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Department of Psychology, University of Delaware, Newark, Delaware 19716-2577; and the Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, University of Leiden, 2300 RA Leiden, The Netherlands
Address all correspondence and requests for reprints to: E. R. de Kloet, Ph.D., Leiden/Amsterdam Center for Drug Research, Division of Medical Pharmacology, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail: e.kloet{at}lacdr.leidenuniv.nl
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Abstract |
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On postnatal day 9 or 12, pups were deprived (DEP) of the dam for 24 h; nondeprived pups of the same age served as controls. The pups were adrenalectomized (ADX) at the end of deprivation and given corticosterone (CORT) replacement by either injection or pellet implants using the following two designs: first at the time of adrenalectomy (ADX) to test the role of CORT in the maintenance of basal ACTH levels, and then 3 h after ADX, to investigate CORT suppression of elevated ACTH levels induced by prior ADX.
Regarding proactive feedback, the results showed that injection of CORT at the time of ADX was only partially effective in preventing ACTH elevations, whereas CORT pellets maintained basal levels of ACTH in all ADX pups. The reactive mode of negative feedback in nondeprived pups was resistant to CORT injection, whereas the CORT pellet resulted in a return to basal levels within 60 min. Maternal deprivation did not affect proactive feedback, but caused a more sustained increase in ACTH levels and a failure to return to basal levels 3 h after ADX despite significantly higher levels of circulating CORT in these DEP pups.
It is concluded that 1) proactive and reactive modes of negative feedback are operative, provided the pups are maintained on chronic replacement with CORT; 2) DEP impairs the reactive, rather than the proactive, mode of feedback inhibition in the neonate.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After maternal deprivation (DEP), the pup’s basal and stress-induced elevations of CORT and ACTH levels are elevated, and the sensitivity of its adrenal to ACTH stimulation is increased. Moreover, when the HPA axes of these DEP pups are activated in response to a challenge, ACTH and CORT rise more slowly than in adults and are sustained at an elevated level (7, 8, 9). Negative feedback seems to be a late developing process. Studies in weanling rats (once they are capable of responding) have shown that the ACTH responses to stress are not terminated as efficiently as in adults (10). Yet, from the animals that are in SHRP, we know that the low basal levels of CORT are necessary to maintain basal levels of ACTH. This is further supported by studies using 10-day-old adrenalectomized (ADX) pups, which have demonstrated that basal plasma ACTH levels are markedly and acutely increased compared with those in sham-operated and nontreated controls (11). However, ADX pups with CORT pellets implanted at the time of surgery fail to show elevated ACTH levels (11). This suggests that some aspect of the negative feedback regulation is functional very early in development.
Negative feedback has two modes of operation (12). 1) The proactive mode involves the maintenance of basal levels of HPA activity. This process ultimately determines the sensitivity or threshold of the response to stress and involves the functions of high affinity mineralocorticoid receptors (MR) for CORT localized in higher brain regions. 2) The function of the reactive mode is to suppress ACTH and CORT levels induced by stress. This phenomenon is well documented and involves the lower affinity glucocorticoid receptors (GR) localized in parvocellular neurons of the paraventricular nucleus and the pituitary corticotrophs. GRs are also localized in abundance in cortical regions, hippocampus, and ascending aminergic pathways, where they mediate a modulatory influence of CORT on HPA activity (12).
During development, these two modes of negative feedback mature differentially. Whereas proactive inhibition can occur early in ontogeny, reactive inhibition is a late developing process. The present studies were designed to examine both proactive and reactive negative feedback in rat pups during the SHRP and to test the hypothesis that the development of CORT negative feedback is dependent on the dam-pup interaction. For this purpose, we examined both modes of negative feedback by replacement of CORT in ADX pups that experienced DEP. The results demonstrate that both proactive and reactive CORT feedback may be functional in the neonate, but are dependent upon a constant exposure to CORT achieved by the use of CORT pellets. CORT injections are only partially effective in the maintenance of ACTH levels after adrenalectomy (ADX) and are ineffective in suppressing elevated ACTH levels. Maternal deprivation further impairs reactive feedback inhibition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Deprivation procedure
In each experiment half of the litters were maternally deprived
(DEP) for 24 h on pnd 8 or 11. The mother was removed, and the
litter remained in the home cage, which was placed on a heating pad
(General Electric) set at 30–33 C in the deprivation room, adjacent to
the main colony room. The deprivation room was kept under the same
temperature and lighting conditions as the main colony room. Neither
food nor water was available during the deprivation period. The
nondeprived (NDEP) litters remained with their dams until the time of
testing.
