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Endocrinology Vol. 143, No. 5 1717-1725
Copyright © 2002 by The Endocrine Society

CRH-ACTH-POMC-ADRENAL

Differential Expression of c-fos and Tyrosine Hydroxylase mRNA in the Adrenal Gland of the Infant Rat: Evidence for an Adrenal Hyporesponsive Period

Darren K. Okimoto, Alison Blaus, Mathias Schmidt, M. Kathleen Gordon, Gersham W. Dent and Seymour Levine

Department of Biological Sciences (D.K.O., M.S.), University of Delaware, Newark, Delaware 19716; Division of Medical Pharmacology (M.S.), Leiden/Amsterdam Center for Drug Research (LACDR), University of Leiden, 2300 RA Leiden, The Netherlands; Department of Psychology (M.K.G.), University of Delaware, Newark, Delaware 19716; CNS Diseases (G.W.D.), Bristol-Myers Squibb Pharma Company, Glenolden, Pennsylvnia 19036; and Center for Neuroscience (S.L.), University of California, Davis, California 95616

Address all correspondence and requests for reprints to: Seymour Levine, Center for Neuroscience, University of California, 1544 Newton Court, Davis, California 95616. E-mail: . slevine{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rats exhibit a stress hyporesponsive period from postnatal day (PND) 4–14 in which the neonate displays a minimal corticosterone response to stress. We used the maternal deprivation model to test whether this adrenocortical hyporesponsiveness to stress results from a decrease in adrenal sensitivity to ACTH. Neonates (PND 6, 9, and 12) were injected ip with dexamethasone to block endogenous ACTH release, and 4 h later injected with graded doses of ACTH and killed. In another experiment, neonates were injected with isotonic saline and adrenal glands were collected at 30, 60, and 120 min post injection to examine c-fos and tyrosine hydroxylase mRNA levels using in situ hybridization. Maternally deprived pups demonstrated elevated corticosterone levels at the two highest ACTH doses and showed a greater magnitude in glucocorticoid secretion compared with the nondeprived pups. Maternally deprived pups given a saline injection exhibited elevated basal and stress-induced levels of corticosterone, in contrast to the nondeprived pups that showed a minimal response. Strikingly, maternally deprived pups exhibited elevated levels of adrenocortical c-fos mRNA, whereas the nondeprived pups did not. In contrast, the pattern of c-fos gene expression in the adrenal medulla in both groups did not display any correlation with glucocorticoid secretion. Tyrosine hydroxylase gene expression in the adrenal medulla was observed in both nondeprived and maternally deprived pups, with the latter exhibiting an earlier response of greater magnitude. These results demonstrate that the suppression of steroidogenesis occurs directly in the adrenal cortex and provide further evidence for an adrenal hyporesponsive period in the rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING POSTNATAL DEVELOPMENT, there is a period from d 4–14 during which the hypothalamic-pituitary-adrenal (HPA) axis of the rat is hyporesponsive to stressors that would normally cause a robust corticosterone response in the adult. During this stress hyporesponsive period (SHRP), it has been suggested that corticosterone is maintained at the low levels required for normal growth and development of the central nervous system during ontogeny (1, 2, 3, 4).

In recent years, several investigators (5, 6) hypothesized that the SHRP may in part result from an adrenocortical hyporesponsiveness to ACTH. It has been demonstrated that, following acute adrenalectomy, there is a marked increase in ACTH that demonstrates the relevance of a feedback mode during the SHRP (7). Thus, the pituitary component of the HPA axis does not appear to be down-regulated during the SHRP. Bacterial endotoxins such as lipopolysaccharides are capable of eliciting a robust ACTH response in the neonate that resembles that of the adult. However, corticosterone is still markedly suppressed (8). Furthermore, neonates administered exogenous ACTH by injection at ages both within and outside of the SHRP, exhibit a significant and persistent elevation in plasma corticosterone in only those ages outside of the SHRP (9). Taken together, these data support the argument that, during the SHRP, the reduction in corticosterone secretion following stress may be due to an inability or reduced capacity of adrenal cortex to respond to ACTH. Therefore, during the SHRP, the evidence suggests that the adrenal gland of the rat becomes less sensitive or insensitive to ACTH.

Maternal behaviors play an important role in maintaining the quiescence of the neonatal HPA axis during the SHRP. Rat pups that have been separated from their dams for 24 h (= maternal deprivation) have elevated basal levels of corticosterone and stress-induced elevations of ACTH and corticosterone compared with their nondeprived (NDEP) counterparts (10, 11, 12). However, different aspects of maternal behavior appear to act on different components of the HPA axis. Whereas feeding is required to suppress the corticosterone response to stress, suppression of stress-induced ACTH can be achieved by stroking the anogenital area of the pup (13, 14). Thus, the maintenance of the SHRP appears to be dependent upon specific behaviors that occur during normal mother-infant interactions.

