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Department of Biology, University of Delaware (G.W.D., S.L.), Newark, Delaware 19716-2577; and DuPont Pharmaceutical Co., Experimental Station (G.W.D., M.A.S.), Wilmington, Delaware 19880
Address all correspondence and requests for reprints to: Seymour Levine, Ph.D., Department of Psychology, University of Delaware, Newark, Delaware 19716-2577. E-mail: glevine{at}udel.edu
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Introduction |
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The central component of the HPA axis may be stress responsive during the SHRP; mild stimuli enhance c-fos and nerve growth factor I-B expression in the paraventricular nucleus (PVN) (8, 9). However, although stress seems to amplify neural activity in the neonatal PVN, stimulus- induced CRH gene expression has rarely been reported. During the first week of life, adrenalectomy (10), cold, (11), glucocorticoid receptor antagonism and surgical stress (12) reportedly did not increase CRH gene transcription. We recently reported that endotoxin treatment during the SHRP results in a hormonal response, without an increase in PVN-CRH messenger RNA (mRNA) levels. To the contrary, we and others have observed a decrease in CRH mRNA after an endotoxin challenge (13) or repeated exposure to maximally tolerated cold (14).
These data have led to the idea that during the SHRP the peripheral stress response may not be reflected centrally by enhanced CRH gene expression in the PVN, and that the cellular regulatory mechanisms for CRH biosynthesis may be deficient early in development. To test this hypothesis we examined the stimulus-induced initiation of CRH gene transcription (hnRNA and mRNA) in the developing animal. The maternal deprivation paradigm was used to render the neonates endocrine responsive to an acute mild stimuli. We compared the effect of mild stress on CRH gene transcription in stress-responsive (DEP) and nonresponsive (NDEP) neonates. Given that deprived animals (DEP) show a significant ACTH response to a mild challenge, we hypothesized that a rapid increase in the primary CRH transcript would readily be detected in DEP animals. Previous work has demonstrated that by using intronic probe technology, stress- induced CRH hnRNA has been detected when no change in mRNA levels was observed (15). Examining CRH hnRNA should provide a more direct view of rapid stimulus-induced transcriptional activation in the neonate. A single injection of isotonic saline was used as the stressor, and three ages were chosen for this examination. Six-day-old neonates were included to represent an age near the onset of the SHRP. On pnd 6 the ACTH response to a mild challenge is barely detectable in NDEP pups and is minimal in DEP pups. In 12-day-old animals, a mild challenge evokes a marked consistent ACTH response in DEP pups compared with their NDEP counterparts (5, 7). Eighteen-day-old animals were examined to represent a developmental period outside of the SHRP. By examining animals on pnd 18, we can compare the central stress response of more mature preweanlings to the response within the SHRP.
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Materials and Methods |
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Deprivation procedure and stress
Male and female pups on pnd 6, 12, and 18 were examined.
Twenty-four hours before testing (days 5, 11, and 17) the dam was
removed from the home cage. The entire litter remained in the home cage
and was placed on a heating pad (30–33 C) in an adjacent room that had
the same temperature and light conditions as the main colony room. Food
and water were unavailable during the deprivation period. The
nondeprived litters remained undisturbed with their mothers until the
time of testing. At the time of testing two pups (male and female) from
each litter were killed immediately; they were the noninjected
controls. The remainder of the pups received a single injection of
isotonic sterile saline (0.9%; volume, 0.1 ml/10 g BW, ip; 27-gauge
needle), which served as the mild stressor. Sodium chloride solution
was nonpyrogenic and endotoxin free (Sigma, St. Louis,
MO). After the saline injection, pups (six pups remaining) were not
returned to the mother; each litter was placed on a heating pad until
decapitation. Pups (one male and one female) were then decapitated at
15, 30, and 240 min after the saline injection.
