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Max Planck Institute of Psychiatry (J.P., M.B.M., A.G., J.M.H.M.R., I.S., M.M.V.G., J.L., F.H., W.W.), 80804 Munich, Germany; GSF Research Center, Institute for Mammalian Genetics (W.W.), 85764 Munich, Germany; and Department of Molecular Microbiology and Immunology, Oregon Health Sciences University (M.S.-P.), Portland, Oregon 97201
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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Abstract |
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Introduction |
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The biological actions of CRH, UCN, and UCN II are mediated by
specific, high affinity, G protein-coupled seven
trans-membrane receptors, which mediate ligand-dependent
stimulation of intracellular cAMP. The cloning of distinct receptor
subtypes, CRHR1, CRHR2
, and CRHR2ß, facilitates the separation of
the many physiological effects of these peptides. CRHR1 and CRHR2
display markedly different pharmacological specificity and tissue
distribution. Although the expression of CRHR1 is high in neocortical,
cerebellar, and sensory relay structures, the expression of CRHR2 mRNA
is generally confined to subcortical structures, in particular within
the choroid plexus, specific septal and amygdaloid nuclei, and
various hypothalamic nuclei (for review, see Ref. 9).
Differences in ligand affinities of CRH and UCN for CRHR1 and CRHR2
support the proposition that UCN/UCN II and CRHR2 may represent a
functionally different circuit to the CRH/CRHR1 system (6, 8). Especially UCN II may be of value in dissociating functions
mediated by the two receptors, as it binds selectively to CRHR2
(6).
CRHR1 has been proposed to mediate the effects of CRH on HPA system function and anxiety-related behavior (10, 11, 12, 13, 14). Mice deficient for CRHR1 display a severe impairment of stress-induced ACTH release from pituitary corticotropes, marked glucocorticoid deficiency, and significantly reduced anxiety-like behavior (15, 16). In line with these findings in rodents, a clinical study using a selective CRHR1 antagonist found alleviation of psychopathology in patients with severe major depression (17). The neuroendocrine analyses of CRHR2-deficient mice suggest that CRHR2 has a modulatory function on the HPA stress response involving the maintenance of ACTH release during stress and the recovery of plasma CORT after the end of the stressor (18, 19).
Thus, the detailed investigations in mouse lines deficient for either CRHR1 or CRHR2 together with recent data from rat models (20) suggest that the CRH neuronal system may comprise two separate, but interrelated, subdivisions that can be coordinately and inversely activated in stress-induced HPA response and anxiety-like behavior.
We generated compound CRHR1/CRHR2 mutant mice to investigate the HPA system regulation in the absence of known CRHRs under basal conditions and in response to different ethologically relevant stressors. To elucidate possible gene dose effects in the action of both CRHRs, our analysis included all nine different genotypes, i.e. heterozygous and homozygous CRHR1- or CRHR2-deficient mice, mice lacking both CRH receptors, compound mutants with homozygous and heterozygous deficiency for either of the receptors and their wild-type littermates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heterozygous CRHR1-deficient male mice (129Ola/CD1) originally generated by Timpl et al. (15) and heterozygous CRHR2-deficient female mice (129SvJ/C57BL/6J) created by Coste et al. (19) were chosen as the first generation parents. Pairs of their Crhr1+/-Crhr2+/- offspring were mated to produce the F2 generation. To obtain a relevant number of all possible genotypes within the same age range, the following pairs of F2 were mated: male Crhr1-/-Crhr2+/- mutants with female Crhr1+/-Crhr2-/- mutants, male Crhr1+/-Crhr2-/- and Crhr1-/-Crhr2+/- mutants with female Crhr1+/-Crhr2+/- mutants, male Crhr1+/-Crhr2+/- mutants with female Crhr1+/-Crhr2+/- mutants, and male Crhr1+/-Crhr2+/- mutants with female Crhr1+/-Crhr2+/- mutants.