Surgical procedure
The pups were first weighed, and then the pups that were to
undergo surgery were anesthetized. For ADX pups, the adrenals were
quickly removed through two small dorsal incisions. The wounds were
sealed with tissue glue, after which the pups were placed into their
home cage and kept there for the appropriate period of time, according
to the experiments described below. For sham-ADX (SHAM) subjects the
same procedure was performed, except the adrenals were merely exposed
instead of removed.
CORT injections and pellets
The pups that were injected received ip injections of 1 mg/kg BW
CORT. CORT (Steraloids, Wilton, NH) was dissolved in saline-100%
ethanol (75:25) to obtain a 1 mg/ml stock solution.
The pups were implanted sc in the neck area with 10-mg pellets, and the incision was sealed with tissue glue. The CORT pellets contained 5% CORT (11) and 95% cholesterol. Pure cholesterol pellets were used as controls (Hormone Pellet Press, Kansas City, KS).
Blood sampling procedure
Between 0900–1300 h, pups were decapitated, and trunk blood
from each pup was collected in precooled plastic vials containing
11–14 drops of 60 mg/ml EDTA kept on crushed ice. The samples were
centrifuged at 2 C for 20 min at 2000 rpm, and the plasma was collected
and placed in a marked, precooled sample tube that was kept at -20 C
until RIA for ACTH (Incstar, Stillwater, MN) and CORT (ICN Biomedicals,
Costa Mesa, CA). The sensitivities of the ACTH and CORT assays were 15
pg/ml and 0.125 µg/dl, respectively.
Data analysis
Data were analyzed by ANOVA (13), with the level of significance
set at P < 0.05. Initial ANOVAs were performed
separately for each age by means. Once sex was determined not to be a
significant variable, data were collapsed across sexes. When
appropriate, post-hoc analysis for simple main and
interaction effects was analyzed by Newman-Keuls procedure.
Experimental design
Table 1
presents the time table of
the designs of Exp I–IV used to distinguish proactive and reactive
feedback modes of CORT.
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Twenty-four hours after the onset of deprivation, two intact pups (one male and one female) from each litter were sampled immediately (NT), and two intact pups were placed in individual compartments and sampled at the end of a 3-h period (NT3). For NDEP litters, mothers were removed immediately before the onset of the procedure. The remainder of each litter was subjected to ADX or SHAM surgery. At this time the CORT injection (1 mg/kg BW ip) was also administered to the ADX+CORTi pups. The pups were placed in individual compartments maintained at 30–33 C. Blood was sampled 3 h later, 27 h after start of the experiment (onset of DEP).
Exp II. Nine-day-old infants were taken from 18 litters. This experiment had the following design: one age (9 days) x two sexes x two conditions (DEP and NDEP) x four treatments [NT, SHAM, ADX and cholesterol pellet (ADX+CHOL), and ADX and CORT pellet (CORTp)]) x one time point (3 h after pellet implantation). Samples from the 9-day-old DEP animals were pooled to provide sufficient plasma for ACTH and CORT assays. Sex and condition were intralitter variables.
The selected dose (5% CORT and 95% cholesterol) was found to be effective in other studies using infant rats (11). Furthermore, only animals 9 days of age were used because of the similar data obtained in the previous experiment for the 9- and 12-day-old infants.
NDEP and DEP pups received their operations and pellets 3 h before sampling, which for the DEP animals meant after 24 h of DEP. The NDEP pups were returned to their mothers after their operation, and the DEP pups were returned to their home cage on the heating pad in the deprivation room. After 27 h of deprivation for the DEP pups and the equivalent amount of time for the NDEP pups, the mothers from the NDEP animals were removed, and blood was collected.
Exp III. Nine- and 12-day-old infant rats were taken from 64 litters. The design was two ages (pnd 9 and 12) x two sexes x two conditions (NDEP and DEP) x four treatments [NT, SHAM, ADX and saline, and ADX and CORT injection (ADX+CORTi)]) x four time points (5 min, 1 h, 2 h, and 3 h). Sex and time points were intralitter variables.