The high levels of plasma corticosterone observed in maternally deprived (DEP) rats during the SHRP may be a result of two factors. That is, either a decrease in corticosterone clearance from the circulation and/or an increase in adrenal corticosterone secretion (15). In rat pups, glucocorticoids that are secreted from the adrenal gland exist in the circulation mainly as free or unbound corticosterone. The neonate appears to have little or no corticosterone-binding globulin (CBG) during the SHRP (16). Maternal deprivation does not affect CBG levels in the neonate during the SHRP (16), suggesting that the free fraction of corticosterone that is available to bind to glucocorticoid receptors may be de facto higher in the neonate compared with the adult (Okimoto, D. K., and S. Levine, unpublished observations). Walker (17) suggests a mechanism that could account for the change in glucocorticoid output. She observed that the enhanced sensitivity of the adrenal cortex to ACTH in DEP 10-d-old pups was abolished by chemical sympathectomy, suggesting a role for the adrenal medulla in regulating steroidogenesis from the adrenal cortex (17).

In this study, we use the maternal deprivation model to test the hypothesis that elevated basal and stress-induced levels of corticosterone in DEP pups during the SHRP result from changes in adrenocortical sensitivity to ACTH. Consequently, we employed in situ hybridization to examine c-fos gene expression in the adrenal cortex of developing NDEP and DEP rats during the SHRP. This immediate-early gene and its product (Fos protein) have been shown in several studies to be up regulated in response to ACTH administration and stress (18, 19, 20, 21). Furthermore, we address the role of the adrenal medulla in modulating changes in adrenal sensitivity to ACTH induced by maternal deprivation by examining adrenomedullary c-fos and tyrosine hydroxylase (TH) gene expression in rats during the SHRP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The subjects of this study were pups bred in the laboratory from Sprague Dawley females and Long-Evans males (Harlan Sprague Dawley, Inc., Indianapolis, IN). Hybrid offspring were used because they have a lower mortality rate than purebreds (6). The date of birth was determined by checking pregnant females daily between 0900 and 0930 h for births. If a litter was found, the day of birth was defined as d 0. On the following day (d 1), litters were culled to 8–10 pups with equal numbers of both sexes in each litter. Each dam and her offspring were transferred to a clean cage where they remained undisturbed until the time of testing or maternal deprivation. Litters were randomly assigned to a condition (NDEP or DEP) and tested at PND 6, 9, or 12. A total of 60 litters (n = 24 for Exp 1, n = 36 for Exp 2) were used in this study. Food (Agway Rat 2000) and tap water were provided ad libitum. Animals were maintained on a 12-h light and 12-h dark cycle (lights on at 0700 h EST) and 25 C. All protocols in this study were approved by the Lab Animal Care and Use Committee at the University of Delaware, and were conducted in strict accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Maternal deprivation procedure
Twenty-four hours before testing, dams were separated from their litters, and the home cages containing these litters were placed in another room for 24 h on a heating pad set at nest temperature (31–33 C). Room temperature and lighting conditions in the maternal deprivation room were identical to those of the maternity room. No food or water was available to the pups during maternal deprivation. NDEP animals remained with the dams in the maternity room and these dams were removed immediately before testing.

Exp 1: ACTH-dose response in dexamethasone-blocked pups
On testing days, the timing of the experiment was conducted as follows: at 0800 h, both NDEP and DEP pups were given an injection of dexamethasone (DEX; Cortrasyn, Organon, West Orange, NJ) at a dose of 100 µg/kg BW. DEX was dissolved in a 0.9% saline solution and injected ip (0.1 ml/10 g BW). NDEP pups were reunited with the dams in their home cages following the DEX injection, whereas the DEP pups remained separated from the dams. Four hours (at 1200 h) after the DEX administration, a male and a female pup were taken from each litter, weighed, and decapitated (basal group). Trunk blood was collected in EDTA-coated tubes and kept on ice. Another male and female pup from the same litter were weighed and then injected ip with a 0.9% saline solution (0.1 ml/10 g BW) immediately after the 24-h deprivation period, and killed 60 min following the injection (vehicle-injected group). Trunk blood was also collected from this group as previously described. The remaining six pups in the litter were injected ip with ACTH {1–39} (Sigma; St. Louis, MO; ACTH-injected group). ACTH was dissolved in a 0.9% saline solution and administered to each pair (one male and one female) at one of the three following doses: 0.01, 0.1, and 1.0 IU/100 g BW in a volume of 1% of the BW (0.1 ml/10 g BW). Sixty minutes after the ACTH injection, pups were decapitated and the trunk blood taken for corticosterone analysis. After the experiment, blood samples were centrifuged for 20 min at 2000 rpm and 5 C. Plasma was stored at -20 C until RIA.