The present experiments were performed with 8- to 12-wk-old male and female mice, weighing between 25–35 g. 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 0600–1900 h, 22 ± 1 C, 40–60% humidity) and received standard pelleted food and water ad libitum.
Measurement of food intake
To assess basal feeding, 24-h food consumption was measured for
7 consecutive d in male mice (n = 10/genotype). Mice were singly
housed and were fed ad libitum with a standard purified,
water-based liquid diet (Lieber-DeCarli diet, Dyets, Inc., Bethlehem,
PA), that allowed exact quantification of food intake in each animal
(21). Basal food intake (milliliters of daily prepared
liquid diet in graduated feeding tubes) was measured once a day (0630
h).
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 ACTH and CORT,
naive male and female mice (n = 6–10/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 s.
Poststress levels of hormones
Restraint stress. Male and female animals were subjected to
restraint stress as a combined emotional and physical stressor
(22). On the day of testing, between 0800–1200 h, each
mouse (n = 6–10/genotype) was placed into a restrainer (diameter,
3 cm; length, 11 cm) for 2, 5, and 10 min, respectively. Blood samples
were obtained by retroorbital bleeding immediately after stress
exposure. The study was performed in three independent experiments (for
each duration of stress) using male and female mice.
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 home cage. Usually within three tests the resident reliably attacked the intruder within less than 2 min (adapted with modifications from Ref. 23). Social defeat stress consisted of introducing a naive, singly housed experimental mouse (intruder; male Crhr1-/-Crhr2+/+, Crhr1+/+Crhr2-/-, and Crhr1-/-Crhr2-/- mutants and wild-type mice; n = 6–10/group) into the resident’s home cage. 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 home cage. 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 15 min. Blood collection was performed by retroorbital bleeding immediately after the end of the stressor.
CRH challenge
After 1 wk of daily handling, male
Crhr1-/-Crhr2+/+
and
Crhr1-/-Crhr2-/-
mutants and wild-type littermates (age, 8–12 wk; n = 6–9/group)
were sc injected with either vehicle or 1 µg CRH (both delivered from
Ferring Pharmaceuticals Ltd., Malmo, Sweden; injection
time, between 0800–0830 h). Blood samples were taken by rapid
retroorbital bleeding 30 min after the injection for determination of
plasma ACTH and CORT levels.
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
C (ACTH) and -20 C (CORT) until assay. Plasma ACTH (50 µl sample)
and CORT (10 µl sample) levels were measured using commercially
available kits (Biochem, Freiburg, Germany) according to the respective
protocols. All samples were measured twice. The inter- and intraassay
variabilities were less than 7%.
Histological and immunohistochemical analyses
Animals (n = 3/genotype; wild-type mice,
Crhr1-/-Crhr2+/+,
Crhr1+/+Crhr2-/-,
and
Crhr1-/-Crhr2-/-
mutants) were deeply anesthetized with phenobarbital and transcardially
perfused with phosphate-buffered 4% paraformaldehyde. Brains were
removed from the skull, postfixed for 3 h in 4% paraformaldehyde,
and then transferred to 20% sucrose in PBS (pH 7.4). Serial 30-µm
coronal frozen sections were cut in a cryostat, transferred into PBS,
and processed as free-floating sections. For each animal, all sections
spanning the respective region of interest were analyzed to allow for
exact comparisons 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, preincubations with 5%
normal goat serum (AVP) and normal donkey serum (CRH), respectively,
for 2 h were 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) and
1:5,000 (polyclonal goat-anti-CRH antibody; Sc 1761, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), respectively, at 4 C.
The specificities of these antibodies have been tested by the
manufacturer. The sections were then incubated with a biotinylated goat
antirabbit (AVP) or donkey antigoat (CRH) 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
(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. CRH staining
was intensified with heavy metals according to the method described by
Adams (24). The slides were then washed in PBS, mounted on
glass slides, air-dried, and lightly counterstained with hematoxylin.