DEP and NDEP received surgery 3 h before sampling, i.e. for DEP animals after 21 h of deprivation. The NDEP pups were returned to their mothers after their operations, and the DEP pups were returned to their home cages on the heating pads in the deprivation room. After 24 h of deprivation for the DEP pups and the equivalent amount of time for the NDEP pups, the mothers of the NDEP animals were removed, and the pups received either saline or CORT treatment. One male and one female were sampled for each time point. For the ADX+CORTi treatment, the pups received a CORT injection ip at 24 h, and the first pair of pups was sampled 5 min later. The sampling then continued for all animals 1, 2, or 3 h later. The last time point was 27 h after the start of the experiment (onset of DEP).
Exp IV. Nine- and 12-day-old infants were taken from 30 litters. The design was two ages (pnd 9 and 12) x two sexes x two conditions (NDEP and DEP) x five time points (NT and 5, 60, 120, and 180 min).
NDEP and DEP animals were ADX 3 h before pellet implantation, which for the DEP animals meant after 21 h of DEP. The NDEP pups were returned to their mothers after their operations, and the DEP pups were returned to their home cages on the heating pads in the deprivation room. After 24 h of deprivation for the DEP pups and the equivalent amount of time for the NDEP pups, the mothers from the NDEP animals were removed. Pellets were implanted, and blood sampling was begun for the time-course study. One male and one female were sampled immediately (NT); the remainder received a CORT pellet. The first pair of implanted pups was sampled 5 min later. Subsequent samples were obtained 1, 2, and 3 h after implantation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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presents the time table of the designs of Exp I–IV used
to distinguish proactive and reactive feedback modes of
corticosterone.
Exp I
To test the proactive mode of negative feedback, animals were ADX
after 24 h of DEP and immediately treated with CORT injection or
vehicle control. SHAM and NT pups served as controls. Blood sampling
began 3 h after CORT injection, coinciding with 27 h DEP for
the DEP animals.
Plasma ACTH levels. 9 days: Figure 1a shows the ACTH data of the 9-day-old
infants. A clear CORTi treatment effect was observed. There
were no differences as a result of deprivation observed.
Post-hoc analysis revealed that for both NDEP and DEP pups,
3-h separation from the mother (NT3) did not elicit an ACTH
response, nor did SHAM operation, as these ACTH levels were equivalent
to basal levels (NT). These levels were significantly lower than those
in ADX animals, which showed a significant increase in ACTH levels
after all other procedures (P < 0.05). ACTH levels in
ADX+CORTi animals were higher than those in NT,
NT3, and SHAM pups, but were significantly lower than those
in ADX animals.
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). Post-hoc analysis showed that in NDEP and DEP pups, the
ACTH levels for NT3 and SHAM were equivalent to basal (NT)
levels. ACTH levels in the ADX+CORTi group were elevated,
but still lower than those in ADX animals, which were significantly
elevated above those after all other treatments. ACTH levels in NDEP
CORT-replaced animals were higher than those in DEP pups
(P < 0.05).
Plasma CORT levels. 9 days: Both a deprivation and
a treatment effect were observed (Fig. 1c). In addition, an interaction
was present. Post-hoc analysis revealed that DEP SHAM and
DEP ADX+CORTi animals had CORT levels that were much higher
than those in all other groups. The CORT level in the CORTi
group was even higher than that in the SHAM group. Differences between
NDEP and DEP occurred only in these two groups. Furthermore, NDEP SHAM
and NT3 CORT values were similar to basal levels in the NT
group. Concentrations in NDEP CORTi animals were
significantly elevated above the basal level (P <
0.05). All ADX animals had CORT levels below the detection limit of the
assay.
12 days: A deprivation and treatment effect as well as an
interaction were observed (Fig. 1d). Post-hoc analysis
showed that the highest CORT levels occurred in the DEP SHAM and
CORTi group. In these DEP animals, CORT levels in the SHAM
and ADX+CORTi group were similar, but significantly
elevated over those in NT and NT3 groups, which were
equivalent. At this age, CORT values were higher in DEP than in NDEP
pups (P < 0.05), except for the ADX group. In NDEP
pups, all treatment groups had CORT levels that were similar to basal
NT values, except for the CORTi group. ADX animals had CORT
levels below the detection limit of the assay.
Exp II
To test the proactive mode of negative feedback, animals were ADX
after 24 h of DEP and immediately treated with CORTp
or vehicle. SHAM and NT pups served as controls. Blood sampling was
performed 3 h after CORTp coinciding with 27 h of
DEP for the DEP animals.
Plasma ACTH levels. Figure 2a
depicts the ACTH data of Exp II. An effect of treatment was observed.
Post-hoc analysis indicated that the ADX+CHOL group had ACTH
levels elevated above those in the NT, SHAM, and ADX+CORTp
groups, which were all similar to each other. There were no differences
in ACTH levels between NDEP and DEP animals.
