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Endocrinology Vol. 148, No. 10 4585-4591
Copyright © 2007 by The Endocrine Society

Identifying Early Behavioral and Molecular Markers of Future Stress Sensitivity

Nirupa Goel and Tracy L. Bale

Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Tracy L. Bale, University of Pennsylvania, 201E Vet, 6046, 3800 Spruce Street, Philadelphia, Pennsylvania 19104. E-mail: tbale{at}vet.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Puberty is a plastic period of neurological development when critical maturation of stress pathways occurs. Abnormal maturation may be predictive of future stress sensitivity and affective disorder risk. To identify potential early markers of stress-related disease predisposition, we examined physiological and behavioral stress responses in male pubertal mice compared with adults, using a genetic model of elevated stress sensitivity, CRF receptor-2 (CRFR2)-deficient mice. Juvenile mice of both genotypes exhibited greater basal and stress-induced corticosterone levels than adult mice, indicating that overall hypothalamic-pituitary-adrenal axis sensitivity diminishes in adulthood. However, juvenile CRFR2-deficient mice displayed a delayed stress recovery typical of adults of this genotype, suggesting an early marker of stress sensitivity. The adult phenotype of reduced hippocampal glucocorticoid receptor expression in these sensitive mice was also detected during puberty. This reduction may account for an impaired hypothalamic-pituitary-adrenal axis negative feedback and as such be an early indicator of a stress-sensitive phenotype. Examination of behavioral responses to stress revealed that CRFR2-deficient mice show exaggerated postpubertal maturation. Although wild-type mice did not alter their burying response to stress-provoking marbles after puberty, CRFR2-deficient mice showed a dramatic increase in burying behavior. We conclude that identification of abnormal pubertal stress pathway maturation may be predictive of adult heightened stress sensitivity and future susceptibility to stress-related affective disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DEVELOPMENT OF affective disorders such as anxiety and depression is highly linked with stressful life events. The stress response is important for survival and maintenance of homeostasis but in a dysregulated state may be a predisposing factor for these disorders (1). Affective disorders increase in prevalence after puberty, a period when gonadal hormones and organization of neural circuits leads to the full presentation of adult behaviors and physiology, including those related to stress and anxiety (2, 3, 4). Biological transitional states during which major changes in hormone levels occur, such as puberty or pregnancy, are associated with psychological vulnerability and may be related to dysfunction of the stress system (5, 6). Thus, examining the maturation of stress neurocircuitry in maladaptive animal models may provide valuable insight into possible markers of affective disorder susceptibility and identify new targets for therapeutic development.

Physiological and behavioral responses to stress normally change around the time of puberty, resulting in the mature adult phenotype. In both humans and rodents, stress-induced corticosteroid release decreases with progression through puberty (7, 8, 9). Hippocampal glucocorticoid receptor expression gradually increases during development and peaks at puberty, concurrent with the emergence of receptor autoregulation (10). As a further indication of the maturating stress system, negative feedback inhibition of the hypothalamic-pituitary-adrenal (HPA) axis reaches adult levels of sensitivity after puberty (11). Behaviorally, alterations in anxiety-related responses also emerge during puberty, including decreased social interactions in a novel environment, decreased anxiety-like behavior in the light-dark test, and decreased novelty-induced arousal (12, 13, 14).

Maturation of stress responsivity is likely a result of morphological and synaptic modifications that occur during pubertal development. The amygdala and hippocampus, which are both limbic regions with an abundance of androgen and estrogen receptors, grow disproportionately in volume during puberty in both rodents and humans (15, 16, 17). Dendritic spine density increases in the hippocampus at puberty onset and decreases in late puberty in male mice. Gonadectomy before puberty prevents this change (18). Substantial pruning of dendrites and spines also occurs in the medial amygdala, whereas projections from the basolateral amygdala to the prefrontal cortex increase in density (19, 20). Gonadal steroids can alter the morphology of dendrites and synapse density directly in hormone-sensitive cells as well as transsynaptically (21). Thus, the connectivity and function of networks important in the regulation of emotion and stress are poised to be modified by the pubertal rise in gonadal hormones. However, these processes may fail to occur or occur incorrectly due to genetic, epigenetic, or environmental influences, resulting in heightened stress sensitivity and an increased susceptibility to affective disorders.