Exp 2: plasma corticosterone and adrenocortical c-fos mRNA determination in saline-injected rat pups
At the time of testing, a male and female pup from each litter were weighed, killed by decapitation, and their trunk blood collected in EDTA-coated microcentrifuge tubes for a basal measurement of corticosterone. Adrenal glands were then quickly removed from each animal, frozen in 2-methylbutane (-45 C), and stored on dry ice. The remaining six pups in each litter were weighed, marked for identification, and injected ip with a 0.9% saline solution (0.1 ml/10 g BW). Injected pups were returned to their home cages and placed in an adjacent room until they were killed. A pair (male and female) of pups from each litter was killed at 30, 60, and 120 min after the saline injection, with trunk blood and adrenal glands collected as previously described. Blood samples were centrifuged for 20 min at 2000 rpm and 5 C. Plasma was collected, transferred to a clean microcentrifuge tube, and stored at -20 C until assayed. Adrenal glands were stored at -70 C until processing for in situ hybridization.

Riboprobe in situ hybridization
Frozen adrenal glands were sectioned at 15 µm in a cryostat and then mounted onto slides (Superfrost, VWR Scientific, West Chester, PA). 35S-Labeled cRNA probes were used to hybridize with complementary c-fos or TH mRNA in adrenal sections. The hybridization procedures used in this study were performed as previously described (6) with slight modifications. Tissue sections (n = 4–6 sections/animal) were fixed in 4% paraformaldehyde and then exposed to 0.25% acetic anhydride in 0.1 M triethanolamine. The sections were then washed in increasing concentrations of ethanol before being rinsed in chloroform. Fifty microliters of hybridization mix containing 1.2–1.5 x 106 cpm 35S-labeled riboprobes [{alpha}-35S-UTP (<1,000 Ci/mmol); NEN Life Science Products, Boston, MA] were then placed on each slide, and a cover slip was added. The c-fos probe was an 860-bp fragment containing the 3' untranslated region of cDNA (22). The TH probe contained the full-length coding regions of rat TH complementary DNA (23). Slides were incubated overnight at 55 C and then washed the next day with 4x SSC, before being treated with RNAase A (20 mg/liter). Sections were then washed in a series of decreasing SSC concentrations at room temperature before being incubated in a 0.1x SSC for 1 h at 65 C. Following the incubation, sections were exposed to a graded series of alcohol washes to remove any remaining water in the tissues. In the final stage, sections were exposed for 14 d for c-fos mRNA and for 3 d for TH mRNA to Kodak Biomax MR film (Eastman Kodak Co., Rochester, NY).

Image analysis
Autoradiographs were digitized and processed using NIH Image (version 1.59, NIH) on a Power Macintosh 7600/132 computer outfitted with a MTI-CCD 72S camera. 14C-Methylmethacrylate standards were used to quantify the optical density values of mRNA levels on the exposed films. Analysis of c-fos mRNA was performed on the adrenal cortex and medulla regions, whereas only the adrenal medulla was analyzed for TH mRNA (mean of 4–6 sections taken for each animal).

Corticosterone RIA
Plasma corticosterone was determined by RIA using a commercially available kit (ICN Pharmaceuticals, Inc., Costa Mesa, CA). The minimal detectable dose for the corticosterone RIA was 0.125 µg/dl.

Data analysis
All data are expressed as means ± SEM. Data were analyzed by ANOVA (Statistical Institute Software package, version 6.11) at a significance of P < 0.05 for each age. Because no significant sex differences were found in plasma corticosterone, c-fos mRNA, and TH mRNA levels, data were collapsed across sexes for each age. When appropriate, post hoc comparisons were conducted using the least squares means test. For the TH mRNA data, the area beneath a curve transform was calculated using the trapezoidal rule (Sigma Plot, version 2.0, Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: plasma corticosterone levels in dexamethasone-blocked pups given exogenous ACTH
In PND 6 rats, the ANOVA showed significant main effects for condition and treatment, and an interaction between condition and treatment (F 9, 98 = 10.31, P = 0.001). Post hoc analysis indicated that DEP pups significantly elevated corticosterone at a dose of 0.1 IU of ACTH (P < 0.001). NDEP pups showed no elevations at this dose. At a dose of 1.0 IU ACTH, although both DEP and NDEP rats elicited a corticosterone response, the magnitude of the response was significantly higher in the DEP pups (P < 0.001, Fig. 1Go).



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  		Figure 1. Plasma corticosterone levels (µg/dl) in PND 6-, 9-, and 12-d-old rats under basal condition (basal) and 60 min after an ACTH injection (0.1 ml/10 g BW) at doses of 0.01, 0.1, and 1.0 IU/100 g BW. Vehicle animals were given saline injections (0.1 ml/10 g BW) at the equivalent time. The open bars represent the NDEP pups, which served as controls. The dark bars represent animals that were DEP 24 h before testing (n = 10/treatment). All animals had been given a dexamethasone injection (100 µg/kg BW) 4 h before being tested to block endogenous ACTH production. **, P < 0.01; ***, P < 0.001, compared with basal corticosterone levels. ##, P < 0.01; ###, P < 0.001, compared with the corresponding NDEP group.

