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Biological Psychiatry Branch (M.A.S., S.-Y.K.), National Institute of Mental Health, Bethesda, Maryland 20892; and Department of Psychology (H.J.J.v.O., S.L.), University of Delaware, Newark, Delaware 19716
Address all correspondence and requests for reprints to: Mark A. Smith, M.D., Ph.D., DuPont Merck, Experimental Station, E400/4448, P.O. Box 80400, Wilmington, Delaware 19880. E-mail: smithma{at}a1.lldmpc.umc.dupont.com
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Abstract |
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Introduction |
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Interestingly, the SHRP depends on normal mother-infant interactions. Following 24 h of maternal deprivation (DEP) during the SHRP, the rat pup responds with significant increases in ACTH and CORT when exposed to novelty or saline injections (7). The endocrine response to these stimuli following DEP is different from that observed in the adult in that the neonate has elevated levels of ACTH and CORT, which remain elevated for a prolonged period following exposure to the stressor (8). These data suggest that the dam is exerting an inhibitory influence on the pup’s HPA axis. Thus, the neural mechanisms required to elicit the appropriate endocrine response following stress appear to be present early in development, but various components of the dam’s behavior (feeding and stroking) seem capable of inhibiting or dampening the response (9).
While the attenuated pituitary and adrenal responses to stress during the SHRP have been well described, very little is known about the acute effects of DEP on the neural correlates of the stress response. A few studies have examined CRH messenger RNA (mRNA) levels in the hypothalamic paraventricular nucleus (PVN) of infant rats. Generally, changes in CRH mRNA levels are not readily observed during the first postnatal week but may increase in response to adrenalectomy (10) or surgical stress (11) during the second postnatal week. At 6 and 9 days of age, DEP and cold stress did not increase CRH expression despite the fact that the CORT response to cold was significantly greater in DEP pups compared with nondeprived pups (12). Thus, changes in CRH mRNA may not correlate with the endocrine response in neonatal rats during the SHRP.
The relative lack of CRH responsiveness during the SHRP raises the question whether the brain is being activated by stress at this time. One way to answer this question is to measure mRNA levels of immediate-early genes (IEGs), such as c-fos and nerve growth factor inducible gene (NGFI-B), in the brain following stress (13). The purpose of the present study then was to determine if brain areas such as the PVN, which are activated in adult rats in response to stress, are likewise activated in the neonate. A second objective was to measure the induction of IEGs in maternally deprived neonates that do exhibit a significant endocrine response to mild stress.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At P11 or P19 rat pups were maternally deprived for 24 h. The mother and litters were removed from the home cage. The litters were deposited in a separate cage placed on a heating pad at 30–33 C in the deprivation room adjacent to the main colony room, under the same temperature and lighting conditions. Neither food nor water were available during the deprivation period. Nondeprived controls were left undisturbed with their mothers until the time of testing.
The stressor consisted of administering a single injection of saline (0.9%, ip, volume = 0.1 ml/10 g BW) and placing the neonate individually in the testing chamber on a heating pad for 30 or 120 min. Neonates were killed either 30 min or 2 h after the saline injection. Trunk blood was collected for determination of ACTH and corticosterone. ACTH and corticosterone (CORT) were measured by RIA [INCSTAR (Stillwater, MN) and ICN Biomedicals, Inc. (Costa Mesa, CA), respectively]. Because of a technical error, CORT values for at 30 min after the stressor were lost from the particular group of animals described here. For comparison sake, we list here CORT values from another experiment run in an identical manner. In that other experiment, the ACTH and CORT values at 0 and 120 min were very similar to those reported here and therefore we believe the CORT values at 30 min after the stressor should be representative.
Brains were removed and frozen in isopentane at -30 C. Brains from rats that were not stressed were processed for measurement of c-fos, NGFI-B, and CRH mRNA levels by in situ hybridization. Brains from rats that were killed 30 min after the saline injection were processed for the immediate early genes, c-fos and NGFI-B. CRH mRNA was analyzed in rats that were killed 2 h after the stressor and compared with the unstressed control group.