CRF is a principal component of the stress response (22). The CRF family consists of several ligands and two receptors, CRF receptor-1 (CRFR1) and CRFR2 (23). After a stressor, CRF activates the HPA axis by acting on CRFR1 in the anterior pituitary (22). CRFR1 may also be involved in behaviors relevant to depression and anxiety, whereas the role for CRFR2 in stress responsivity is less understood (24, 25, 26). Patients with depression and anxiety disorders display alterations in the CRF system including increased CRF expression in the hypothalamus and locus ceruleus, elevated CRF concentrations in cerebrospinal fluid, and reduced CRFR1 in the frontal cortex (27, 28, 29, 30, 31) (for review see Ref. 32). Furthermore, HPA abnormalities such as cortisol hypersecretion are reported in patients with affective disorders and can be normalized after successful antidepressant treatment (33, 34). Additionally, Rett syndrome is a neurological disorder characterized by social behavior deficits and anxiety, and recent research has identified increased CRF expression as a probable mediator of these symptoms (35). Thus, central dysregulation of the CRF system is associated with heightened risk of disease presentation.

Genetic mouse models of elevated stress sensitivity, especially those that involve disruption of CRF pathway function, can aid in the elucidation of mechanisms underlying disease onset. CRFR2-deficient mice display increased stress responsivity and a prolonged stress recovery time (36, 37). These mice also exhibit increased anxiety- and depressive-like behaviors in tests of active and passive coping responses in stress-provoking novel environments (36, 38, 39, 40). In addition, CRF and urocortin I, ligands with high affinity for CRFR1, are basally elevated in these mice (36). Thus, CRFR2-deficient mice provide us with a useful genetic model of CRF dysregulation in which stress pathway maturation can be examined to identify potential markers of future stress sensitivity and disease predisposition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
CRFR2-deficient (KO) and wild-type (WT) mice were generated on a mixed 129:C57BL/6J background from heterozygous breeding as previously described (36). Male mice were divided by age into two groups: juvenile and adult. All mice were weaned at 28 d of age. Juvenile mice were tested during mid-puberty, at d 37–46 (41). This allowed for 1 wk of habituation after weaning. Adults tested were 4–5 months old. Separate cohorts of mice were used for testosterone measurements, corticosterone measurements, and behavioral analyses. Mice were group housed under controlled conditions of a 12-h light, 12-h dark cycle (lights on at 0700 h) with access to food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

Testosterone analysis
Blood samples were collected from male WT and KO mice on postnatal d 42 via a retroorbital bleed to measure plasma testosterone levels at mid-puberty (n = 9). Adult WT and KO plasma was obtained from trunk blood at 4 months of age (n = 5). Plasma was stored at –80 C until assayed for testosterone using a commercial 125I RIA kit (Diagnostic Products Corp., Los Angeles, CA). The minimum detection limit of the assay was 4 ng/dl, and intraassay coefficient of variation was 17%.

Corticosterone analysis
To measure the HPA axis response to acute stress, mice were exposed to a 15-min restraint in a 50-ml conical tube (n = 5). Blood samples were collected from a tail nick at four time points: 1) time 0, immediately upon removal from the cage; 2) time 15, immediately after the restraint stress; 3) time 30, after 15 min of recovery in the home cage; and 4) time 60, after 45 min of recovery in the home cage. Samples were collected into EDTA-treated tubes, immediately centrifuged, and stored at –80 C until assayed for corticosterone. Corticosterone concentrations were measured using a commercial 125I RIA kit (MP Biomedicals, Orangeburg, NY). The minimum detection limit of the assay was 7.7 ng/ml, and intraassay coefficient of variation was 7.3%.

Open-field test
Using a separate cohort (n = 10) of WT and KO, juvenile and adult mice, we examined anxiety states in the open-field test. Testing was performed during the dark phase of the light/dark cycle as previously described (n = 10) (36). Test duration was 5 min. Crosses into the inner four squares, total number of lines crossed, and number of fecal boli were quantified.

Marble-burying test
As another measure of stress-provoked anxiety-like responses, we performed the marble-burying test 48 h after the open-field test. Mice were placed individually in cages (20 x 40 x 15 cm) with 5 cm of bedding and 12 uniform marbles evenly distributed on the surface of the bedding. Testing occurred in the dark phase of the light/dark cycle for a duration of 30 min. Number of marbles buried (two thirds covered by bedding) were binned at 5-min intervals.