 
The ANOVA also revealed significant main effects of condition and treatment in PND 9 rats (F 9, 113 = 35.14, P = 0.001). In contrast to the PND 6 animals, PND 9 pups did not respond to the lower doses of ACTH. However, at the highest dose of ACTH, the magnitude of the corticosterone response in the DEP pups was significantly higher when compared with the NDEP group (P < 0.001, Fig. 1Go).

In PND 12 rats, there were significant main effects of condition and treatment, and an interaction between condition and treatment for the ANOVA (F 9, 118 = 33.52, P = 0.0001). DEP pups at this age responded at a lower dose of ACTH (P < 0 0.01). In addition, the magnitude of the response at the highest dose of ACTH was much greater for DEP group (P < 0. 001, Fig. 1Go).

Exp 2: plasma corticosterone, adrenal c-fos mRNA, and TH mRNA levels in saline-injected rat pups
Plasma corticosterone levels
The ANOVA revealed significant main effects of condition and time for PND 6 rats (F 23, 286 = 44.14, P = 0.0001). DEP pups exhibited a marked increase in basal levels of corticosterone (P < 0.05). In addition, the DEP group demonstrated further elevations in corticosterone following the saline injection (P < 0.05 at all time points). NDEP pups showed a minimal response to stress at 120 min. At the other time points, NDEP pups did not respond to stress and differed significantly from the DEP pups (P < 0.05, Fig. 2Go).



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  		Figure 2. Plasma corticosterone levels (µg/dl) in PND 6-, 9-, and 12-d-old rats under basal condition (Time 0) and 30, 60, and 120 min after a saline injection (0.1 ml/10 g BW). The open bars represent the NDEP pups, which served as controls. The dark bars represent animals that were DEP 24 h before testing (n = 10–12/treatment). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with basal levels. #, P < 0.05; ##, P < 0.01; ###, P < 0.001, compared with the corresponding NDEP group.

 
The corticosterone response to the saline injection in d 9 pups was similar to that observed at the younger age. Consequently, the ANOVA demonstrated significant main effects of condition and time at PND 9 (F 23, 286 = 44.14, P = 0.0001). Basal levels of corticosterone were elevated in DEP pups (P < 0.05). In addition, there were further elevations of corticosterone following stress in these animals (P < 0.05). NDEP pups failed to respond to the injection. At no time were corticosterone levels elevated above basal levels. Consequently, corticosterone levels were significantly lower in the NDEP pups at all time points when compared with their DEP counterparts (P < 0.05, Fig. 2Go).

The ANOVA for the PND 12 rats showed that there were significant main effects of condition and time (F 23, 286 = 44.14, P = 0.0001). Consistent with the data presented for the two younger ages, basal levels of corticosterone were considerably higher in the DEP pups (P < 0.01). In addition, the corticosterone response to stress was greatly amplified at this age (P < 0.05). In contrast, NDEP pups had characteristically low basal levels and showed only a minimal elevation of corticosterone at 120 min post injection (P < 0.05). When compared with the DEP pups, corticosterone levels in the NDEP pups were significantly lower at all time points examined (P < 0.05, Fig. 2Go).

Adrenal cortex c-fos mRNA levels
At PND 6, the ANOVA revealed significant main effects of condition and time, and an interaction between condition and time (F 7, 95 = 10.57, P < 0.0001). There were no differences in the resting levels of c-fos mRNA in the adrenal cortex between the DEP and NDEP rats. However, at 60 and 120 min post injection, there was a striking increase in c-fos gene expression in the adrenal cortex of DEP pups (P < 0.001). At no time was c-fos mRNA elevated in the adrenal cortex of the NDEP pups (Fig. 3Go; also see Fig. 5Go).



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  		Figure 3. c-fos mRNA levels (optical density) in the adrenal cortex of PND 6-, 9-, and 12-d-old rats under basal condition (Time 0), 30, 60, and 120 min after a saline injection (0.1 ml/10 g BW). The open bars represent the NDEP pups, which served as controls. The dark bars represent animals that were DEP 24 h before testing (n = 10–12/treatment). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with basal levels. #, P < 0.05; ##, P < 0.01; ###, P < 0.001, compared with the corresponding NDEP group.

 


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  		Figure 5. Representative autoradiographs depicting the expression of c-fos mRNA in the adrenal gland of PND 6-, 9-, and 12-d-old rats under basal condition (Time 0) and 60 min after a saline injection (0.1 ml/10 g BW). The NDEP pups served as controls. DEP pups were separated from their mothers 24 h before testing (n = 10–12/treatment).

 
The ANOVA showed significant effects of condition and time, and an interaction between condition and time at PND 9 (F 7, 91 = 10.5, P < 0.01). c-fos mRNA levels increased in a manner similar to that observed in the corticosterone response for PND 9 pups. Although there were no differences in resting levels of c-fos mRNA between the DEP and NDEP rats, the DEP pups showed a marked increase in c-fos gene expression in response to the saline injection (P < 0.01). In contrast, the NDEP pups showed no changes in adrenal cortical c-fos mRNA levels (Fig. 3Go; also see Fig. 5Go).

