Neumann Neuroendocrine Responses to Stress in Mice: Hyporesponsiveness in Pregnancy and Parturition Endocrinology, December 1, 2003; 144(12): 5268 - 5276. [Abstract] [Full Text] [PDF]
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 I. Sillaber, G. Rammes, S. Zimmermann, B. Mahal, W. Zieglgansberger, W. Wurst, F. Holsboer, and R. Spanagel Enhanced and Delayed Stress-Induced Alcohol Drinking in Mice Lacking Functional CRH1 Receptors Science, May 3, 2002; 296(5569): 931 - 933. [Abstract] [Full Text] [PDF]
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J. Preil, M. B. Muller, A. Gesing, J. M. H. M. Reul, I. Sillaber, M. M. van Gaalen, J. Landgrebe, F. Holsboer, M. Stenzel-Poore, and W. Wurst Regulation of the Hypothalamic-Pituitary-Adrenocortical System in Mice Deficient for CRH Receptors 1 and 2 Endocrinology, November 1, 2001; 142(11): 4946 - 4955. [Abstract] [Full Text] [PDF]
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 A. E. Kresse, M. Million, E. Saperas, and Y. Tache Colitis induces CRF expression in hypothalamic magnocellular neurons and blunts CRF gene response to stress in rats Am J Physiol Gastrointest Liver Physiol, November 1, 2001; 281(5): G1203 - G1213. [Abstract] [Full Text] [PDF]
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