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Endocrinology Vol. 147, No. 10 4578-4588
Copyright © 2006 by The Endocrine Society

Urocortin 3 Modulates the Neuroendocrine Stress Response and Is Regulated in Rat Amygdala and Hypothalamus by Stress and Glucocorticoids

Pauline M. Jamieson, Chien Li, Christina Kukura, Joan Vaughan and Wylie Vale

The Clayton Foundation Laboratories for Peptide Biology (P.M.J., C.L., J.V., W.V.), The Salk Institute for Biological Studies, La Jolla, California 92037; and Clinical Neurocardiology Section (C.K.), National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Wylie Vale, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The endogenous corticotropin-releasing factor (CRF) type 2 receptor (CRFR2)-selective ligand urocortin 3 is expressed in discrete subcortical brain regions with fibers distributed mainly to hypothalamic and limbic structures. Close anatomical association between major urocortin 3 terminal fields and CRFR2 in hypothalamus, lateral septum, and medial amygdala (MEA) suggest it is well placed to modulate behavioral and hormonal responses to stress. Urocortin 3 was administered intracerebroventricularly to male rats under basal conditions or before a restraint stress, and circulating ACTH, corticosterone, glucose, and insulin were measured. Urocortin 3 activated the hypothalamic-pituitary-adrenal axis under basal conditions and augmented ACTH responses to restraint stress. Elevated blood glucose with lowered insulin to glucose ratios in both groups suggested increased sympathetic activity. Circulating catecholamines were also increased by urocortin 3, providing additional evidence for sympathoadrenomedullary stimulation. Intracerebroventricular urocortin 3 increased vasopressin mRNA expression in the parvocellular division of the hypothalamic paraventricular nucleus, whereas CRF expression was unchanged, providing a possible mechanism by which urocortin 3 mediates its actions. Urocortin 3 mRNA expression was examined after exposure to stress-related paradigms. Restraint increased levels in MEA with a trend to increased expression in the rostral perifornical hypothalamic area, whereas hemorrhage and food deprivation decreased expression in MEA. Adrenalectomy markedly increased expression in the rostral perifornical hypothalamic area, and high-level corticosterone replacement restored this to control levels. The evidence that urocortin 3 has the potential to influence hormonal components of the stress response and the changes in its expression levels after stressors is consistent with a potential function for the endogenous peptide in modulating stress responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORTICOTROPIN-RELEASING FACTOR (CRF) is a key effector of the behavioral and neuroendocrine responses to stressors (1), mediating its effects predominantly via CRF type 1 receptors (CRFR1) within the central nervous system (CNS) (2). The more recently described members of the CRF family of peptides, the urocortins (Ucn 1, 2, and 3), signal preferentially through the CRF type 2 receptor (CRFR2). Within the CNS, both receptors are expressed in regions involved in the neuronal circuitry of stress responses including the amygdala, hippocampus, and the paraventricular nucleus (PVN) of the hypothalamus (3, 4).

Pharmacological studies and studies of CRFR1 null mice have demonstrated the dominance of CRF acting at the CRFR1 in activating the hypothalamic-pituitary-adrenal (HPA) axis and stimulating anxiety-like behaviors (5, 6, 7, 8). The role of the Ucns and CRFR2 is less defined. Within the CNS, CRFR2 is expressed in discrete regions largely restricted to subcortical structures (3, 4), and the receptor has been implicated in modulating stress-related physiological functions including anxiety- and depressive-like behaviors (9, 10, 11), the neuroendocrine stress response (10, 12), and monoaminergic functions (13). Although Ucn 1 has affinity for both CRF receptors, Ucn 2 and Ucn 3 demonstrate preferential specificity for the CRFR2 (14, 15). Pharmacological studies of the biological actions of these ligands have yielded inconsistent data. Ucn 2 has been reported to increase anxiety-like behaviors in some paradigms (16, 17, 18), whereas both Ucns 2 and 3 have been described as anxiolytic in others (19, 20, 21, 22). With regard to the hormonal stress response, Ucns 2 and 3 have been reported to have no effect (18, 22) or to stimulate centrally mediated neuroendocrine responses (12, 13). The role of endogenous Ucns in mediating these distinct responses is unknown.

Ucn 3 is the most highly specific agonist known for CRFR2 (14, 15). Ucn 3-expressing neurons are found predominately within the medial amygdala (MEA), the hypothalamic median preoptic nucleus (MePO), and the rostral perifornical area (PFA) lateral to the PVN (23). The terminal fields of Ucn 3 neuronal projections show substantial congruence with CRFR2 expression in most sites including the intermediate part of the lateral septum, the ventromedial hypothalamus, the posterior bed nucleus of the stria terminalis, and the MEA, supporting the notion that it is an endogenous ligand for the receptor well placed to mediate physiological effects on stress-related endocrine and behavioral responses. The details of what roles Ucn 3 may play in responses to physiological circumstances are entirely unknown. Thus, we have examined the effects of Ucn 3 administered centrally on hormonal aspects of the stress response.