Appropriate negative controls were performed by omission of the primary
antibody. For histological analysis, peripheral organs (pituitary
gland, thymus, lung, blood vessels, heart, liver, spleen, adrenal
gland, and testes) were dissected after perfusion with
phosphate-buffered 4% paraformaldehyde. Subsequently, tissue was
postfixed, dehydrated, and embedded in paraffin, and 8-µm sections
were cut on a microtome. Sections were mounted on glass slides and air
dried. After deparaffination and hydration, routine hematoxylin-eosin
staining was performed.
Statistical analysis
Statistical analysis was performed with a software package
(SigmaStat, version 2.03, Statistical Solutions Ltd., Boston, MA).
Statistical significance of differences between groups was determined
by ANOVA (one-way ANOVA, factor: genotype; or two-way ANOVA, factors:
genotype and duration of stress, where appropriate), followed by
post-hoc Newman-Keuls test. P < 0.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We observed no differences in
adrenal gland size and structure between male CRHR2-deficient mice and
their wild-type littermates. In contrast,
Crhr1-/-Crhr2+/+
mice showed a marked adrenocortical atrophy, yet no obvious difference
in adrenal gland pathology could be observed between male
Crhr1-/-Crhr2+/+
and
Crhr1-/-Crhr2-/-
mutants (Fig. 1). No sexual dimorphism in the degree of adrenocortical
atrophy was detected within
Crhr1-/-Crhr2+/+
and
Crhr1-/-Crhr2-/-
mutants. Furthermore, no obvious differences in adrenal gland pathology
could be observed between female
Crhr1-/-Crhr2+/+
and
Crhr1-/-Crhr2-/-
mutants (data not shown).
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Neuroendocrinology
To investigate the differential effects of CRHR1 and CRHR2 on HPA
system function as well as its gene dose dependence, we examined nine
different genotypes in male animals, i.e.
heterozygous and homozygous CRHR1- or CRHR2-deficient mice
(Crhr1+/-Crhr2+/+,
Crhr1-/-Crhr2+/+,
Crhr1+/+Crhr2+/-,
Crhr1+/-Crhr2-/-),
mice lacking both CRH receptors
(Crhr1-/-Crhr2-/-),
compound mutants with homozygous and heterozygous deficiency for
either of the receptors
(Crhr1+/-Crhr2+/-,
Crhr1+/-Crhr2-/-,
and
Crhr1-/-Crhr2+/-),
and their wild-type littermates
(Crhr1+/+Crhr2+/+)
and four different genotypes in females (wild-types, mice deficient for
either CRHR1 or CRHR2, and mice lacking both CRHRs) under basal
conditions and in response to different ethologically relevant
stressors. Male and female animals (all genotypes) were subjected to
different durations of restraint stress as a combined emotional and
physical stressor. In addition, male mice (wild-type, mice deficient
for either CRHR1 or CRHR2, and mice lacking both CRHRs) were subjected
to social defeat as a predominantly emotional stressor (for review, see
Ref. 25).
Basal levels of ACTH and CORT in male and female animals
Two-way ANOVA including all tested genotypes did not reveal
significant differences between any of the genotypes in
basal morning plasma ACTH and CORT
levels either in male (Table 1) or female (Fig. 2) animals.
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). No significant increase in
plasma ACTH was observed in either
Crhr1-/-Crhr2+/+
and
Crhr1-/-Crhr2-/-
mutants following any of the restraint procedures (P >
0.8). Two minutes of restraint stress induced a significant increase in
plasma ACTH in wild-type mice and
Crhr1+/+Crhr2-/-
mutants compared with basal levels (P < 0.001). In
Crhr1+/+Crhr2-/-
mutants, plasma ACTH peaked after 5-min restraint stress and
significantly decreased after a 10-min period of stress
(P < 0.05). In contrast, wild-type animals showed a
continuous increase in plasma ACTH with increasing durations of
stress.
Plasma CORT levels after restraint stress in female animals.