In situ hybridization
We measured expression of genes important in stress neurocircuitry by in situ hybridization. Mice were killed 36 h after the marble-burying test. Brains were rapidly removed and frozen at –80 C until sectioning. Parallel series of tissue were cut into 20-µm coronal sections. Randomly selected brains were assayed for CRF mRNA in the paraventricular nucleus of the hypothalamus (PVN) and the central nucleus of the amygdala (CeA) (36). Hippocampal tissue from these brains was assayed for type II glucocorticoid receptor mRNA (GR probe kindly provided by Dr. Audrey Seasholtz, University of Michigan, Ann Arbor, MI) (42). Slides were processed and hybridized according to methods previously described (43). After incubation with 35S-labeled riboprobe, slides were washed in 1x saline sodium citrate at room temperature, ribonuclease buffer at 37 C, and 0.1x saline sodium citrate at 65 C. Glucocorticoid receptor hybridized slides were apposed to film for 7 d. Glucocorticoid receptor films were analyzed for mean OD of signal using IPLab Scientific Image Processing (BD Biosciences, San Jose, CA) such that anatomically matched sections for each animal were compared. A background reading of similar area and adjacent to the region of interest for each brain section analyzed was taken and subtracted from the specific signal. Two measurements from adjacent brain sections were averaged for each animal (n = 5).

CRF slides were dipped in NTB liquid nuclear emulsion (Eastman Kodak, Rochester, NY) as described (36). Slides were exposed for 18 d. Slides were counterstained with hematoxylin. The level of CRF labeling was quantified by counting grains per cell using IPLab software. Under bright-field illumination, a region of interest was drawn. For PVN analysis, a circular region including magnocellular and parvocellular divisions was used. For CeA analysis, a rectangular region that encompassed the area with signal was used. All cells within the drawn region were counted for grains (average of 89 cells per section for PVN and 110 cells per section for CeA). Grain counts were multiplied by a factor of 1.6 to correct for discrepancy between the software’s default setting of grain size (1.25 pixels) and measured grain size (0.79 pixels). The grain density was then calculated and corrected for background by subtracting the density measurement from an area of nonspecific signal. Brain sections chosen for analysis were anatomically matched, and measurements from two adjacent sections were averaged for each animal (n = 5).

Statistical analysis
Corticosterone and marble-burying data were analyzed using repeated-measures multivariate ANOVA (time x age x genotype). All other data were analyzed using two-way ANOVA (age x genotype). Significant differences were identified at P ≤ 0.05. Significant main effects and interactions were further analyzed with Student’s t test. For data on CRF expression in the CeA, the interaction term was not significant (P = 0.26) and was therefore dropped from the model to explore significant main effects. All data are reported as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pubertal maturation
To confirm that there were no genotypic differences in pubertal development, we compared body weights and testosterone levels of these animals. No differences were found between genotypes for body weights at 42 d of age (WT, 19.5 g ± 0.8; KO, 20.1 g ± 0.4; P = 0.53). Additionally, two-way ANOVA of testosterone levels indicated no differences between groups (juvenile WT, 283.64 ng/dl ± 108.68; juvenile KO, 177.43 ng/dl ± 143.44; adult WT, 81.96 ng/dl ± 10.49; adult KO, 44.00 ng/dl ± 5.06; F1,25 = 0.30, P = 0.58 for genotype; F1,25 = 1.65, P = 0.21 for age).

HPA axis activity
To examine the maturation of the HPA stress axis, corticosterone levels were measured in juvenile and adult mice after a 15-min restraint stress. The assay revealed a significant increase in corticosterone levels in juveniles and an altered course of stress recovery when compared with adults (Fig. 1Go). A significant main effect of age was found (F1,18 = 4.42, P < 0.05). Juveniles had higher corticosterone levels than adults at baseline and immediately after the stressor (F1,17 = 7.19, P < 0.05 for time 0; F = 7.17, P < 0.05 for time 15). There was no effect of genotype on plasma corticosterone levels. There was a significant main effect of time (F3,15 = 29.03, P < 0.0001) and a time x age interaction (F3,15 = 5.79, P < 0.01). This interaction effect was significant for the time points during recovery (F2,18 = 8.99, P < 0.01 for time 15–60) but not for the initial corticosterone rise (F1,17 = 3.65, P = 0.07). In response to stress, corticosterone release increased in all groups, and during recovery, levels began to decline in juveniles while remaining elevated in adults.


Figure 1
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  		FIG. 1. Juvenile mice exhibited higher basal and stress-induced corticosterone levels. After a 15-min restraint stress, adult mice showed a prolonged stress recovery, whereas juvenile corticosterone levels declined at a faster rate (time x age interaction, P < 0.01). *, P < 0.05, main effect of age at time 0 and 15.