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illustrates the CORT data of Exp
II. An effect of treatment was found. ANOVA also revealed an effect of
condition. Furthermore, an interaction between condition and treatment
was observed. Post-hoc analysis showed that differences
between NDEP and DEP groups occurred in only the SHAM and
ADX+CORTp groups. With these treatments, levels in the DEP
groups were elevated over those in the NDEP groups. Post-hoc
analysis also showed that for the NDEP animals, the
ADX+CORTp group had higher CORT levels than those in the
NT, SHAM, and ADX+CHOL groups. For the DEP animals, the
ADX+CORTp group had higher CORT levels than the SHAM group,
which had higher levels than both the NT and ADX+CHOL groups.
Exp III
To test the reactive mode of negative feedback, animals were ADX
after 21 h of maternal deprivation and 3 h later were treated
with CORT injection or vehicle. SHAM and NT pups served as controls.
Blood sampling was performed 5 min and 1, 2, and 3 h after CORT
injection. The last sample coincided with 27 h of DEP for the DEP
animals.
Plasma ACTH levels. 9 days: Figure 3a depicts the ACTH data for the
9-day-old NDEP infants. This figure illustrates that the ADX and
ADX+CORTi groups had ACTH levels significantly elevated
over those in the NT and SHAM groups at each time point. Levels in the
NT groups, whose ACTH levels represent basal levels, were equivalent to
those in the SHAM groups at each time point. In addition, it was found
that the ADX group had ACTH levels equivalent to those in the
ADX+CORTi group at each time point. Also of note was the
manner in which the levels of ACTH decreased over time in the ADX and
ADX+CORTi groups. In the ADX group, the animals sampled at
5 min had plasma ACTH levels significantly elevated above those at each
of the other time points, which were all statistically not different
from each other. In the ADX+CORTi group, the level at 5 min
was elevated over those at 2 and 3 h, which were similar to each
other. The level at 1 h was also elevated over that at 3 h
(P < 0.05 for each comparison).
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illustrates the ACTH data for the 9-day-old DEP animals; ADX
and ADX+CORTi animals had ACTH levels that were
significantly elevated over those in the NT and SHAM pups at each time
point. Again, NT and SHAM levels were equivalent to each other at each
time point as were ADX and ADX+CORTi levels. The time
course of ACTH levels differed from that in NDEP animals. In the ADX
DEP group, plasma ACTH levels at each time point were similar. The
ADX+CORTi data showed that the level at 5 min was elevated
over those at the other three time points, and the level at 2 h
was elevated over that at 3 h (P < 0.05).
Considering Fig. 3, a and b, together, there was an effect of
condition. ACTH levels in the DEP ADX and ADX+CORTi animals
remained elevated longer than those in the NDEP infants.
Post-hoc analysis indicated a difference only at one time
point within each of these groups. DEP was elevated over NDEP in the
ADX group at 1 h (P < 0.01) and in the
ADX+CORTi group at 2 h (P <
0.05).
12 days: Figure 3c depicts the ACTH data for the 12-day-old
NDEP infants. Similar to the data presented in Fig. 3, a and b, the
plasma ACTH levels in the ADX and ADX+CORTi groups were
significantly elevated over those in the NT and SHAM groups at each
time point; NT and SHAM groups had similar ACTH levels. The plasma ACTH
level in ADX animals was lower than that in ADX+CORTi
animals at 5 min and 1 h. At the other time point the groups were
not different. In the ADX+CORTi group, ACTH was elevated at
5 min over that at each of the other time points, which were all
statistically not different.
Finally, Fig. 3d illustrates the ACTH data for the 12-day-old DEP
animals. Again, the levels in the ADX and ADX+CORTi groups
were elevated over those in the NT and SHAM groups. The NT and SHAM
groups were similar to each other at each time point and remained
constant over time. There was a difference between ADX and
ADX+CORTi at 2 h (ADX < ADX+CORTi).
In terms of differences between time points within treatments, the ACTH
levels in ADX+CORTi animals were higher at 5 min than at
2 h, and values at 1 h were higher than those at 2 and 3
h.
Comparison between DEP and NDEP indicates an effect of condition. ACTH levels in the DEP ADX animals remained elevated longer than those in the NDEP infants. These differences occurred at 1, 2, and 3 h (NDEP < DEP). In the ADX+CORTi groups, the only difference occurred at 5 min (NDEP > DEP).