If Ucn 3 is indeed an endogenous modulator of the stress response, it might be expected that expression levels would be responsive to stress-related changes in homeostatic equilibrium as for CRF and arginine vasopressin (AVP) (24, 25). Therefore, we also examined regulation of Ucn 3 mRNA in the major sites of expression in response to a variety of stress-related paradigms and found that expression levels were indeed subject to regulation in a site- and stressor-specific manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Adult male Sprague Dawley (Harlan Sprague Dawley Inc., Indianapolis, IN) rats (200–225 g) were housed in a temperature-controlled room with controlled lighting (lights on 0600–1800 h) and given free access to laboratory chow and water except where stated. All experimental protocols and procedures were approved by The Salk Institute Animal Use and Care Committee.

Intracerebroventricular (icv) administration of Ucn 3
Nine days before experiments, indwelling 26-gauge stainless steel guide cannulae (Plastics One, Roanoke, VA) were inserted into the right lateral ventricle (0.3 mm posterior, 1.4 mm right, 3.4 mm ventral to bregma skull surface, with incisor bar 3.3 mm below the interaural line) of rats anesthetized with ketamine (100 mg/kg)/acepromazine (4 mg/kg)/xylazine (10 mg/kg) administered sc (26). Indwelling iv catheters (PE 50; Becton Dickinson and Co., Sparks, MD) were inserted into the right jugular vein, filled with heparinized saline, passed through a sc tunnel, and exteriorized at the back of the neck under isoflurane anesthesia 3 d before testing (27) to allow for recovery from surgery with respect to HPA responsiveness (28). All animals were housed individually after surgical procedures.

The procedure for in vivo evaluation of the HPA axis was as previously described (29). On the morning of experiments, rats (n = 8 per group) were moved to a soundproof room and singly housed in opaque sampling buckets (at 0600 h). An internal cannula (Plastics One) was attached to the implanted icv guide cannula, and a catheter containing Ucn 3 or vehicle (5 µl sterile water, pH 7.4) was extended outside the bucket to allow peptide delivery without handling the animal. The iv catheters were also extended with a sampling tube to allow remote sequential blood sampling. Rats were left undisturbed for 3 h before withdrawal of a basal blood sample followed by slow icv infusion of peptide or vehicle over 1 min. For rats injected under nonstressed conditions, blood samples were collected at 5, 15, 30, 60, and 90 min after icv injection. Rats subjected to restraint stress (n = 8 per group) were removed from sampling buckets 10 min after icv injection and placed in Broome Style Rodent Restrainers (Plas-Labs Inc., Lansing, MI) for 15 min. They were then returned to their original sampling bucket for the remaining duration of the experiment. Blood was drawn 5, 15, 30, 60, and 90 min after the onset of the restraint stress. Each blood sample (350 µl volume) was withdrawn and replaced with an equal volume of sterile saline. Samples were mixed with 10 µl of 0.5 M EDTA, placed on ice, and centrifuged at 4 C for 10 min at low speed within 30 min of collection. Plasma was aliquoted and stored at –20 C until assayed.

In a separate experiment, rats were implanted with icv cannulae alone. Injections of 10 µg Ucn 3 or vehicle (n = 6 per group) were made following the procedure described above, and 4 h later, rats were euthanized and the brains removed and frozen on dry ice for subsequent analysis of CRF and AVP mRNA levels.

Intravenous administration of Ucn 3
Three days before experiments, rats (n = 8 per group) underwent jugular catheterization alone. Three hours after removal to sampling buckets, a basal blood sample was collected and iv injection of Ucn 3 or vehicle (100 µl sterile saline) was made via the jugular catheter. Blood samples were drawn at 5, 15, 30, 60, and 90 min post injection and handled as described above.

Catecholamine measurements
Because of the limited blood volume that can be withdrawn from rats without hemorrhage-induced activation of the HPA axis (30), blood sampling for catecholamine measurements was conducted during a separate experiment. Each blood sample (600 µl volume) was withdrawn and replaced with an equal volume of heparinized blood collected from a donor rat not more than 30 min previously. Samples were collected in heparinized 1-ml syringes, transferred to plastic tubes, and immediately centrifuged at 4 C for 5 min at low speed. Plasma was separated, transferred to liquid nitrogen, and stored at –80 C until assay. In addition to the basal blood sample, samples were collected 5 and 15 min after icv injection of Ucn 3 or vehicle (n = 6–8 per group). Catecholamines, including norepinephrine and epinephrine, were extracted from plasma samples using an alumina extraction procedure and quantified by liquid chromatography with electrochemical detection as described elsewhere (31). Intraassay coefficients of variation were 6.8% for noradrenaline and 13.4% for adrenaline.

Stressors and environmental challenges
Restraint stress.
Rats were subjected to restraint stress commencing at 0900 h in a soundproof room and removed from the restrainers and euthanized 30 min or 4 h later. Control animals were left undisturbed in their home cages until euthanasia under stress-free conditions at the same time as the stressed rats.