 
Behavioral responses to novel stressors
To determine how stress-induced coping behaviors mature over puberty and how this may differ in a CRF dysregulated system, mice were tested in the open-field and marble-burying tests. Results from the open-field test showed no differences in crosses into the inner squares (Fig. 2AGo). A main effect of genotype revealed that KO mice displayed reduced arousal, as measured by total line crosses, during the test (F1,36 = 6.99, P < 0.05) (Fig. 2BGo). There were no significant differences in rearing (juvenile WT, 21.3 ± 2.7; juvenile KO, 17.8 ± 3.0; adult WT, 21.4 ± 2.2; adult KO, 19.2 ± 2.9; F1,39 = 1.09, P = 0.30 for genotype; F1,39 = 0.08, P = 0.78 for age). In addition, no differences in grooming were noted. Furthermore, KO mice failed to show the normal maturation of a reduced fecal boli response found in WT mice. WT mice exhibited a significant decrease in fecal matter with age, but this did not occur in KO mice (Fig. 2CGo). A significant main effect of age (F1,36 = 29.84, P ≤ 0.01) and a significant age x genotype interaction (F1,36 = 26.49, P < 0.05) were detected.


Figure 2
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  		FIG. 2. In the open-field test, stress-sensitive KO mice showed alterations in maturation of stress responses. A, Examination of crosses into the inner squares of the box revealed no differences between groups. B, Adult KO mice displayed fewer overall line crosses than WT littermates. *, P < 0.05, main effect of genotype. C, Fecal boli produced during the open-field test decreased with age in WT but remained elevated in KO mice. *, P < 0.05 compared with WT adults; #, P < 0.05 compared with WT juveniles.

 
Stress-coping behaviors were also examined in a 30-min test of marble burying. Burying increased over time in all groups, and adult mice buried significantly more marbles than juveniles (Fig. 3Go). Main effects of time and age were detected (F5,32 = 9.69, P < 0.0001 for time; F1,36 = 5.87, P < 0.05 for age). At time 30, the age x genotype interaction term was nonsignificant (F1,36 = 2.20, P = 0.15), but the age difference in burying appears to be driven by an increased response in adult KO mice.


Figure 3
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  		FIG. 3. Stress-sensitive KO mice showed an augmentation of burying behavior maturation in the marble-burying test. Juvenile mice buried significantly fewer marbles than adults. *, P < 0.05, main effect of age by repeated-measures ANOVA.

 
Gene expression
Puberty-related alterations in stress responses may result from changes in expression of genes involved in CRF signaling. To examine this, we compared expression levels of CRF and glucocorticoid receptors. In the PVN, there were no genotypic or age-related changes in CRF (Fig. 4Go, A and B). The apparent genotypic difference in juvenile animals was not significant (F1,17 = 4.68, P = 0.16). In the CeA, the density of CRF mRNA per cell was elevated in KO mice, as revealed by a significant main effect of genotype (F1,13 = 4.71, P < 0.05) (Fig. 4Go, C and D).


Figure 4
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  		FIG. 4. Increased CRF expression in the CeA is an early marker of stress sensitivity. A and C, Silver grains for CRF mRNA in the PVN (A) and CeA (C) for juvenile (Juv) and adult (Ad) WT and KO mice. B, Analysis of CRF in the PVN revealed no group differences in density of grains per cell. D, Expression of CRF mRNA in the CeA was increased in KO mice. *, P < 0.05, main effect of genotype. Diagrams were adapted from Paxinos and Franklin (60 ). BLA, Basolateral amygdala; MeA, medial amygdala; SCN, suprachiasmatic nucleus; 3V, third ventricle.

 
Glucocorticoid receptor expression in the hippocampus was also investigated (Fig. 5AGo). There was a main effect of genotype in the CA1 and CA3 subregions, indicating that KO mice showed reduced expression (F1,13 = 4.54, P ≤ 0.05 for CA1; F1,14 = 6.57; P < 0.05 for CA3) (Fig. 5Go, B and C). In addition, there was a trend for decreased expression in KO mice in the dentate gyrus (F1,14 = 3.14, P = 0.10) (data not shown).