Plasma CORT levels. 9 days: Figure 4a depicts the CORT data for the
9-day-old NDEP infants. An analysis of the data presented here revealed
no differences between treatments or between the time points
measured.
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illustrates the CORT data for the 9-day-old DEP animals. The
most striking differences were between the treatment groups.
Post-hoc analysis indicated differences between the
treatment groups at each time point for these DEP animals. At 5 min,
the ADX+CORTi group had a higher CORT level than the SHAM
group, which had a higher level than both the NT and ADX groups. At
1 h, the same interactions were observed. At 2 h,
ADX+CORTi and SHAM CORT levels were statistically not
different from each other and were higher than those in the NT and ADX
groups. Finally, at 3 h, the ADX+CORTi and SHAM groups
were similar and higher than those in the ADX group, but values in each
of these groups were also statistically not different from those in the
NT group.
Examining Figure 4, a and b, together, a highly significant effect of
condition is evident. Post-hoc analysis found this to be
present in the SHAM and ADX+CORTi groups only. For the SHAM
groups, the DEP groups had higher CORT levels than the NDEP groups at
each time point. In the ADX+CORTi groups, in all time
points except 3 h, an elevation in the plasma CORT levels was
found in the DEP groups compared with those in the NDEP groups.
12 days: Figure 4c depicts the CORT data for the
12-day-old NDEP infants. As was the case with the NDEP 9-day-old
animals, there were no differences between the time points within each
of the treatments. However, an effect of treatment was observed at 5
min; values in the ADX+CORTi group were elevated over those
in NT, SHAM, and ADX group.
Figure 4d illustrates the CORT data for the 12-day-old DEP animals.
Similar to the 9-day-old DEP infants, there were differences between
the treatments at each time point. At 5 min, the ADX+CORTi
group had a higher CORT level than the SHAM and NT groups, which were
similar to each other and each higher than that in the ADX group. At
1 h, the same pattern was observed. At 2 h, SHAM and NT CORT
levels were statistically not different and higher than that in the
ADX+CORTi group, which was higher than that in the ADX
group. Finally, at 3 h, values in the SHAM and NT groups were
similar and higher than those in the ADX+CORTi and ADX
groups. Turning to differences within treatment groups between the time
points, the only group in which CORT levels were statistically
different from each other was the ADX+CORTi group. In this
group, the CORT level at 5 min was higher than that at 1 h, which
was higher than those at 2 and 3 h.
Considering Fig. 4, c and d, together, an effect of condition was again
observed. Post-hoc analysis found this to be present in the
NT, SHAM, and ADX+CORTi groups only. For the NT and SHAM
groups, the DEP groups had higher plasma CORT levels than the NDEP
groups at each time point. In the ADX+CORTi groups at all
time points except 3 h, an elevation in plasma CORT levels was
found in the DEP groups compared with those in the NDEP groups.
Exp IV
To test the reactive mode of negative feedback, animals were ADX
after 21 h of DEP and 3 h later were treated with CORT
implants. NT pups served as controls. Blood sampling was performed 5
min and 1, 2, and 3 h after CORTp. The last sample
coincided with a 27-h DEP for the DEP animals.
Plasma ACTH levels. 9 days: Figure 5a shows the ACTH data for the 9-day-old
pups. Clear condition and treatment effects were observed as was an
interaction between condition and treatment. In both conditions, ACTH
levels decreased over time. Post-hoc analysis showed that
basal levels were similar for NDEP and DEP animals. Five minutes after
pellet implantation, ACTH levels were significantly elevated, but were
not significantly different from each other. At all the other time
points, DEP ACTH levels were significantly elevated over levels in the
NDEP animals. In the NDEP animals, the levels were still elevated at 60
min, but 120 min after pellet implant, ACTH levels had returned to
basal values. However, in the DEP animals, ACTH levels decreased, but
failed to return to basal values after 3 h.
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shows the ACTH data for the 12-day-old
animals. Results similar to those for the 9-day-old animals were
observed, with condition and treatment effects plus an interaction
between condition and treatment. Post-hoc analysis revealed
the same pattern of results as that in the 9-day-old animals. The only
difference was that values in NDEP and DEP groups were also different 5
min after pellet implantation. The DEP group was not different from
NDEP group at basal levels and was elevated over the NDEP group at
every time point after pellet implantation. Again, in NDEP animals, the
ACTH levels had returned to basal values at 120 min, whereas in the DEP
pups, they were still elevated over basal at 180 min.