Hemorrhage.
Seven days before the experiment, indwelling iv catheters were inserted as described above and flushed with heparinized saline daily to habituate the animals to the process. On the day of the experiment, 25% of blood volume (16 ml/kg body weight) was withdrawn via the catheter at 0900 h. Control rats had their catheters flushed with no blood withdrawn. Rats were euthanized 30 min or 4 h post hemorrhage.

Food deprivation.
Rats were food deprived from 0900 h until 0900 h 48 h later. They were housed in cages with wire mesh floors to prevent ingestion of bedding material. They had free access to water at all times. Control animals were similarly housed but had access to chow ad libitum.

Adrenalectomy and glucocorticoid replacement.
Rats were bilaterally adrenalectomized (ADX) or sham-operated (sham ADX) through dorsal incisions under isoflurane anesthesia. A subgroup of ADX rats were sc implanted with a 100-mg slow-release corticosterone pellet (Innovative Research, Sarasota, FL) (ADX+CORT). The control group was not operated on. All animals were housed individually after the surgical procedures and were euthanized at 0900 h 10 d after surgery.

At the end of experiments, rats were euthanized by decapitation and trunk blood collected for hormonal analyses and handled as described above. Brains were rapidly removed, frozen on dry ice, and stored at –80 C until sectioning (n = 5–7 per experimental group).

RIAs and glucose assay
Plasma ACTH (Nichols Institute Diagnostics, San Juan Capistrano, CA), corticosterone (ICN Biomedicals Inc., Costa Mesa, CA), and insulin (Sensitive Rat Insulin RIA Kit; Linco Research Inc., St. Charles, MO) levels were measured in unextracted samples using commercially available immunoassay kits. ACTH, corticosterone, and insulin intraassay coefficients of variation were 3.2, 7.1, and 4.8%, respectively. Duplicate samples at multiple time points repeated from individual rats were analyzed within the same assay. The ACTH assay has been validated for the measurement of rat ACTH (32). Plasma glucose was enzymatically determined using Trinder’s reagent (Sigma Diagnostics, St. Louis, MO). The intraassay coefficient of variation was 1.0%.

In situ hybridization
Coronal brain sections (20 µm thick) were cut on a cryostat, thaw-mounted onto glass slides, and stored at –80 C until use. Antisense cRNA probes were transcribed from a 230-bp fragment of the AVP-specific 3' end of a rat cDNA, a 1.2-kb fragment of rat CRF cDNA, or a 600-bp mouse Ucn 3 full-length cDNA. Probes were labeled with [35S]UTP (PerkinElmer Life Sciences, Emeryville, CA). The specific activity of the probe was approximately 1–3 x 108 dpm/µg cRNA. The saturating concentration for the probe used in the assay was 0.3 µg/ml·kb. The procedure for in situ hybridization has been described previously (33). Briefly, the brain sections were fixed in 4% paraformaldehyde and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) followed by a rinse in 2x standard saline citrate (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air dried. The slides were exposed to the cRNA probe overnight in humidified chambers at 55 C. After incubation, the slides were washed in SSC of increasing stringency, in RNase, and then in 0.1x SSC at 63 C, dehydrated through a graded series of alcohols, and dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak, Rochester, NY), exposed for 14 d (Ucn 3) or 10 d (AVP and CRF) at 4 C, and developed. After development, the slides were counterstained with cresyl violet.

In situ data analysis
To analyze CRF, AVP, or Ucn 3 mRNA levels, coronal brain sections were anatomically matched across animals from all groups. The mRNA signal was quantitated using the Image Pro image analysis system by Media Cybernetics (Carlsbad, CA), which identified silver grains by the brightness of the image. An estimate for silver grains over the entire area of interest on each tissue section was given as the area occupied by silver grains within the marked area. The marked area was constant for each reading. For CRF and AVP, this included just the area encompassing the entire mRNA signal within the PVN. The cresyl violet counterstain allowed us to identify subregions within the PVN. For Ucn 3, mRNA was quantitated in the PFA, the MEA, and the MePO.