Riboprobe in situ hybridization
Frozen brain sections (15 µm) through the level of the
hypothalamic PVN (-1.8 mm bregma) were cut on a cryostat and mounted
onto gelatin-coated slides. In situ hybridization using
riboprobes labeled with 35S-UTP was performed as described
(14, 15). Briefly, the sections were fixed in 4% paraformaldehyde,
dehydrated in increasing concentrations of ethanol, and delipidated in
chloroform. Antisense cRNA probes were transcribed from appropriate
linearized plasmids using T7, Sp6, or T3 polymerases according to the
manufacturer’s instructions (Ambion, Austin, TX). The c-fos
probe consisted of an 860 by fragment containing the 3' untranslated
portion of the complementary DNA (cDNA), which was generously donated
by T. Curran (16). The NGFI-B probe was a 1-kb fragment containing the
coding region of NGFI-B (17) and was kindly provided by Guoqiang Xing,
NIMH. The cRNA probe for rat CRF was transcribed from a 1-kb cDNA
insert in pGEM 4 containing the full length coding region of rat CRF
[kindly provided by Dr. K. Mayo, Northwestern University, and
characterized previously (18)]. For each slide containing two brain
sections, a saturating amount of 35S-labeled riboprobe
(1–2 x 106 cpm) was added to 50 µl hybridization
buffer containing 20 mM Tris-HCl (pH 7.4), 50% formamide,
300 mM NaCl, 1 mM EDTA (pH 8), 1 x
Denhardt’s, 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. Hybridization
took place overnight at 54 C. The next day the sections were rinsed in
4 x SSC, treated with 20 µg/ml RNAse A, rinsed in decreasing
concentrations of SSC at room temperature and finally washed in
0.1 x SSC for 1 h at 65 C. The slides were apposed to Kodak
Biomax MR film for approximately 2 (NGFI-B), 5 (CRF), or 10
(c-fos) days. Some sections were dipped in photographic
emulsion (Kodak NTB-2) and developed after 4 weeks in Kodak D19
developer.
Data analysis
Autoradiograms were analyzed using image analysis software
developed by Wayne Rasband at NIH as described (15). Briefly, the gray
scale values were corrected for film nonlinearity using 14C
standards and expressed in dpm/mg for the cortical areas. In the case
of the PVN, gray scale values were multiplied by the area of the PVN
(to take into account increases in the number of cells expressing the
mRNA species) and expressed in arbitrary units.
Measurements were taken from two to six sections from each animal. Data are given as the mean ± SEM. Differences between groups were tested statistically by ANOVA followed by least squares analysis with Bonferroni correction for multiple groups using SuperANOVA software. In situ hybridization assays were carried out at different times for the P12 and P20 experiments, so the absolute values cannot be compared between the two ages.
As no differences in the mRNA levels of c-fos, NGFI-B, or CRH between male and female rats were observed, the data from the two sexes were combined for statistical analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2. However, in rats that had been
maternally deprived for the preceding 24 h, saline injection
caused marked elevations in ACTH and especially corticosterone beyond
that induced by maternal deprivation alone. (The increased basal
plasma CORT levels in DEP animals may have been due in part to
dehydration. Cortisol binding globulin is decreased by DEP (2) and
therefore may not explain the increased CORT levels.) There was a trend
toward an interaction between stress and deprivation on ACTH
(F2,72 = 2.80, P = 0.067). For
corticosterone there was a potentiation of the stress effect by DEP
(F2,64 = 8.73, P < 0.0001). In the DEP
group, despite the brief nature of the stressor, corticosterone levels
continued to be elevated 2 h later. This may have been partially
due to a decreased clearance rate of CORT in DEP rats (Levine et
al., unpublished observations).
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c-fos and NGFI-B mRNA in the PVN and cortex
P12.
To address whether the hypothalamic PVN and other brain
regions were activated by maternal deprivation and stress in infant
rats during the SHRP, we measured mRNA levels of two immediate-early
genes, c-fos and NGFI-B. Despite the minimal endocrine
response in NDEP P12 rats, we observed a significant increase in
c-fos mRNA levels in the PVN 30 min after the saline
injection. See Figs. 3 and 4. After 24 h of DEP,
c-fos levels were slightly but significantly elevated.
However, stressing the DEP rats resulted in a large increase in
c-fos mRNA levels, the magnitude of which was significantly
greater than that seen in the NDEP stressed group (F2,68 =
4.17, P = 0.045 for the interaction between stress and
DEP). Similar results were observed for NGFI-B. See Fig. 5.
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). However, in
contrast to the PVN, maternal deprivation did not significantly
facilitate the ability of stress to induce c-fos and NGFI-B
in these cortical areas.
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CRH mRNA in the PVN
Despite the fact that maternal deprivation facilitated the
endocrine response to stress at P12, steady-state CRH mRNA levels in
the PVN were reduced. This was also true at P20. See Figs. 6 and 7. We
were unable to detect an increase in CRH mRNA levels in the infant rats
at 2 h following a saline injection regardless of whether they had
been previously deprived.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2). However, when we
examined the PVN, we found significant increases in c-fos
and NGFI-B mRNA levels in response to stress. In the cerebral cortex,
the fold increases in c-fos and NGFI-B during the SHRP (P12)
were similar to those seen at P20. The fact that stress induces IEGs in
the brain of neonatal rats during the SHRP is perhaps not surprising
given the well known behavioral observation that infant rats vigorously
respond to stress in the form of ultrasonic vocalizations (20). Therefore, the brain seems capable of being activated by stress during the SHRP, but this activation is not translated into a pituitary-adrenal endocrine response. Why during the SHRP, stress-induced brain activation does not result in significant ACTH and CORT secretion is still unclear. Perhaps the dissociation between c-fos/NGFI-B induction and release of ACTH is due to a defect in transduction of PVN stimulation into neurosecretion of CRH in immature rats. However, CRH can be released from the median eminence of 5-day-old pups in response to endotoxin (4). Alternatively, CRH may have been released but cannot stimulate ACTH secretion during the SHRP. This also seems unlikely, as injection of ovine CRH into 10-day-old pups causes a robust secretion of ACTH (although, interestingly, the CORT response is minimal) (19).