Figure 5
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  		FIG. 5. Stress-sensitive KO mice showed reduced hippocampal glucocorticoid receptor expression. A, Autoradiographs of glucocorticoid receptor mRNA for juvenile and adult WT and KO mice. B and C, In the CA1 (B) and CA3 (C) subregions, KO mice displayed decreased glucocorticoid receptor expression. *, P < 0.05, main effect of genotype. Diagram was adapted from Paxinos and Franklin (60 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The prevalence of many stress-related psychiatric disorders rises markedly after puberty. The lifetime prevalence of mood disorders increases from 1% at age 11 to 21% at age 18 (44). As a critical period of neural reorganization, puberty leads to the maturation of anxiety-related responses and stress physiology (3, 4, 13). Use of a stress-sensitive genetic mouse model to examine maturation of stress neurocircuitry may elucidate early markers of stress sensitivity and possible future affective disorder predisposition. We investigated behavioral and physiological stress responses in CRFR2-deficient mice during puberty and adulthood and compared these responses with alterations in gene expression in critical stress-regulatory brain regions.

Neurobiological transitions during puberty lead to changes in HPA stress axis reactivity. In our studies, we found that juveniles exhibited higher basal and stress-induced corticosterone levels that were blunted in adulthood. Previous studies have also shown postpubertal decreases in corticosterone, both basally and after stress (8, 9, 45). The maturation of the HPA stress axis may be partially due to decreased stress-induced neuronal activation in the PVN after puberty (8, 46). It is interesting to note that the magnitude of the corticosterone response in juvenile males was comparable to levels normally found in adult females after a stress exposure (47). Because HPA axis hyperactivity is associated with major depression, and because women are more susceptible to stress-related affective disorders, our data may support that puberty is a time of heightened emotional instability. The use of antidepressant drugs in juveniles is associated with an increased risk of suicidal ideation, further emphasizing the psychological vulnerability of this age group (48).

Despite the higher stress-induced corticosterone levels, juvenile mice showed a faster rate of stress recovery, suggesting a more sensitive negative feedback of the HPA axis. However, by 60 min, corticosterone levels did not fully return to baseline levels, suggesting that the procedure of taking blood repeatedly from the animal may be an additional mild stressor. Although not significant, KO mice appeared to have elevated corticosterone levels during stress recovery. Increased corticosterone at 10 min of restraint stress and during recovery 90 min after stress has been previously reported in adult KO mice, using different methods and separate mice for each time point (36, 37). The reduced glucocorticoid receptor levels seen in both age groups in the CA1 and CA3 hippocampal regions may contribute to delayed stress recovery because these receptors are important in negative feedback of the HPA axis (49). Thus, CRF pathway dysregulation can potentially exacerbate the stress phenotype of juveniles and may be an early contributor to disease vulnerability.

Testosterone rises dramatically during puberty in males and may partially mediate the maturation of the HPA axis. Numerous studies have reported an increased stress response after gonadectomy in males, suggesting an inhibitory action of testosterone on stress-induced corticosterone release (50, 51, 52). However, prepubertal rats given physiological doses of testosterone continued to exhibit heightened stress-induced HPA activity compared with adults (9). Therefore, neural organization during puberty appears necessary for the inhibitory actions of testosterone to be functional.

In addition to the physiological HPA stress response, our studies also identified pubertal effects on behavioral responses to novel stress-provoking environments. Examination of anxiety-like responses and passive coping in the open-field test revealed that arousal in the novel environment remained unchanged in WT mice from puberty to adulthood. However, KO mice exhibited a significant reduction in stress-stimulated activity that appeared to be more pronounced in adulthood. This reduction in activity was not explained by a genotypic difference in rearing or grooming and thus may be an indication of increased fear and anxiety in the novel environment (53). Because we have previously shown that there are no genotypic differences in locomotor activity in a habituated environment, these results indicate that the decrease in line crosses seen in the present study is specific to settings of novel exploration (38). CRF is known to reduce activity in unfamiliar environments, supporting a possible increased action of the CRF system in KO mice (54).

Examination of fecal boli production in the open-field test is another indicator of an anxiety-like state in rodents (53, 55). As expected, fecal boli production in WT mice decreased significantly in adulthood. However, this reduction was not found in stress-sensitive KO mice. Postpubertal decreases in stress sensitivity likely involve an influence of testosterone because previous studies have shown that gonadectomy of adult males leads to increased fecal boli in the open field (56). These results support a failure of KO mice to display a characteristic stress reduction and ability to passively cope to an anxiogenic environment as adults. Our findings from the open-field test show that stress sensitivity increases in KO mice from puberty to adulthood, but not in WT mice, suggesting an abnormal maturation of stress pathways during puberty.