There were also significant main effects of condition and time, and an interaction between condition and time in the ANOVA at PND 12 (F 7, 95 = 13, P < 0.0001). The DEP pups showed an elevated expression of c-fos mRNA at all time points following the injection (P < 0.05). However, the NDEP pups did not exhibit any discernable changes in c-fos gene expression (Fig. 3Go; also see Fig. 5Go).

Adrenal medulla c-fos mRNA levels
The ANOVA demonstrated significant condition and time effects, and an interaction between condition and time at PND 6 (F 7, 95 = 9.72; P < 0.0001). c-fos gene expression in the adrenal medulla of the DEP pups was significantly increased at 30, 60, and 120 min following injection (P < 0.001), with a peak observed at 60 min (P < 0.001). In contrast, c-fos mRNA levels in the NDEP pups significantly increased at 30 min post injection (P < 0.01). There was a marked reduction in c-fos gene expression compared with the 30 min time point at 60 and 120 min following the injection (P < 0.05 and P < 0.01, respectively). Resting levels of c-fos mRNA did not differ between the NDEP and DEP rats, but significantly higher amounts of transcript were seen in the DEP pups at the last two time points (P < 0.001, Figs. 4Go and 5Go).



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  		Figure 4. c-fos mRNA levels (OD) in the adrenal medulla of postnatal day (PND) 6-, 9-, and 12-d-old rats under basal condition (Time 0) and 30, 60, and 120 min after a saline injection (0.1 ml/10 g BW). The open bars represent the NDEP pups, which served as controls. The dark bars represent animals that had been DEP 24 h before testing (n = 10–12/treatment). **, P < 0.01; ***, P < 0.001, compared with basal levels. #, P < 0.05; ##, P < 0.01; ###, P < 0.001, compared with the corresponding NDEP group. a, P < 0.05; b, P < 0.01, compared with the NDEP group at 30 min post injection.

 
At PND 9, the ANOVA demonstrated a significant main effect of time and an interaction between condition and time (F 7, 91 = 6.3; P < 0.0001). There were no discernable differences in c-fos gene expression in the adrenal medulla of the DEP pups at this age, although c-fos mRNA levels tended to increase 30 min after the injection, followed by a decrease and subsequent increase at the last time point. For some unknown reason(s), the resting level of c-fos mRNA in the adrenal medulla of the NDEP pups was initially very high, which resulted in a significantly lower basal level of c-fos gene expression in the DEP pups (P < 0.001). c-fos mRNA levels in the NDEP pups decreased thereafter with significantly lower expression observed at 60 and 120 min post injection (P < 0.001). In contrast, c-fos mRNA levels were significantly higher in the DEP pups 120 min after the injection (P < 0.001, Figs. 4Go and 5Go).

There were significant main effects for condition and time in the ANOVA at PND 12 (F 7, 95 = 4.97; P < 0.0001). The pattern of c-fos gene expression in the DEP pups at this age was similar to that observed at PND 9, but with a significant increase observed at 30 min following injection (P < 0.01). In the NDEP pups, there were no observable differences in c-fos gene expression compared with the resting levels, although c-fos mRNA levels at 60 and 120 min after injection were significantly reduced compared with the 30-min time point (P < 0.05, Figs. 4Go and 5Go).

Adrenal medulla TH mRNA levels
At PND 6, the ANOVA indicated significant main effects of condition and time (F 7, 92 = 4.94; P < 0.001). The DEP pups showed a significant increase in TH gene expression in the adrenal medulla at 30 and 60 min post injection (P < 0.01), with the highest amount observed at the earlier time point. Interestingly, the NDEP pups also exhibited significant increases in TH mRNA levels, but at all time points following the injection (P < 0.05). There were no differences in basal levels of TH mRNA between the NDEP and DEP rats, although the DEP animals exhibited significantly higher levels of TH gene expression 30 min after the injection (P < 0.05, Figs. 6Go and 7Go). Overall TH gene expression when represented as an area beneath the curve was significantly elevated in the DEP pups (P < 0.05, Fig. 8Go).



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  		Figure 6. TH mRNA levels (OD) in the adrenal medulla of PND 6-, 9-, and 12-d-old rats under basal condition (Time 0) and 30, 60, and 120 min after a saline injection (0.1 ml/10 g BW). The open bars represent the NDEP pups, which served as controls. The dark bars represent animals that were DEP 24 h before testing (n = 10–12/treatment). *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with basal levels. #, P < 0.05, compared with the corresponding NDEP group.

 


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  		Figure 7. Representative autoradiographs depicting the expression of TH mRNA in the adrenal medulla of PND 6-, 9-, and 12-d-old rats under basal condition (Time 0) and 30 min after a saline injection (0.1 ml/10 g BW). The NDEP pups served as controls. DEP pups were separated from their mothers 24 h before testing (n = 10–12/treatment).