Statistical analysis
To exclude prestressed rats in the Ucn 3-injected animals, for ACTH and corticosterone analyses, data from animals with an ACTH level of greater than 50 pg/ml or corticosterone greater than 100 ng/ml in the basal sample were excluded, except in the experiment for catecholamine measurements in which ACTH was not measured and basal sample levels of catecholamines were all found to be within the expected range for unstressed rats (34). The number of experimental animals included in each group for statistical analyses after this exclusion is stated in the figure legends. The area under the curve (AUC) was calculated as a measure of total release of hormones over each time course using the trapezoidal rule (Kinetica; Innaphase, Philadelphia, PA). Statistical analyses were first performed using one-way ANOVA for comparison between groups with the Student-Newman-Keuls multiple comparison test employed to make post hoc comparisons. One-way ANOVA with repeated measures was used for within-group comparison at individual time points, and Dunnett’s multiple comparison test was used to make post hoc comparisons between the basal time point and subsequent time points within the group. Because plasma concentrations of catecholamines are not normally distributed (35), for analysis of catecholamine data, a nonparametric method was employed (Mann-Whitney U test). For AVP, CRF, and Ucn 3 mRNA expression, the Student’s t test or one-way ANOVA followed by the Student-Newman-Keuls multiple comparison test was employed. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of icv Ucn 3 on the hormonal response to restraint stress
To test the hypothesis that a CRFR2 ligand could modulate the HPA axis response to stressors, Ucn 3 was administered icv 10 min before a 15-min restraint stress and plasma levels of ACTH and corticosterone measured at intervals for 90 min after the onset of the stressor. ACTH was significantly elevated by restraint stress in all groups (Fig. 1AGo). ACTH levels in vehicle and 2 µg Ucn 3-treated groups had returned to basal levels at 60 and 90 min, whereas after 10 µg Ucn 3, ACTH levels were mildly but persistently elevated at these time points. The total ACTH released (AUC) over the time course was significantly increased by both doses of Ucn 3 (Table 1Go) with no quantitative difference between the 2- and 10-µg doses of Ucn 3. Corticosterone levels were elevated by stress at all time points in all groups, and levels between groups were not affected by Ucn 3 administration at any time point (Fig. 1BGo) as would be expected given that the ACTH levels reached in all groups should result in a maximal adrenocortical response (36, 37, 38).


Figure 1
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  		FIG. 1. Effects of Ucn 3 on the hormonal response to restraint stress. Ucn 3 was injected icv 10 min before the onset of a 15-min restraint stress. Plasma levels of ACTH (A), corticosterone (B), and glucose (C) and the insulin to glucose ratio (D) were measured at time points for 90 min after the onset of the stressor. Data are expressed as mean ± SEM. *, P < 0.01 for 10 µg Ucn 3 vs. vehicle; {dagger}, P < 0.05 for 2 µg Ucn 3 vs. vehicle; n = 8 for vehicle; n = 5 for 2 µg Ucn 3; n = 8 for 10 µg Ucn 3.

 

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  		TABLE 1. AUC for plasma ACTH

 
Increased sympathetic output is associated with stimulation of hepatic glucose production and inhibition of insulin release resulting in elevated blood glucose levels and a decreased circulating insulin to glucose ratio (39, 40). As expected, plasma glucose levels were elevated above basal levels in all groups by restraint stress, and this was augmented and prolonged by Ucn 3 (Fig. 1CGo) such that glucose levels were higher in Ucn 3-treated (2 and 10 µg) animals vs. vehicle at 5 and 15 min and remained elevated at 30 min by 2 and 10 µg Ucn 3 and at 60 min by the 10-µg dose. Plasma insulin was not significantly different between groups at any time point, although Ucn 3 tended to decrease levels over the time course (data not shown). Insulin to glucose ratios were calculated as an indirect indicator of sympathetic activity (40). The ratio was decreased below basal levels at all time points in Ucn 3-treated (2 and 10 µg) groups with no significant effect in the vehicle group. The insulin to glucose ratio was decreased with respect to the vehicle group by Ucn 3 at 60 min (2 and 10 µg) and 90 min (10 µg) (Fig. 1DGo).

Ucn 3 administered icv activates the HPA axis under basal conditions
Because Ucn 3 augmented the hormonal stress response to an exogenous stressor rather then inhibiting it, the effects of Ucn 3 on the HPA axis under basal conditions were investigated. Vehicle injection resulted in transient elevation of ACTH above basal levels at 5 and 15 min. An icv injection of 0.1 µg Ucn 3 was not significantly different from the vehicle group at any time point (Fig. 2AGo). Doses of 1 and 10 µg Ucn 3 significantly elevated ACTH levels above the basal blood sample level over the time course of the study and above the vehicle group level at 5 (10-µg dose only), 15, and 30 min post injection. Total ACTH release (AUC) was significantly elevated by 1 and 10 µg Ucn 3 with respect to vehicle and 0.1 µg Ucn 3, and 10 µg had a significantly higher AUC than 1 µg Ucn 3 (Table 1Go). Changes in corticosterone levels reflected the changes seen in ACTH levels with 10 µg Ucn 3 icv significantly elevating corticosterone levels above the vehicle group levels at 30 and 60 min post injection (Fig. 2BGo).


Figure 2
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  		FIG. 2. Ucn 3 effects on plasma ACTH and corticosterone levels. Ucn 3 was administered icv (A and B) or iv (C and D), and plasma ACTH (A and C) and corticosterone (B and D) levels were measured for 60 min after injection. Data are expressed as mean ± SEM. *, P < 0.05 for 10 µg Ucn 3 vs. vehicle; {dagger}, P < 0.05 for 1 µg Ucn 3 vs. vehicle; §, P < 0.05 for 1 µg Ucn 3 vs. 10 µg Ucn 3. For icv experiments, n = 8, n = 5, n = 5, and n = 8 for vehicle and 0.1, 1, and 10 µg Ucn 3, respectively. For iv experiments, n = 8, n = 7, n = 5, and n = 6 for vehicle and 0.1, 1, and 10 µg Ucn 3, respectively.