Two-way ANOVA revealed a significant effect of both genotype
(P < 0.001) and duration of restraint stress
(P < 0.001) as well as a significant interaction of
both factors (P < 0.001; Fig. 2B). No significant
differences between the genotypes could be detected in plasma CORT
levels after 2 min of restraint stress. In wild-type mice and
Crhr1+/+Crhr2-/-
mutants, plasma CORT levels significantly increased after 5-min stress
compared with basal levels (P < 0.01). Both wild-type
animals and
Crhr1+/+Crhr2-/-
mutants showed a further increase in plasma CORT after 10-min restraint
stress (P < 0.01). In contrast to plasma ACTH levels,
CORT response curves were not significantly different between wild-type
animals and
Crhr1+/+Crhr2-/-
mutants. A significant increase in plasma CORT levels was observed in
female
Crhr1-/-Crhr2+/+
mutants after 5- and 10-min restraint stress (P <
0.01). However, female mice deficient for both CRHRs did not show
elevated plasma CORT levels compared with baseline after any of the
stress conditions.
Plasma ACTH levels after restraint stress in male animals. In
male mutants lacking functional CRHR1, plasma ACTH and CORT
concentrations did not increase significantly after any
of the stress conditions (different
lengths of restraint stress and 15-min social defeat stress; see Fig. 3 and 4).
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). All genotypes other than mutants
lacking CRHR1 showed a significant increase in plasma ACTH after
restraint stress (P < 0.001). Plasma ACTH response
curves did not significantly differ among animals having two intact
alleles for CRHR1. Among heterozygous CRHR1-deficient mice, however, an
earlier onset of ACTH release was observed in
Crhr1+/-Crhr2-/-
mutants. These animals showed significantly higher plasma ACTH levels
after 2-min restraint stress (P < 0.05) compared
with
Crhr1+/-Crhr2+/+
and
Crhr1+/-Crhr2+/-
mutants. However, plasma ACTH concentrations in all three
Crhr1+/- genotypes were similar after 5-
and 10-min stress (see Fig. 3). In contrast to females, ACTH response
curves in male
Crhr1+/+Crhr2-/-
mutants did not show a rapid peak and early decline. No significant
differences were observed among the six genotypes with at least one
copy of the CRHR1 gene after 5- and 10-min stress, respectively.
Plasma CORT levels after restraint stress in male animals.
Two-way ANOVA revealed a significant effect of both genotype
(P < 0.001) and duration of stress (P
< 0.001), with a significant interaction of both factors
(P < 0.001; Fig. 4). In wild-type animals and
Crhr1+/+Crhr2-/-
mutants only, a significant increase in plasma CORT concentrations was
observed after 2-min restraint stress compared with basal levels
(P < 0.001).
Crhr1+/+Crhr2+/-
mutant mice did not show elevated CORT levels after 2-min stress. Among
the three genotypes with two copies of the CRHR1 gene, the plasma CORT
response curve in homozygous CRHR2-deficient mice showed the most rapid
increase in plasma CORT levels (see Fig. 4). In contrast,
Crhr1+/+Crhr2+/-
mutants showed a delayed onset of CORT release and slowly increasing
plasma CORT levels after different lengths of stress. In heterozygous
CRHR1-deficient mice, plasma CORT levels rose after 5-min, but not
2-min, restraint stress and were further increased after a 10-min
period of stress (P < 0.001). After increasing length
of stress,
Crhr1+/-Crhr2-/-
mutant mice showed significantly elevated CORT concentrations compared
with
Crhr1+/-Crhr2+/+
and
Crhr1+/-Crhr2+/-
mutants (see Fig. 4). Apart from the impaired CORT release in
Crhr1+/+Crhr2+/-
mutant mice, plasma CORT levels in mice with two copies of the CRHR1
gene were significantly higher than CORT levels in
Crhr1+/- mutants after any of the stress
conditions (P < 0.05).
Stress hormone response after social defeat stress in male
animals
Plasma ACTH levels (Fig. 5A).