 


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  		Figure 8. Area beneath the curve for TH mRNA expression in the adrenal medulla of PND 6-, 9-, and 12-d-old rats. Data were collapsed across time for each age. The open bars represent the NDEP pups, which served as controls. The dark bars represent animals that had been DEP 24 h before testing (n = 10–12/treatment). *, P < 0.05.

 
There were also significant main effects of condition and time in the ANOVA at PND 9 (F 7, 89 = 3.32; P < 0.01). TH mRNA levels in the DEP rats at this age were significantly elevated at 30, 60, and 120 min after the injection (P < 0.05). In the NDEP pups, TH mRNA levels also increased, but only at the last two time points (P < 0.05). As with the previous age, there were no observed differences in the resting levels of TH mRNA between the NDEP and DEP rats, but the DEP pups also demonstrated significantly higher levels of TH mRNA at 30 min post injection (P < 0.05, Figs. 6Go and 7Go). Overall TH gene expression as a function of the area beneath the curve was also significantly increased in the DEP pups at this age (P < 0.01, Fig. 8Go).

As in the previous two ages, the ANOVA revealed significant main effects of condition and time at PND 12 (F 7, 93 = 4.50; P < 0.001). TH mRNA levels were significantly increased in the DEP pups at all time points tested following the injection (P < 0.05), with the highest levels observed at the last two time points (P < 0.001). The NDEP pups also showed a significant increase in TH mRNA levels at all time points (P < 0.05), with the last time point being the highest level seen (P < 0.01). TH mRNA levels at any of the observed time points (Figs. 6Go and 7Go), as well as overall TH gene expression as a function of the area beneath the curve did not differ between the NDEP and DEP rats at this age (Fig. 8Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During postnatal development, the HPA axis has been shown to be hyporesponsive. Although it was originally believed that all the components of the HPA axis were down-regulated, in recent years it has been demonstrated that several components of the HPA axis can respond in an adult manner, such as the brain (6) and the pituitary gland (5). In the hypothalamus, CRH gene expression is in fact hyperresponsive during the so-called SHRP (6). There is general agreement, however, that during the SHRP, glucocorticoid secretion from the adrenal gland of rats is down regulated. Basal levels of corticosterone are maintained at low levels and the response to even severe challenges is blunted (2, 24). Specific aspects of the mother-infant interaction modulate the blunted adrenal response during the SHRP. Following 24 h of maternal deprivation, basal levels of corticosterone are markedly elevated and in response to novelty (9), restraint (25) and a saline injection (11), ACTH and corticosterone are elevated. Stroking in the form of anogenital stimulation results in the suppression of the ACTH response, reduces c-fos mRNA response in the paraventricular nucleus (PVN) of the hypothalamus, and normalizes basal levels of CRH mRNA in the PVN of the DEP pups (14). In contrast, feeding appears to regulate the responsiveness of the adrenal gland. Pups that were deprived of the mother for 24 h but fed during the period of maternal deprivation show reduced basal levels of corticosterone and are stress hyporesponsive (13, 14). Thus, it appears as though feeding is responsible for rendering the adrenal hyporesponsive to ACTH during the so-called SHRP.

The hypothesis that the rat adrenal is relatively insensitive to ACTH during the SHRP is supported by previous studies. Rosenfeld et al. (16) showed that the DEP pups at PND 4 responded with increased corticosterone to a lower dose of exogenously-administered ACTH compared with the NDEP pups. Further, Suchecki et al. (13) reported that although the ACTH response to stress can be reduced by anogenital stroking in the absence of food, the DEP pups still respond with a robust secretion of corticosterone. Although these data strongly support the notion that feeding inhibits the neonatal adrenal, there is some evidence that suggests another mechanism that could explain the high levels of circulating corticosterone in the DEP pups—that is, a reduced clearance of corticosterone from the circulation in the neonate (15). Maternal deprivation appears to further reduce the clearance of corticosterone that would result in persistently higher circulating levels, and would not necessarily reflect a greater response in the rat adrenal. One of the purposes of the present study was to examine the hypothesis of increased adrenal sensitivity following maternal deprivation by examining cellular activation in the adrenal cortex of the DEP and NDEP pups.

In the first experiment, we examined the adrenocortical response to graded doses of exogenously administered ACTH. Although this experiment was in part a replication of a study by Rosenfeld et al. (16), there were several important differences. Whereas Rosenfeld et al. (16) examined only pups at PND 4, the present study examined three different ages (PND 6, 9, and 12). Second, in the previous study (16), which examined the adrenal response to ACTH, the pups were not prevented from responding to the injection per se. In the present experiment, the adrenocortical response to the injection was prevented by pretreatment with DEX, which lowered the basal levels of corticosterone in both the DEP and NDEP rats, and eliminated the stress-inducing response to the saline injection in the DEP pups. Insofar as DEX presumably blocks endogenous ACTH release, any adrenocortical response could be attributed to the steroidogenic action of ACTH. In general, DEP pups responded to lower doses of ACTH and at the highest dose, the magnitude of the corticosterone response in the DEP pups was significantly higher. Although differences in clearance rates could have contributed to these differences, these data are consistent with the hypothesis that adrenal sensitivity is increased as a consequence of maternal deprivation.