 
Ucn 3 stimulates sympathetic activity
The icv injection of 0.1 or 1 µg Ucn 3 had no effect on plasma glucose levels when compared with the vehicle group. However, 10 µg Ucn 3 resulted in significant elevations of glucose at 5, 15, and 30 min when compared with all other groups (Fig. 3AGo). Within-group analysis revealed that 10 µg Ucn 3 elevated glucose levels above basal levels at all time points, whereas levels were unchanged over the time course in the other groups.


Figure 3
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  		FIG. 3. Ucn 3 effects on circulating glucose and insulin to glucose ratios. Ucn 3 was injected icv (A and B) or iv (C and D), and plasma glucose (A and C) and insulin to glucose ratios (B and D) were measured for 60 min after injection. Data are expressed as mean ± SEM. *, P < 0.05 for 10 µg Ucn 3 vs. vehicle. For icv experiments, n = 8, n = 5, n = 5, and n = 8 for vehicle and 0.1, 1, and 10 µg Ucn 3, respectively. For iv experiments, n = 8, n = 7, n = 5, and n = 6 for vehicle and 0.1, 1, and 10 µg Ucn 3, respectively.

 
The icv injection of vehicle or Ucn 3 did not affect plasma insulin levels between or within groups over the time course (data not shown). However, 10 µg Ucn 3 tended to decrease plasma insulin levels over time (1.03 ± 0.11 ng/ml at 0 min vs. 0.77 ± 0.05 ng/ml at 60 min; P = 0.07), and insulin to glucose ratios were significantly decreased by 10 µg Ucn 3 at 5 and 60 min with respect to basal levels and at 5, 30, and 60 min with respect to vehicle, providing indirect evidence of increased sympathetic activity (Fig. 3BGo).

To obtain direct evidence of this sympathetic stimulation, plasma catecholamines were measured after icv administration of 10 µg Ucn 3 in an additional experiment. Epinephrine and norepinephrine were increased significantly at 5 min post injection, providing evidence of increased sympathoadrenomedullary and sympathoneural activity, respectively (Fig. 4Go). Levels had returned to baseline by 15 min.


Figure 4
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  		FIG. 4. Ucn 3 administered icv elevates plasma catecholamines. Ten micrograms of Ucn 3 were injected icv, and plasma levels of epinephrine (A) and norepinephrine (B) were measured and were elevated at 5 min post injection. Data are expressed as mean ± SEM. *, P < 0.05 vs. vehicle; n = 8 for vehicle; n = 7 for Ucn 3.

 
Effects of Ucn 3 are centrally mediated
An icv infusion generally results in significant exposure of the pituitary gland to peptide as well as variable amounts of peptide accessing the general circulation. Therefore, to determine whether the observed effects were mediated centrally or via peripheral mechanisms, Ucn 3 was injected iv at the same doses as had been employed for icv injections. The iv injection of vehicle or 0.1 or 1 µg Ucn 3 did not alter ACTH or corticosterone levels over the time course tested (Fig. 2Go, C and D). The 10-µg dose of Ucn 3 elevated ACTH above basal levels and above vehicle group levels at 5, 15, and 30 min post injection (Fig. 2CGo), and the AUC was also significantly greater (Table 1Go). Corticosterone levels for 10 µg Ucn 3 followed the expected pattern based on ACTH results (Fig. 2DGo).

Comparison between iv and icv groups revealed significantly increased ACTH release in icv-injected animals for 1 and 10 µg Ucn 3 as calculated by (AUC icv Ucn 3 – AUC icv vehicle) compared with (AUC iv Ucn 3 – AUC iv vehicle) (Fig. 5Go). For 0.1 µg Ucn 3, icv and iv groups were significantly different but with iv animals having the greater value.


Figure 5
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  		FIG. 5. Ucn 3 activation of the HPA axis is centrally mediated. Ucn 3 was administered icv or iv, and the AUC for ACTH release was calculated over 60 min after injection. Data are expressed as mean ± SEM. *, P < 0.05 for (icv Ucn 3 – icv vehicle) vs. (iv Ucn 3 – iv vehicle).

 
Likewise, plasma glucose levels were increased with respect to the other groups or to basal levels (within-group comparison) only in response to 10 µg Ucn 3 (Fig. 3CGo). The effect was of lesser magnitude and duration than with icv Ucn 3 injection. The insulin to glucose ratios were lowered significantly by 10 µg iv Ucn 3 with respect to vehicle and to basal levels at 5 min (Fig. 3DGo). Otherwise, there were no significant differences in insulin to glucose ratios between groups or within groups over the time course.

AVP expression levels are increased in the PVN after icv Ucn 3
Expression levels of CRF in the hypothalamic PVN 4 h after icv injection of 10 µg Ucn 3 were unchanged with respect to vehicle injection (Fig. 6AGo). In contrast, AVP expression was increased by Ucn 3, and this was specifically a result of increased expression levels in the parvocellular division PVN (Fig. 6Go, B and C).