Sampling and hormone assays
At the designated time after the saline injection, trunk blood
was collected into precooled EDTA-coated microfuge tubes. Blood samples
were centrifuged for 20 min at 2000 rpm at 2 C. Plasma was stored at
-20 C until RIA was performed. Plasma ACTH (INCSTAR Corp., Stillwater, MN) and CORT (ICN Biomedicals, Inc., Cleveland, OH) were measured with commercially available
kits. The sensitivities of the ACTH and CORT were 15 pg/ml and 0.125
µg/dl, respectively. At the designated time after the saline
injection (15, 30, and 240 min), brains were rapidly removed and frozen
by immersion in 2-methylbutane at -40 C, then stored at -70 C until
processing for in situ hybridization.
Riboprobe in situ hybridization
Frozen brains were sectioned (16 µm) in the coronal plane
through the hypothalamic PVN and then mounted on SuperFrost slides (VWR
Scientific, West Chester PA). Using
[35S]UTP-labeled riboprobe, hybridization and
histochemical localization of the CRH hnRNA and mRNA were performed as
described previously (16). Briefly, sections were fixed in 4%
paraformaldehyde and acetylated in 0.25% acetic anhydride in 0.1
M triethanolamine. Subsequently, brain sections were
dehydrated in increasing concentrations of ethanol and delipidated in
chloroform. The CRH hnRNA probe (453 bp; subcloned into a pCRII vector)
was localized entirely with the one intron of the rat CRH gene. The
complementary RNA probe for CRH was transcribed from a 1-kb
complementary DNA insert in pGEM4 containing the full-length coding
region of rat CRH. The antisense complementary RNA probe was
transcribed from a linearized plasmid using SP6 polymerase according to
instructions supplied with the kit (Ambion, Inc., Austin,
TX). Tissue sections (two or three brain sections per slide) were
saturated with 100 µl hybridization buffer [20 mM
Tris-HCl (pH 7.4), 50% formamide, 300 mM NaCl, 1
mM EDTA (pH 8), 1 x Denhardt’s solution, 250 µg/ml
yeast transfer RNA, 250 µg/ml total RNA, 10 mg/ml salmon sperm DNA,
10% dextran sulfate, 100 mM dithiothreitol, 0.1% SDS, and
0.1% sodium thiosulfate] containing 1.5 x
106 cpm 35S-labeled
riboprobe ([
-35S]UTP, <1000 Ci/mmol;
NEN Life Science Products, Boston, MA). Brain sections
were coverslipped and incubated overnight at 54 C. The following day
the sections were rinsed in 4 x SSC (standard saline citrate),
treated with ribonuclease A (20 mg/liter), and washed in increasingly
stringent SSC solutions at room temperature. Finally, sections were
washed in 0.1 x SSC for 1 h at 65 C and dehydrated through
graded concentrations of alcohol. The slides were apposed to
Kodak Biomax MR film (Eastman Kodak Co.,
Rochester, NY) for 5 weeks (CRH hnRNA) or 3–4 days (CRH mRNA).
Data analysis
All data are expressed as the mean ± SEM, and
significance was accepted at P < 0.05. Data were
analyzed by ANOVA for each age. No gender differences were found for
plasma hormones or CRH RNA expression; therefore, data were collapsed
across this variable. Statistical significance between groups was
determined by ANOVA, followed by post-Newman-Keuls comparisons.
Autoradiographs were digitized, and relative levels of hnRNA and mRNA
were determined by computer-assisted optical densitometry (NIH Image,
Bethesda, MD). To correct for film nonlinearity,
[14C]methylmethacrylate standards were used,
and the mean of four to six measurements was taken from each animal.
In situ hybridization assays for each probe were carried out
at the same time for all three ages; therefore, the values between the
ages are comparable.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). Compared with NDEP pups, stress levels
of CORT were greater at 15 and 30 min in DEP on pnd 18. The magnitude
of the CORT response was significantly greater in the DEP animals
compared with the NDEP animals at all ages.