It may be that the small increases in c-fos and NGFI-B we observed in NDEP rats are simply not reflective of CRH neurosecretion from the median eminence. Indeed, we did not observe any increase in CRH mRNA levels in response to stress, but this may be because the stressor was too mild to induce CRH mRNA or because we examined the brains too early after the application of the stressor (12). Alternatively, CRH may have been induced, but only in a small subpopulation of vasopressin-containing PVN neurons (21). Future studies examining CRH protein levels and/or using probes for CRH heteronuclear RNA may clarify this issue.
The fact that 24 h of maternal deprivation enables the neonate to respond to mild stressors during the SHRP demonstrates that the mother normally exerts a strong inhibitory effect on the central response to stress during this period of time (9). In the absence of normal mother-infant interactions, this inhibition is compromised, thus enabling the neonate to respond to mild stressors such as a saline injection and enhancing the response to robust stressors such as interleukin-1 (22). The locus of this inhibitory effect of the mother is not known. However, our results suggest that one level of inhibition may occur in the hypothalamic PVN. This is based on our observation that stress induced c-fos and NGFI-B mRNA levels in the PVN to a much larger degree in the DEP animals at P12 compared with the NDEP group. This effect of maternal deprivation appeared to be specific for the PVN as DEP did not have a synergistic effect on stress-induced IEGs in cortical areas. The relatively large increase in c-fos and NGFI-B in the DEP rats that were stressed at P12 may reflect a level of stimulation sufficient to release CRH and thus result in the observed increases in ACTH and CORT during the SHRP. This is not to say that extrahypothalamic areas might not also be involved in the release from inhibition. For example, Vasquez et al. (23) have reported that following DEP there was a down regulation of mineralocorticoid receptors in the CA1 region of the hippocampus. These authors suggested that the relative ratio of mineralocorticoid and glucocorticoid receptors in the CA1 region of the hippocampus influences PVN sensitivity and results in an enhanced and sustained ACTH and CORT response to a mild stress stimulus in DEP pups.
It was surprising to find that maternal deprivation decreased CRH mRNA levels in the PVN while potentiating the stress response, at least at P12. One other group also found a decrease in CRH mRNA in response to chronic stress and disturbed mother-infant interactions (24), but why DEP should decrease CRH levels remains a mystery. DEP is apparently decreasing CRH mRNA transcription or increasing CRH mRNA degradation. It is possible that other factors such as vasopressin may play a role in potentiating the stress response following DEP.
The effects of maternal deprivation were markedly different between the SHRP (P12) neonates and rats that were more mature (P20). The most striking difference was that DEP potentiated the ability of stress to induce IEGs in the PVN at age P12 but attenuated the effects of acute stress at P20. It may be that, at P20, the brain has matured to the point where it can respond to the increased corticosterone levels present during the 24 h of DEP and thus have a smaller response to a subsequent stressor. It is also noteworthy that, at P20, ACTH and CORT returned to unstressed baseline levels at 2 h after the saline injection only in the DEP rats. This is unusual because glucocorticoid levels typically remain elevated in response to stress in neonatal rats (25). The NDEP P20 neonates, as well as both NDEP and DEP P12 rats, continued to have elevated ACTH and CORT levels at 2 h, which is more typical. Perhaps DEP at P20 results in higher and more prolonged glucocorticoid feedback that subsequently curtails the HPA axis response to stress. Alternatively, 24 h of food deprivation, which is an unavoidable component of the DEP (9), may attenuate the subsequent response to mild stress at P20 just as it does in the adult (26). However, while fasting may be at least partially responsible for the decreased endocrine and IEG responses to stress in DEP P20 rats, the role fasting plays in the potentiation of the stress response in DEP P12 rats is less clear. Perhaps at P12, fasting leads to more dramatic physiological changes that cause a facilitation of the stress response.
In conclusion, we have demonstrated that several brain areas, including the PVN, are activated in response to stress in NDEP neonatal rats during the SHRP despite a negligible endocrine response. However, DEP at P12 facilitates the ability of stress to induce c-fos and NGFI-B in the PVN. Thus, part of the inhibitory effect of the mother-infant interaction during the SHRP may occur at the level of the hypothalamic PVN. Prolonged separation from the mother during the SHRP may result in elevated (27) or reduced (28) CRH mRNA levels in the PVN of adult rats depending on the age when they were separated from their mother. These early environmental experiences may lead to permanently altered stress responsivity and be a significant risk factor for adult psychopathology.
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Acknowledgments |
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Received May 27, 1997.
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