Figure 6
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  		FIG. 6. CRF and AVP mRNA levels in the PVN after icv injection of Ucn 3. A, CRF mRNA levels in the PVN are unchanged by Ucn 3 vs. vehicle; B, AVP levels are increased in the parvocellular PVN (pv) and unchanged in the magnocellular PVN (mg) by Ucn 3 vs. vehicle; C, silver grains by in situ hybridization for AVP mRNA in the PVN. 3V, Third ventricle. Data are expressed as mean ± SEM. *, P < 0.05 vs. vehicle; n = 5 for vehicle; n = 6 for Ucn 3.

 
Ucn 3 expression is differentially regulated by stressors and environmental challenges
Ucn 3 mRNA expression was significantly increased in the MEA 30 min (174 ± 8% of control levels) after the onset of restraint stress with a trend to increased expression in the PFA, but this did not reach statistical significance (Fig. 7Go, A and B). Expression had returned to basal levels in animals subjected to restraint stress for 4 h. ACTH and corticosterone were maximally elevated at 30 min as expected (Fig. 7Go, D and E). However, ACTH had returned to basal levels after 4 h stress, whereas corticosterone was still elevated. In contrast, hemorrhagic stress produced a small but significant decrease in Ucn 3 expression (70 ± 6% of control) at 30 min post hemorrhage in the MEA (Fig. 8BGo). The activation of the HPA axis was of lesser magnitude with this challenge as measured by ACTH levels at 30 min and the return of corticosterone to basal levels by 4 h (Fig. 8Go, D and E).


Figure 7
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  		FIG. 7. Ucn 3 mRNA expression levels are increased in the MEA after restraint stress. Ucn 3 mRNA was quantified in PFA (A), MEA (B), and MePO (C) after 30 min or 4 h restraint stress. Circulating plasma ACTH (D) and corticosterone (E) were measured. Data are expressed as mean ± SEM. *, P < 0.05 vs. control; n = 5 for control and restraint stress at 30 min; n = 6 for control and restraint stress at 4 h.

 

Figure 8
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  		FIG. 8. Ucn 3 mRNA expression levels are decreased in the MEA after hemorrhage stress. Ucn 3 mRNA was quantified in PFA (A), MEA (B), and MePO (C) 30 min or 4 h after hemorrhage stress. Circulating plasma ACTH (D) and corticosterone (E) were measured. Data are expressed as mean ± SEM. *, P < 0.05 vs. control; n = 5 for control and n = 6 for hemorrhage at 30 min; n = 6 for control and hemorrhage at 4 h.

 
Rats that underwent 48 h food deprivation were subjected to a metabolic homeostatic challenge. Similar to hemorrhage, there was a decrease in Ucn 3 mRNA in the MEA (68 ± 10% of control) with no effect in other areas examined (Fig. 9AGo; see also Fig. 11Go, A and B). The HPA axis was activated with a relatively small increase in ACTH but high circulating corticosterone levels (Fig. 9Go, B and C). Plasma glucose and insulin were significantly lower than in control animals, confirming the effectiveness of the fast (Fig. 9Go, D and E).


Figure 9
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  		FIG. 9. Ucn 3 mRNA expression levels are decreased in the MEA after food deprivation. Ucn 3 mRNA was quantified in PFA, MEA, and MePO (A) after 48 h food deprivation. Circulating plasma ACTH (B), corticosterone (C), glucose (D), and insulin (E) were measured. Data are expressed as mean ± SEM. *, P < 0.05 vs. control; n = 6 for control and food deprivation.

 

Figure 11
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  		FIG. 11. Ucn 3 mRNA expression after food deprivation and ADX. Silver grains by in situ hybridization for Ucn 3 mRNA in the MEA of control animals (A) and after food deprivation (B) and in the rostral PFA of control (C), sham ADX (D), ADX (E), and ADX plus corticosterone (ADX+CORT) (F) animals. opt, Optic tract; fx, fornix. Scale bar, 50 µm.

 
To examine the potential role of glucocorticoids in mediating these regulatory effects, Ucn 3 expression after ADX or ADX with high-dose corticosterone replacement was examined. Ucn 3 mRNA expression in the PFA was increased dramatically by ADX (185 ± 15% of control levels or 195 ± 16% of sham ADX levels) and was completely restored to control or sham ADX levels by corticosterone (Figs. 10AGo and 11Go, C–F). Corticosterone did not, however, reduce expression levels further to below controls. The increase in expression in ADX rats was most marked in the rostral part of the PFA (Fig. 11Go, C–F). Expression in the MEA was unaffected by these manipulations (Fig. 10BGo). Measurement of corticosterone and ACTH levels confirmed the effectiveness of ADX and high-dose corticosterone replacement (Fig. 10Go, D and E).