Two-way ANOVA revealed a significant effect of both genotype
(P < 0.001) and treatment condition (P
< 0.001), with a significant interaction of both factors
(P < 0.001). As observed after restraint stress,
social defeat induced a significant increase in plasma ACTH in
wild-type mice and
Crhr1+/+Crhr2-/-
mutants compared with basal levels (P < 0.001). In
Crhr1-/- mutants, however,
plasma ACTH did not increase significantly after either social defeat
or restraint stress compared with basal levels. In contrast to
restraint stress,
Crhr1+/+Crhr2-/-
mutants showed a significantly greater increase in plasma ACTH compared
with wild-type mice after social defeat (P <
0.05).
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). Two-way ANOVA revealed a
significant effect of both genotype (P < 0.001) and
treatment conditions (P < 0.001), with a significant
interaction of both factors (P < 0.001). Both
Crhr1+/+Crhr2-/-
mutants and wild-type mice showed a significant increase in plasma CORT
levels after social defeat (P < 0.001). Consistent
with ACTH levels, plasma CORT concentrations were significantly higher
in
Crhr1+/+Crhr2-/-
mutants compared with wild-type mice (P < 0.05).
However, no effects of the different stressors on plasma CORT levels
were observed in either
Crhr1-/-Crhr2+/+
or
Crhr1-/-Crhr2-/-
mutants.
CRH challenge
In wild-type mice only, administration of CRH under basal
conditions significantly increased plasma ACTH and CORT levels compared
with those in vehicle-treated animals (P < 0.01; Fig. 6). There was no statistically
significant difference among the three vehicle-treated groups. CRH did
not exert any significant effect on plasma ACTH and CORT levels
in mice lacking CRHR1
(Crhr1-/-Crhr2+/+
and
Crhr1-/-Crhr2-/-
mutants).
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). In mice homozygously deficient for
CRHR1, the zona externa of the median eminence exhibited a considerable
increase in CRH-like immunoreactivity compared with wild-type
littermates (Fig. 7B). This region displayed many prominent and
strongly immunoreactive axons accumulating in the neurosecretory
vesicles, whereas in wild-type mice and
Crhr2-/- mutants, only weakly
immunoreactive structures were present in the zona externa. No
difference in CRH-like immunoreactivity was observed between
CRHR2-deficient mice and wild-type animals.
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). In
Crhr1-/-Crhr2-/-
mutant mice, both nuclei contained a higher number of AVP-positive
neurons that displayed many strongly labeled axons. However, in
wild-type mice, a smaller number of less intensely stained axons were
observed along the different projection pathways.
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Discussion |
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Deficiency of CRHR1 and CRHR2 does not affect regulation of basal
food intake and body weight
Because CRH and both UCN and UCNII have been shown to influence
feeding behavior (6, 7, 28), we measured weight and basal
food intake in
Crhr1-/-Crhr2-/-
mutants and wild-type animals. In the present series of experiments
Crhr1-/-Crhr2-/-
mutants did not show any significant difference in body weight compared
with age-matched wild-type littermates. Accordingly, the total food
intake per 24 h did not differ between the genotypes. These
findings are consistent with previous reports of feeding regulation in
CRHR1-deficient mice (29) and mice lacking a
functional CRHR2 (18, 19). Thus, neither CRHR is likely to
play a critical role in the basal regulation of body weight and the
total amount of food intake. Rather, they may be involved in biphasic
control of UCN-mediated feeding behavior (19, 30) as well
as in cross-talk among central leptin, melanocortin, and CRH pathways
(31, 32).
The endocrine phenotype of
Crhr1-/-Crhr2-/- mutant mice is determined
by the functional lack of CRHR1
CRHR1 was found to be absolutely required for a normal endocrine
stress response. Mice lacking a functional CRHR1 display a severe
impairment of stress-induced HPA system activation and marked
glucocorticoid deficiency (15, 16). In CRHR2-deficient
mice, changes in the stress-induced time course of ACTH and CORT
recovery indicate a role for CRHR2 in adaptation of the stress response
initiated by CRHR1 (18, 19). The present study shows that
the phenotype of mice lacking both CRHR1 and CRHR2 is similar to that
of mice deficient for CRHR1.