CRH hnRNA and CRH mRNA in the PVN
Time-course analysis of CRH hnRNA revealed a stress-induced
expression in NDEP pups at all three ages. Despite the minimal
endocrine response in NDEP 6- and 12-day-old neonates 15 min after
stress, CRH hnRNA expression was markedly enhanced (Fig. 2). In DEP 12-day-old neonates, hnRNA CRH
levels were significantly increased at 15 min after stress compared
with control levels. On pnd 6 and 12, stress-induced CRH hnRNA levels
at 15 min were significantly greater in NDEP pups compared with the 15
min expression in their DEP counterparts. Thirty minutes after stress
in both NDEP and DEP pups CRH hnRNA expression had returned to or below
control expression. During the SHRP, maternal deprivation tended to
facilitate the endocrine stress to this mild challenge (Fig. 1A). In
contrast to this peripheral response, DEP neonates showed an overall
reduced expression of CRH hnRNA compared with their NDEP counterparts
(Figs. 2 and 3). In animals that had
matured beyond the SHRP, CRH hnRNA expression was enhanced at 15 and 30
min after stress in both NDEP and DEP pups. The overall magnitude of
basal and stress-induced CRH hnRNA expression was also lower in the DEP
pups compared with that in the NDEP pups on pnd 18 (Fig. 2).
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and 5).
In DEP neonates the pattern of CRH mRNA expression was also similar to
that of CRH hnRNA. On pnd 6, CRH mRNA levels in DEP pups were not
enhanced by the challenge; however, on pnd 12, CRH mRNA was elevated at
30 min. During the SHRP, CRH mRNA expression at 15 min was
significantly greater in NDEP neonates than in DEP pups at 15 min
poststress. In pups outside the SHRP, stress resulted in a marked
increase in CRH mRNA throughout the 240-min period in the NDEP pups and
at 30 min in the DEP pups. Similar to the response of the CRH primary
transcript, the magnitude of cytoplasmic RNA was less in the deprived
pups than in the nondeprived pups (Figs. 4 and 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concurrent with the rapid increase in the CRH primary transcript, we observed a significant increase in CRH mRNA in NDEP pups at all three ages. These results were quite surprising for two reasons. First, based on our original hypothesis we did not predict a CRH gene response in NDEP animals, because they showed a minimal ACTH stress response. Second, such rapid stress-induced CRH mRNA expression has not been observed in the adult. Stimuli such as immobilization (18), swimming, restraint, and hypertonic saline (19) all require 2–4 h to measure an appreciable accumulation of CRH mRNA in the PVN of adult rats. Although the temporal relationship between a given stimulus and the transcription of neuroendocrine peptides has not been well established, these data suggest that in adult animals there is a temporal lag between the stimulatory event and detectable changes in the CRH mRNA pool. Based on the time course reported for the adult, we and others have consistently examined CRH mRNA levels at 3–4 h after an acute stimulus in neonatal rats (8, 9, 11, 12, 13). If the dynamics for stress-induced changes in CRH mRNA are indeed more rapid in the developing brain, this temporal difference might explain why stimulus-induced CRH-mRNA is rarely detected in neonates. The present data clearly show that early in ontogeny the regulatory mechanisms to rapidly up-regulate CRH gene transcription are functional. Thus, these data suggest that the developmental regulation of CRH biosynthetic processes are stress responsive during the so-called SHRP.
The findings in the present study are even more remarkable when one considers the intensity of the stimulus used to elicit the CRH gene response. An isotonic saline injection is a mild stressor, because it is minimally invasive, physiologically inactive, and of short duration. Although the time points are not directly comparable to those used in the present study, an isotonic saline injection in adult rats fails to induce CRH mRNA when examined 1–8 h postinjection (19, 20). Further, in response to a vehicle injection (dimethylsulfoxide), CRH hnRNA expression was not enhanced at 15 min poststimulus in the adult rat (17). These data suggest that although a saline/vehicle injection does elicit an ACTH response in adult rats, the intensity is insufficient to induce CRH gene transcription. Thus, considering stimulus intensity, neonates during and outside of the SHRP appear to be more stress responsive at the hypothalamic level of the HPA axis than the adult (19, 20).