Figure 10
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  		FIG. 10. Ucn 3 mRNA expression levels are increased in the PFA after ADX. Ucn 3 mRNA was quantified in PFA (A), MEA (B), and MePO (C) 7 d after ADX, sham ADX, or ADX plus corticosterone (ADX+CORT). Circulating plasma ACTH (D) and corticosterone (E) were measured. Data are expressed as mean ± SEM. *, P < 0.05 vs. control; n = 5 in all experimental groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ucn 3 and the hormonal stress response
Ucn 3 is to date the most specific agonist for CRFR2 described. Although showing similar binding affinity and activation of CRFR2 to Ucn 2, it demonstrates higher functional selectivity as demonstrated by its inability to activate adenylate cyclase in cells expressing CRFR1 at micromolar concentrations (15). Therefore, the effects of Ucn 3 in this study are discussed mainly within the context of its proposed action at the CRFR2 because the amount of peptide employed in these studies should not reach concentrations above those known not to activate CRFR1. The present study shows that exogenous icv administration of Ucn 3 stimulates ACTH and subsequently corticosterone secretion under both basal conditions and in animals exposed to a restraint stress. Under basal conditions, ACTH release was stimulated in a dose-dependent manner. In contrast, a lower 2-µg dose of Ucn 3 augmented the ACTH response to stress to the same levels as a 10-µg dose, possibly because of an enhanced effect in an already activated system (41), suggesting that activation of CRFR2 is stimulatory to the HPA axis.

CRF and AVP are the main physiological secretagogues for ACTH. CRF expression in the parvocellular PVN increases rapidly in response to acute stress with maximal levels seen around 2–4 h after stress (42). Increases in AVP expression are slower in onset but are detectable by 4 h (42). At 4 h post icv Ucn 3, AVP but not CRF expression was increased specifically within the parvocellular PVN, providing a possible link between Ucn 3 actions and the HPA axis. A mechanism involving CRF release or an effect on mRNA expression levels at an earlier time point cannot, however, be excluded. Exogenously administered Ucn 3 may act on CRFR2 in PVN (3, 4) to increase AVP expression. However, there are few Ucn 3 neuronal terminals in PVN (23), suggesting an endogenous effect would be likely mediated by Ucn 1 or Ucn 2 or indirectly via projections to PVN from major Ucn 3 terminal fields including the lateral septum, bed nucleus of the stria terminalis, and amygdala (23). Likewise, exogenous Ucn 3 could act indirectly on CRFR2 in these areas (3, 23), and this may explain the higher doses of Ucn 3 required to elicit responses compared with CRF in similar studies (43). Indeed, there is consistency across studies in that CRFR2-specific ligands require higher doses and have shorter-lived effects than CRF or Ucn on the same physiological parameters, suggesting a modulatory rather than a primary function. Two previous studies have described Ucn 3 actions on the HPA axis and reported no effects (18, 22). However, dose, experimental design, and method of administration differed from our model such that animals were stressed with elevated ACTH and corticosterone levels at the time of peptide administration, making comparisons with our data difficult.

Increased sympathetic output is associated with stimulation of hepatic glucose production and inhibition of insulin release resulting in elevated blood glucose levels and a decreased circulating insulin to glucose ratio (39, 40). This indirect measurement of the sympathoadrenal response also indicated activation by Ucn 3 under both basal and stressed conditions. CRFR2 in the area postrema and the nucleus of the solitary tract are well placed to mediate effects on glucose and insulin via the autonomic nervous system as seen with other peptide hormones (3, 44). Direct evidence of sympathetic activation in this study was provided by increased circulating catecholamines after icv Ucn 3, and this increase was rapid and transient as previously reported for this type of stressor (45). Ucn 3 administered icv has been reported to increase blood pressure and heart rate and to increase circulating epinephrine (12), whereas centrally administered Ucn 2 elevates portal pressure through CRFR2 and sympathetic-noradrenergic pathways (46). The icv Ucn 3 has also been found to elevate hippocampal corticosterone and norepinephrine metabolites via CRFR2 (13). The increase in circulating catecholamines in the present study additionally demonstrates the potential for Ucns acting within the CNS to elicit effects in the periphery via sympathoadrenomedullary responses as well as via direct autonomic innervation of specific organ systems.

The higher doses of Ucn 3 used in the present study have the potential to access the pituitary gland and may diffuse across the blood-brain barrier to the periphery (47). We demonstrated that the same dose of Ucn 3 administered iv was significantly less effective than icv Ucn 3 at activating the HPA axis, raising glucose and lowering glucose to insulin ratios, indicating the observed effects were likely predominantly centrally mediated. The small elevation in ACTH after 10 µg iv Ucn 3 was likely because of the homeostatic stress of hypotension. CRFR ligands including Ucn 3 cause vasodilation, attributable to peripheral activation of vascular CRFR2 (48, 49, 50). CRFR2 agonists have potential effects on pancreatic physiology. Peripherally administered Ucn 3 sequentially elevates plasma glucagon, glucose, and then insulin, which appears to be a CRFR2-mediated effect on pancreatic islet secretion (51). The icv Ucn 3 did not increase insulin levels at any dose in this study, again suggesting the effects on glucose to insulin ratio effects are mediated via a different mechanism.