No significant differences between wild-type and Crhr1-/-Crhr2-/- mutant mice were detected in basal plasma levels of ACTH and CORT in male or female animals. Recently, we demonstrated a selective activation of the hypothalamic vasopressinergic system to maintain basal ACTH release in CRHR1-deficient mice (29). The increased AVP-like immunoreactivity in the hypothalamic neurosecretory system, in particular in the PVN, suggests a similar compensatory regulation of stress hormone homeostasis in Crhr1-/-Crhr2-/- mutants under basal conditions.
In contrast to the basal situation, where AVP possibly maintains ACTH secretion, none of the stressors applied (different durations of restraint stress and social defeat) led to an increase in circulating ACTH and CORT in male Crhr1-/-Crhr2+/+ and Crhr1-/-Crhr2-/- mutant mice. This confirms the fundamental role of CRHR1 in initiating the stress-induced HPA response (15, 16).
CRH-like immunoreactivity in the PVN and the zona externa of the median eminence was clearly increased in Crhr1-/-Crhr2-/- and Crhr1-/-Crhr2+/+ mutant mice, whereas no difference in hypothalamic CRH-like immunoreactivity could be detected between wild-type animals and CRHR2-deficient mice. Consistent with this finding, an increase in CRH-like immunoreactivity (15) as well as in the expression of CRH mRNA (16) was detected in the PVN of CRHR1 deficient mice, whereas no change in the CRH mRNA level was seen in the PVN of nonstressed Crhr2-/- mutants (18, 19). As only single CRHR1-deficient mice and Crhr1-/-Crhr2-/- mutants display a significant glucocorticoid deficiency with basal plasma CORT levels being hardly detectable, the increased CRH expression in the PVN may be due to the reduced negative feedback effects of corticosteroids. Supporting this hypothesis, adrenocortical steroids are known to feedback directly on the paraventricular neurons predominantly regulating CRH release (33).
It is well known that different types of stressors exert specific patterns of neuroendocrine activation (for review, see Ref. 25). Social defeat, for example, induces a stress response pattern that almost exclusively activates the sympathetic nervous system, reflected by elevated plasma epinephrine and norepinephrine concentrations (34). To compare the effects of different stressors, we subjected male Crhr1-/-Crhr2-/- and Crhr1-/-Crhr2+/+ mutants to social defeat stress and examined whether a different neural stress response might lead, in contrast to the restraint paradigm, to significant HPA activation. However, social defeat failed to induce a significant increase in plasma ACTH and CORT levels in either Crhr1-/-Crhr2-/- or Crhr1-/-Crhr2+/+ mutant mice.
Despite this severe impairment of HPA system function in Crhr1-/-Crhr2-/- mutants, physical and vegetative adaptations promote normal growth and body weight in response to at least mild environmental stressors such as novel cage stress, reproduction, or social life. Therefore, it would be of particular interest whether other stress response systems, in particular the locus coeruleus/noradrenergic-sympathetic system, might compensate for the impairment of the CRH system in animals lacking functional CRH receptors.
CRHR1 and CRHR2 may be indirectly involved in fine-tuning of
adrenocortical CORT release
It is important to note that plasma ACTH and CORT concentrations
did not differ between male
Crhr1-/-Crhr2-/-
and
Crhr1-/-Crhr2+/+
mutants after any of the stress conditions. Thus, neither CRHR2 is
likely to compensate for a functional loss of CRHR1, nor does its
deficiency exacerbate the CRHR1-dependent impairment of the HPA system
function in male animals.