That maternal deprivation increased adrenal sensitivity is clearly supported by the results obtained by examining c-fos gene expression in the adrenal cortex. The corticosterone response to a saline injection in animals in which adrenal c-fos gene expression was examined replicated the results of many previous experiments (16, 26). At every age tested, basal levels of corticosterone were elevated in the DEP animals and there was a significant elevation of corticosterone following stress. In the NDEP pups, only minimal elevations of corticosterone were seen only at 120 min following stress on PND 6 and 12. The expression of c-fos also unambiguously discriminated between the DEP and NDEP pups. The effect was almost absolute. DEP pups showed striking increases in c-fos mRNA in the adrenal cortex. At no time was c-fos gene expression altered following stress in the NDEP pups. There is a remarkable concordance between the corticosterone results and the expression of c-fos in response to a saline injection in the neonate.

In the adult rat, it has been demonstrated that c-fos mRNA and Fos protein are reliable indices of adrenocortical activation (18, 19, 20, 21, 27). Fos activation in the adrenal cortex of the adult rat has been shown following exposure to ACTH or restraint (20, 21). Although some portion of the elevated circulating corticosterone in the DEP pups may be attributable to reduced hormone clearance (15), the data presented here demonstrate emphatically that the neonatal adrenal is insensitive during the SHRP and that removal of the inhibitory influence of the mother renders the adrenal more sensitive to ACTH.

There are at least three possible mechanisms that could account for the decreased adrenal sensitivity during development. Zilz et al. (28) have proposed a role for the peripheral benzodiazepine receptors (PBR) in regulating adrenal sensitivity. This intracellular molecule is believed to assist in the transport of cholesterol from intracellular stores to inner mitochondrial membranes for steroidogenesis. Cholesterol transport is initiated in adrenocortical cells in response to cAMP formation that results from activation of the ACTH receptor by its ligand. In this in situ hybridization study, PBR binding capacity and immunoreactive PBR content directly paralleled that of ACTH-induced steroidogenesis (28). During the period of development when the adrenal is insensitive to ACTH, PBR are considerably reduced compared with the adult. It is therefore possible that PBR may be increased during maternal deprivation. Insofar as it has been demonstrated that fasting is responsible for the increased sensitivity of the adrenal gland in the DEP pups, this would be a nutritionally mediated process.

Recently, there have been several reports (29, 30, 31, 32, 33, 34) that suggest a role for leptin in the regulation of the HPA axis. Leptin, a product of the ob gene in adipocytes, appears to affect the HPA axis at multiple levels. In the neonate, increased leptin levels have been shown to reduce pituitary ACTH secretion (32) and stress-induced expression of CRH mRNA in the PVN (33, 34). Germane to this discussion is the fact that leptin appears to directly inhibit secretion of glucocorticoids in vitro from primary cell lines derived from dispersed human and rat adrenal glands through binding to leptin receptors on these adrenocortical cells (30). In addition, neonates are exposed to high levels of leptin postnatally (35, 36). This surge of leptin during this period of development corresponds with the down-regulation of adrenal sensitivity in the rat. Presumably one source of the increased leptin would be derived from the maternal milk (35). Thus, the 24-h fast that accompanies maternal deprivation would be expected to result in a marked decrease in circulating leptin, which would remove the inhibitory influence of leptin on the neonatal adrenal gland. Fasting might also induce down-regulation of the leptin receptor in the DEP pups.

As mentioned earlier, Walker (17) provided evidence of adrenal sympathetic involvement in the regulation of glucocorticoid synthesis. PND 10 pups that were chemically sympathectomized by treatment with guanethidine show a reduction in resting and stress-induced levels of corticosterone, but not of ACTH, compared with the saline-injected pups. Furthermore, guanethidine treatment also abolished the increase in adrenal sensitivity to ACTH induced by maternal deprivation in these animals. Thus, the data by Walker suggests that removal of sympathetic inputs to the adrenal cortex may affect corticosteroid output by altering adrenal sensitivity to ACTH. The results of this study indicate that the adrenal medulla is also capable of responding to stress during neonatal development. In contrast to the adrenal cortex, temporal profiles of c-fos gene expression in the adrenal medulla of rat pups given a saline injection did not show a strong correlation with the corticosterone data, except for the PND 6 DEP pups. Both the NDEP and DEP rats appear to show an initial c-fos response that is transient in the NDEP pups but remains elevated in the DEP pups. This differentiation in the temporal expression of c-fos mRNA in the adrenal medulla of the DEP pups may indicate prolonged sympathetic activity in these animals. It is unclear why basal c-fos mRNA levels were exceedingly high in the PND 9 NDEP pups.