Regulation of Ucn 3 in the brain by stressors and environmental challenges
If endogenous Ucn 3 has a role in modulating the stress response, its expression levels are likely to be regulated in response to relevant physiological circumstances as for CRF (52). We found that expression levels are indeed regulated by environmental challenges in a stressor- and site-specific manner as for other CRF family members and receptors (52, 53, 54, 55).

Restraint stress has been consistently reported to increase expression of CRF ligands (55, 56, 57), and accordingly, we observed robust induction of Ucn 3 expression. Ucn 3 expression has been reported to increase in MEA, but not the PFA, after restraint stress in mice (22). In fact, the trend was to increased expression in both sites, which is consistent with our data. Thus, Ucn 3 mRNA expression appears to be rapidly responsive to psychological stressors in keeping with its proposed role in modulating anxiety-like behaviors (20, 22). Indeed, the rostral PFA projects to the lateral septum, the posterior division of the bed nucleus of the stria terminalis, and the ventromedial hypothalamus, areas associated with behavioral responses to stressors including anxiety and anorexia (58, 59), whereas stimulation of the MEA is excitatory to the HPA axis (60).

In contrast, a small but significant decrease in expression in MEA was observed after hemorrhage and food deprivation. This discrepancy has previously been reported for Ucn 1 (61) and may reflect differences in circuitry between stressors (62). Many investigators categorize stressors into psychological and homeostatic stimuli. Restraint stress has a strong psychological component and preferentially activates neurons in the MEA, whereas hemorrhage and other homeostatic stressors recruit neurons in the central amygdala (63, 64). Alternatively, additional behavioral or metabolic responses required by a given challenge may modify the effects on Ucn 3 expression. Food deprivation is a homeostatic stressor but also requires appetite stimulation, an effect that may be facilitated by reduced Ucn 3 expression, thus overriding regulation due to the stressful aspects of the manipulation. Indeed, CRF expression is also attenuated by food deprivation (65, 66), an appropriate pattern of regulation given the anorectic properties of both peptides (18, 67). CRFR2 is also down-regulated in ventromedial hypothalamus by starvation (68). Likewise, hemorrhage requires vasomotor responses that might be best served by a reduction in a vasodilatory peptide such as Ucn 3.

Changes in Ucn 3 expression after restraint and hemorrhage were rapid but transient. Reul and Holsboer (69) proposed a model whereby acute activation of the CRFR2 potentiates the stress response in the short term but exerts a delayed anxiolytic role in the stress recovery phase, which could explain this observation. The cDNA probe used in this study spanned an intronless region (15) and therefore recognizes heteronuclear RNA in addition to mature mRNA allowing detection of early changes in transcriptional activity.

Significant induction of Ucn 3 mRNA levels was observed chronically after ADX specifically in PFA with no change in amygdalar levels. In ADX, loss of negative feedback on the HPA axis increases CRF levels in the PVN (70) and suppresses expression in the amygdala and bed nucleus of the stria terminalis (71, 72), and Ucn 3 expression may be regulated similarly. In agreement with the previous report, where glucocorticoids did not alter Ucn 3 mRNA levels (22), high-level corticosterone restored expression to control levels but decreased them no further. Levels in the amygdala were unaffected by corticosterone. Therefore, the effects of ADX on Ucn 3 mRNA expression are likely more complicated than a direct regulatory effect of glucocorticoids.

In summary, these data add to the considerable evidence that Ucn 3 and CRFR2 play an important but complex role in regulating central responses to stressors. The adaptive significance of the stress response depends upon achieving a balance between mounting an adequate coping response and limiting the detrimental consequences of prolonged or overwhelming stress. The importance of the system is likely to be in the fine tuning required to achieve the most appropriate response to any given challenge to maximize the benefit to the organism.


   Acknowledgments
 
We thank Dr. Graeme Eisenhofer for advice on catecholamine analyses and interpretation of data and Dr. Ruth Andrew for assistance with data handling.


   Footnotes
 
This work was supported by National Institutes of Health Grant DK26741, The Adler Foundation, The Clayton Foundation, and the Kleberg Foundation.

Disclosure statement: P.M.J., C.L., C.K., and J.V. have nothing to disclose. W.V. is a Senior Foundation for Research Investigator and consults for Neurocrine Biosciences Inc. and Acceleron Pharma Inc. W.V. has equity interests in Neurocrine Biosciences Inc. and Acceleron Pharma Inc.

First Published Online June 29, 2006

Abbreviations: ADX, Adrenalectomized; AUC, area under the curve; AVP, arginine vasopressin; CNS, central nervous system; CRF, corticotropin-releasing factor; CRFR, CRF receptor; HPA, hypothalamic-pituitary-adrenal; icv, intracerebroventricular; MEA, medial amygdala; MePO, median preoptic nucleus; PFA, rostral perifornical area; PVN, paraventricular nucleus; SSC, standard saline citrate; Ucn, urocortin.

Received April 25, 2006.

Accepted for publication June 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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