The maternal deprivation paradigm was used in the present study as to tool to render the neonate endocrine responsive to an acute mild challenge during the SHRP (7). Because DEP neonates display an augmented ACTH stress response, we hypothesized that compensatory CRH gene transcription would readily be detected in DEP pups and that the magnitude would be greater than their NDEP counterparts. The present results fail to support this hypothesis. Surprisingly, the magnitude of stimulus-induced CRH gene expression was greater in the NDEP pups compared with the DEP pups at all ages. In fact, in DEP 6-day-old pups, CRH transcription was not significantly elevated. One possibility for the dampened CRH gene response in DEP pups may result from the prolonged 24 h of maternal deprivation. Perhaps during the deprivation period the neuroendocrine system was activated that resulted in repeated (or sustained) release of CRH peptide from the median eminence. Consistent with this premise, DEP pups have elevated CORT for at least 16 h of the 24-h deprivation period (6). Consequently, after 24 h of deprivation, the neonate has depressed basal or stress-induced CRH gene expression compared with NDEP animals. Supportive of this interpretation, reduced basal CRH gene expression after 24 h of deprivation has been previously reported (8, 9). Another possibility for the reduced CRH gene response in the DEP pups may result from the inhibitory influence of CORT on CRH mRNA expression. In adults, CORT treatment (2 days) (16) and CORT-treated adrenalectomized animals (21, 22) result in decreased CRH mRNA. However, a recent study demonstrated that elevated peripheral CORT may not be responsible for reduced CRH mRNA after deprivation. Dexamethasone-treated DEP pups had reduced basal CRH mRNA; however, expression was not suppressed in dexamethasone-treated NDEP pups (9). It is unclear why deprivation results in a blunted stimulus-induced CRH gene response. It is conceivable that during the deprivation period various brainstem and limbic inhibitory circuits become active and thereby result in reduced CRH neuronal activity.
Although secretagogue release can only be inferred from changes in CRH transcription, during the SHRP the pituitary stress response may be uncoupled from CRH up-regulation. NDEP neonates (pnd 6 and 12) showed meager stress- induced ACTH and CORT, whereas CRH gene transcription was markedly increased. This dissociation between stress-induced CRH gene activation and pituitary secretion might suggest reduced pituitary responsiveness early in development. However, this seems unlikely, because pituitary CRH receptors are functional during the SHRP. It has also been repeatedly demonstrated that DEP neonates can release ACTH in response to a saline injection (5, 6, 7, 8). Alternatively, the lack of a pituitary response in the presence of enhanced CRH mRNA activity may indicate that an additional ACTH-releasing factor is required to elicit ACTH release during the SHRP. Notably, arginine vasopressin exerts a synergistic action on ACTH release in adult animals (23, 24). Chronic stress paradigms have implicated vasopressin as the essential peptide for sustaining pituitary activation (25, 26, 27). If maternal deprivation represents a chronic stimulus during the SHRP, and additional stress is imposed, the activation of both CRH and vasopressin may be required to evoke a pituitary response. Thus, during the SHRP, a mild challenge might activate the neuroendocrine cascade by stimulating CRH neuronal activity and, depending upon the deprivation condition, may or may not result in a pituitary-adrenal response. In contrast, animals that have matured beyond the SHRP display a pituitary stress response that is concordant with the CRH gene response. Thus, the preweanling pup might represent a transitional stage of development, as indicated by a rapid central response coupled with stress-induced ACTH secretion.
In summary, the current study demonstrates that the CRH component of the HPA axis is indeed stress-responsive during the so-called SHRP and in weanling pups. Despite a minimal endocrine response in NDEP pups there was a robust induction of the CRH primary transcript. Quite remarkably, pups during and outside the SHRP display a rapid increase in CRH mRNA in response to a challenge that reportedly does not elicit a CRH gene response in adult rats. These results suggest that during development the dynamic profile of the stimulus-induced gene response is unique. Thus, during ontogeny the cellular mechanisms controlling CRH gene expression respond rapidly to a mild challenge. In this regard, the hypothalamic level of the HPA axis during development may be stress hyperresponsive rather than hyporesponsive.
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Footnotes |
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Received August 27, 1999.
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