Chromaffin cells in the adrenal medulla of adult rats are know to express c-fos mRNA and Fos protein in response to a variety of stressors that include immobilization, capsaicin, deoxy-D-glucose, nicotine, angiotensin, and insulin-induced hypoglycemia (19, 37, 38, 39, 40). In addition, there is a body of molecular evidence that supports the notion that induction of the TH gene in chromaffin cells is in part regulated by the proteins produced by c-fos in combination with other immediate early genes, e.g. c-jun (41, 42, 43). Thus, immediate early genes such as c-fos participate in the transcriptional regulation of the TH gene, and hence, catecholamine biosynthesis during the stress response.

The gene that encodes for TH, the initial and rate-limiting enzyme of catecholamine synthesis (44), is expressed not only in the DEP pups at all ages tested, but in the NDEP pups as well. However, TH gene expression in the DEP pups appears to occur more rapidly and is of greater magnitude in two of the three ages tested (e.g. PND 6 and 9). Taken together, these data provide additional support for a role of the adrenal medulla in mediating changes in adrenocortical sensitivity to ACTH following maternal deprivation. There is very little or no information regarding the effects of maternal deprivation on the adrenomedullary-catecholamine response during neonatal development. The expression of the TH gene increases severalfold following birth in the rat (45), coincident with a surge in catecholamine release at birth (46), which is believed to aid the newborn in the transition at birth. These events occur in the absence of a functional sympathetic innervation of the chromaffin cells in the rat adrenal at birth, which is not fully established until PND 10 (47). In addition, a number of different stimuli that include hypoxia, reserpine, guanethidine, endogenous opiates, and hypoglycemia have been shown to elicit catecholamine release from the immature (noninnervated) adrenal, suggesting that the regulation of catecholamine biosynthesis occurs via a nonneural mechanism in the neonate (47, 48, 49, 50, 51). Holgert et al. (52) reported levels of TH mRNA in rat chromaffin cells that stayed relatively constant at PND 6 and 8 (96.3% and 97.1% of adult levels, respectively), but were still very low compared with TH mRNA levels seen at birth. At PND 10 and 16, the levels of TH message in the chromaffin cells of these pups significantly dropped to 77.5% and 81.7% of adult TH mRNA levels, respectively (52). These results do not appear to support those of Lau et al. (53) who observed a direct increase in the activity of the TH enzyme with age. In neonates (PND 2–29), the increase in TH enzyme activity occurred in conjunction with the age-dependent increase in basal catecholamine levels over the same developmental period.

In conclusion, these experiments have demonstrated that during the period of development referred to as the SHRP, the adrenal cortex has a reduced sensitivity to ACTH. This process appears to be largely regulated by maternal factors (13, 14). For the adrenal gland, the critical element of the mother-infant interaction is related to feeding (13, 14). The neonatal adrenal does have the capacity to synthesize and secrete glucocorticoids in response to exogenous and endogenous ACTH as evidenced by an increased sensitivity to ACTH and a marked increase in intracellular activation in the adrenal cortex following maternal deprivation. Thus, the adrenal during the SHRP is actively inhibited by some as yet unspecified mechanism(s) related to milk intake. In recent years, evidence has been accumulating that has challenged the concept of an SHRP that assumes an overall lack of or reduction in the response to stress in most components of the HPA axis. What is clear is that regardless of the stimulus used to provoke the HPA axis, the adrenal response is invariably reduced or nonexistent. Thus, there have been several attempts (5, 6) to more accurately describe this period of development as an adrenal hyporesponsive period. Exposure to high levels of glucocorticoids has been shown to affect the development of the brain (1, 2, 3, 4). It should be noted that during this period of postnatal development CBG is extremely low (16). Thus, even small elevations of corticosterone would exist largely as free corticosterone and therefore be more biologically active. Active suppression of steroidogenesis by whatever mechanism(s) would represent a fail-safe mechanism to prevent exposure of the developing central nervous system to excessive levels of adrenal steroids. Suppression of steroidogenesis can be a function of the failure of the pituitary to release ACTH. The results of the present study demonstrate that the suppression of steroidogenesis can occur directly in the adrenal gland of the rat.


   Acknowledgments
 
We are grateful to Dr. Mark A. Smith for his gracious assistance with the c-fos and TH in situ hybridizations and to Dr. Jim Herman for his helpful suggestion, which inspired this study.


   Footnotes
 
This work was supported by Grant MH-45006 from the National Institutes of Mental Health to Seymour Levine.

Abbreviations: CBG, Corticosterone-binding globulin; DEP, maternally deprived; HPA, hypothalamic-pituitary-adrenal; NDEP, nondeprived; PBR, peripheral benzodiazepine receptors; PND, postnatal day; PVN, paraventricular nucleus; SHRP, stress hyporesponsive period; TH, tyrosine hydroxylase.

Received October 26, 2001.

Accepted for publication February 1, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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