To further examine the progression of typical male active coping strategies during stress provocation, we measured behaviors in a marble-burying test. Overall, adult mice buried significantly more marbles than juveniles, supporting a postpubertal maturation of active coping responses. CRF increases defensive burying of an aversive stimulus, suggesting a possible increased potency of the CRF system in adulthood (57). This higher degree of active coping corresponds to the lower sensitivity of the HPA axis, perhaps indicating that adults are less emotionally vulnerable than juveniles. Although the interaction between age and genotype was not significant, it appeared that the greatest age-related alteration in behavior is in the stress-sensitive KO mice, due to low burying activity in KO juveniles. Depleted active coping in KO juvenile mice is again suggestive of a maladaptive stress state during puberty that can be exaggerated by CRF dysregulation. It is unknown whether the pubertal rise in testosterone plays a role in the progression of these behaviors because the effects of testosterone on defensive burying are unclear and have not been well studied. To summarize, CRF dysregulation seems to contribute to the heightened emotional vulnerability of puberty by diminishing the ability to actively cope with stress provocation. Furthermore, as seen in the open-field test, CRF dysregulation causes an altered pubertal maturation in passive coping that leads to a stress-sensitive phenotype in adulthood.

To examine possible underlying mechanisms for a failure in stress system maturation, we investigated gene expression levels for hippocampal glucocorticoid receptors and CRF in the PVN and CeA. As expected, glucocorticoid receptors appeared to increase in adult WT mice in the CA1 region of the hippocampus, but expression did not change with age in stress-sensitive KO mice. Glucocorticoid receptor levels are known to increase during development, concurrent with rising sensitivity to negative-feedback inhibition of the HPA axis (10, 11). Thus, the failure of this progression in the KO mice may be a marker of stress sensitivity and may, in part, explain the prolonged stress recovery detected in these mice (37). Another early marker was the reduced glucocorticoid receptor expression in the CA3 region, which was found in the KO mice at both ages. A similar trend was detected in the dentate gyrus. Thus, the KO mice show a dysregulation of the glucocorticoid system that may lead to alterations in stress and behavioral responses. Prolonged exposure to corticosterone due to delayed stress recovery can lead to dendritic atrophy in CA3 neurons and sensitization to the neurotoxic effects of future stress insults (58).

We also examined CRF expression in the PVN and CeA to further identify alterations in key stress-regulatory brain regions. CRF in the PVN was not significantly altered with age, in accordance with findings from previous reports, although there was a trend for reduced expression in juvenile KO mice (46). In the CeA, CRF was elevated in KO mice, consistent with our previous studies (36). This effect appeared to be more pronounced in the juvenile KO mice and may serve as an early marker of behavioral stress sensitivity. The alterations in CRF expression correlate with the increase in the CeA and decrease in the PVN that are known to occur in response to corticosterone (59). Additionally, our behavioral results may be partially mediated by increased CRF in the CeA, which has been implicated in suppression of exploration and increases in anxiety and fear-related behaviors (54). Thus, early alterations in glucocorticoid receptor and CRF expression may be indicators of abnormal stress pathway maturation.

In conclusion, these data show that interactions between a genetic vulnerability, modeled here as CRFR2 deficiency, and maturation during puberty shape the adulthood expression of physiological and behavioral stress responses. Puberty is a key transition period in the development of affective disorders, and there is a critical need for the investigation of the impact of stress in juvenile animals. Using our stress-sensitive mouse model, we have identified early markers of stress sensitivity that may be predictive of future stress-related disease. CRF dysregulation appears to increase the pubertal phenotype of a heightened HPA stress response. In addition, these mice showed abnormal maturation of anxiety-like behavioral responses as well as exhibiting characteristically low burying activity as juveniles. Underlying these physiological and behavioral markers, we found that juvenile KO mice expressed reduced levels of glucocorticoid receptors in the hippocampus and elevated CRF in the CeA. Our results emphasize that the juvenile period is a critical time for stress system maturation and that the early emergence of stress sensitivity may be associated with a predisposition for affective disorders.


   Acknowledgments
 
We thank Kendall Carlin for technical assistance.


   Footnotes
 
This work was supported by the University of Pennsylvania Research Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: CeA, Central nucleus of the amygdala; CRFR1, CRF receptor-1; HPA, hypothalamic-pituitary-adrenal; KO, CRFR2-deficient; PVN, paraventricular nucleus of the hypothalamus; WT, wild type.

Received April 13, 2007.

Accepted for publication July 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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N. Goel and T. L. Bale
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