However, female Crhr1-/-Crhr2+/+ mutants were still able to elicit a rudimentary CORT response after 5 min of restraint stress, whereas female Crhr1-/-Crhr2-/- mutants did not show a significant hormone increase after any of the restraint periods. Indeed, there is evidence that CRHR1 deficiency may be at least partly compensated in female mice. In agreement with our results, significant CORT release in response to stress was also reported for female mice lacking CRHR1 and CRH, respectively (16, 26). As the ACTH response is markedly blunted in both the Crhr1-/-Crhr2-/- and Crhr1-/-Crhr2+/+ mutant mice, the more distinct impairment of CORT response in Crhr1-/-Crhr2-/- mutants might suggest an additional effect of CRHR2 on CORT release. In male Crhr1-/-Crhr2-/- and Crhr1-/-Crhr2+/+ mutants, however, CRH administration failed to illicit a CORT response. Thus, at least in males, CRHR2 alone is not likely to stimulate CORT release at the adrenocortical level, which is consistent with our observations in male compound heterozygous mutants. Rather, the sexual dimorphism in CORT response may suggest a role for sex steroids in regulation of the HPA system that is not mediated through CRH (26). Furthermore, a different degree of adrenocortical atrophy between female Crhr1-/-Crhr2+/+ and Crhr1-/-Crhr2-/- mutants might be another reason for the different CORT response. In accordance with this hypothesis, in CRH-deficient mice, a sexually dimorphic adrenal response to stress has been correlated with a different degree of adrenocortical atrophy between the genders (26). However, we observed no sexual dimorphism in the degree of adrenocortical atrophy within the groups of Crhr1-/-Crhr2+/+ and Crhr1-/-Crhr2-/- mutants. Corresponding to male animals, no obvious differences in adrenal gland pathology could be detected between female Crhr1-/-Crhr2+/+ and Crhr1-/-Crhr2-/- mutants. Consistent with our findings, no differences in adrenal gland size or structure between male and female Crhr1-/-Crhr2+/+ mutants have been reported, although both genders show different CORT responses to stress (15, 16). Apparently, there is no constant correlation between the degree of adrenocortical atrophy and CORT release in CRHR-deficient mice.
It is well known that CRH may influence adrenocortical steroidogenesis independently of pituitary function (for review, see Ref. 35). Recently, CRHR1 and CRHR2 mRNA expression has been demonstrated in the adrenal gland of humans (36). Accordingly, within the adrenal gland itself, CRH-like immunoreactivity with ACTH-releasing activity has been detected in the adrenal glands of humans (37), rats (38), and other species (39, 40). The possibility of adrenocortical CRH/CRH receptor signaling is further supported by our findings in male compound heterozygous mice. Although plasma ACTH concentrations did not significantly differ among the six genotypes with at least one copy of CRHR1, CORT concentrations clearly depended on the gene dose of CRHR1 and CRHR2.
In heterozygous CRHR1 mutant mice, the CORT response curve showed a later onset compared with those in wild-type controls and Crhr1+/+Crhr2-/- mutants. This shift was independent of the gene dose of CRHR2. In addition, plasma CORT levels were significantly lower in Crhr1+/-Crhr2+/+ mutants compared with wild-type mice as well as in Crhr1+/-Crhr2-/- mutants compared with Crhr1+/+Crhr2-/- mutant animals after any of the stress conditions applied. In contrast, plasma CORT levels were significantly increased in CRHR2-deficient mice compared with animals having both copies of the CRHR2 gene despite having one or two copies of CRHR1. Additionally, CORT and ACTH response curves in Crhr1+/-Crhr2-/- mutants confirm the initial description of the single CRHR2 knockout with a rapid increase, but early decline, of ACTH release in these mutants (19). In summary, our data suggest that CRH is likely to stimulate adrenocortical CORT release through CRHR1, whereas CRHR2 might mediate inhibitory CRH actions on adrenocortical function.
To further investigate whether CORT can be released through adrenal CRHR2, we compared the ACTH and CORT responses to a CRH challenge in wild-type, Crhr1-/-Crhr2-/-, and Crhr1-/-Crhr2+/+ mutant mice. CRH did not exert any significant effect on circulating ACTH and CORT in Crhr1-/-Crhr2+/+ mutants. Thus, a direct effect of CRH via CRHR2 on adrenocortical steroidogenesis seems unlikely.
Most recently, a third novel CRHR has been cloned from catfish that is highly homologous to its mammalian counterparts. The novel receptor has been found to be highly expressed in pituitary and brain (41). However, the failure to activate the HPA system after CRH administration in Crhr1-/-Crhr2-/- mutant mice suggests that a third receptor binding CRH with high affinity is not likely to be involved in stress hormone release, at least not via direct effects on pituitary corticotropes or adrenocortical cells.
CRH signaling may play a role in adrenal gland development
In our previous study (15) no significant difference
in the size of the adrenal cortex could be observed between
CRHR1-deficient mice and their wild-type littermates. Here, we report a
similar decrease in size of the CORT-producing zona fasciculata in
Crhr1-/-Crhr2-/-
and
Crhr1-/-Crhr2+/+
mutants. Considering the well known influence of genetic background on
the morphological phenotype of transgenic mice (for review, see Ref.
42), it is likely that the difference in adrenocortical
size between our Crhr1-/-
mouse line (129Ola/CD1 background) (15) and the single
CRHR1-deficient progeny from our breeding of CRHR double mutants (mixed
129Ola/CD1–129SvJ/C57BL/6J background) is attributed to their
different genetic background. Correspondingly, Smith et al.
(16) observed significant adrenocortical atrophy in their
CRHR1-deficient mouse line (129SvJ/C57BL/6J background) compared with
wild-type animals. These researchers could restore normal gland
morphology by ACTH replacement during early postnatal life, suggesting
that a functional hypothalamic-pituitary system is required for normal
development of the adrenal gland. According to this finding, one reason
for the histopathological defect in the adrenal cortex in mice lacking
functional CRHR1 might be a marked impairment of ACTH secretion during
the neonatal period. Correspondingly, no adrenocortical atrophy could
be detected in CRHR2-deficient mice (18, 19). On the other
hand, there is evidence that intraadrenal CRH and ACTH exert trophic
effects on adrenocortical cells. Their actions may be mediated via
locally produced peptide growth factors, such as members of the
fibroblast growth factor and IGF families (43). The loss
of CRHR-dependent pathways might also lead to a severe impairment of
normal adrenocortical growth and differentiation, suggesting an
indirect involvement of the CRHR system in adrenal gland
development.
In summary, we show that the endocrine phenotype of mice lacking both CRHR1 and CRHR2 is dominated by the functional loss of CRHR1. CRHR2 does not appear to compensate for CRHR1 deficiency, nor does the lack of CRHR2 synergistically exacerbate the CRHR1-dependent impairment of HPA system function. Pointing to a dual organization of the CRHR system, our results confirm the fundamental role of CRHR1 in initiating the stress-induced HPA response and the modulation of its recovery by CRHR2. Delineation of compensatory contributions of other neural stress systems, in particular maintenance of physical and vegetative homeostasis, will require a precise analysis of their components in mice with mutations in CRH signaling pathways. In addition, our data add to the complexity of adrenocortical function (44) and suggest different roles of CRHR1 and CRHR2 in fine-tuning adrenocortical CORT release.
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Acknowledgments |
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Footnotes |
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1 J.P. and M.B.M. contributed equally to this work. 
Abbreviations: CORT, Corticosterone; CRHR, CRH receptor; HPA, hypothalamic-pituitary-adrenal; UCN, urocortin.
Received March 29, 2001.
Accepted for publication August 3, 2001.
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, Crhr2+/+ genotypes 
,
Crhr2+/- mice; 
,
Crhr2-/- genotypes. Each triple set of
columns represents a particular CRHR1 genotype group. *, Statistically
significant differences between genotypes; #, no significant effect of
the experimental condition vs. basal levels within the
same genotype; +, significant effect of the experimental condition
vs. hormone levels after 5 min of the restraint
procedure.