Plasma CORT levels. Figure 5, c and d, illustrates the CORT
data from Exp IV.
9 days: ANOVA revealed condition and treatment effects. An interaction between condition and treatment was observed as well. Post-hoc analysis showed that CORT levels in DEP animals were significantly higher than those in NDEP animals. In addition, it was shown that all of the implanted animals had higher CORT levels than those in the NT group.
12 days: The CORT values in the 12-day-old animals were very similar to those in the 9-day-old animals. Condition and treatment effects as well as an interaction between condition and treatment were observed again. Post-hoc analysis showed that in all of the implanted animals, CORT levels were elevated over those in the basal NT condition. In all implanted groups, except the 5 min group, DEP levels were elevated over those in the NDEP group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies in rat pups (11) have shown that ADX provoked an immediate large increase in plasma ACTH that peaked about 3 h after surgery. In addition, pituitary ACTH content was depleted. After this initial rise, there was a marked decrease in circulating levels of ACTH that persisted for 48 h, at which time ACTH levels returned to the levels seen 3 h after ADX. It is presumed that this initial increase is due to the release of CRH stored in the median eminence and that once these stores are depleted, there is a refractory period during which CRH is being resynthesized to provide the required impetus to once again stimulate the pituitary to release ACTH in the absence of CORT negative feedback. Exp I and II demonstrated that proactive feedback is operational, given that the initial rise in ACTH levels induced by ADX was significantly attenuated by CORT replacement immediately after ADX regardless of the administration route. In addition, deprivation condition did not modify these results. However, replacement with CORT pellets that provide a constant release of CORT was more efficacious at completely preventing the ADX-induced rise in ACTH levels in both DEP and NDEP animals. Thus, the ACTH values of the nontreated control, sham-operated, and pellet replaced pups were virtually identical regardless of DEP condition.
There were, however, several differences between NDEP and DEP pups. Although 3 h postsham surgery, CORT is elevated in both NDEP and DEP pups, the elevations were 2- to 3-fold higher in DEP pups. CORT elevations after both injection and pellet implant were also significantly higher in DEP animals. Such elevations of CORT can be achieved in several ways. First, CORT elevations are a consequence of increased ACTH stimulation and increased adrenal sensitivity to ACTH. Second, increases in circulating CORT can be a result of increased CBG, decreased clearance of CORT, or decreased blood volume. Regarding the first possibility, after a saline injection or exposure to ether, ACTH levels are significantly elevated (1, 9) in the DEP neonate, whereas NDEP pups failed to elicit an ACTH response. In addition, increased adrenal sensitivity to exogenous ACTH has been observed in DEP neonates (1). However, in the current experiment the CORT-replaced animals were ADX; therefore, the sensitivity of the adrenal cannot solely account for the CORT differences between NDEP and DEP. In addition, increased CBG is not likely to be an explanation for the high CORT levels in the DEP animals because the marked diminution of CBG levels during SHRP is not altered by DEP (1).
Another characteristic of the neonate is reduction in the clearance of CORT (16). DEP appears to further reduce the clearance of CORT from the circulation. Accordingly, an identical amount of exogenously administered CORT results in persistently higher CORT levels in DEP pups. This was consistently seen in all of the experiments in which CORT was exogenously administered. Furthermore, one of the obvious consequences of being deprived of the dam for 24 h is that the pup is fasted for that period. Fasting has been found to reduce blood flow and to contribute to the reduction in CORT metabolism (17, 18). Under normal rearing conditions, rat pups will gain approximately 2–3 g/day between 9–12 days of age. However, after DEP, the pups will lose about 2 g after 24-h fasting from a body weight of 18–20 g. Such a drastic reduction in body weight could cause a reduced blood volume, resulting in an increased concentration of plasma CORT. Although we cannot dismiss this possibility, data from other experiments reported here do not support this hypothesis. Therefore, decreased metabolism and clearance of CORT can explain the elevated CORT levels in DEP animals.
Whereas DEP did not appreciably affect the expression of proactive inhibition, there were striking consequences of the deprivation manipulation on reactive inhibition. These effects are manifested in at least two ways. First, the elevations of ACTH induced by ADX persisted for several hours longer in DEP than in NDEP pups. Second, the metabolism of CORT was markedly affected in DEP pups. There is a pronounced difference in CORT levels 5 min after a CORT injection. This difference persists for at least 1 h. However, by 3 h the CORT levels are equivalent to those in NDEP pups. This would support the hypothesis of reduced clearance and not support the idea that increased CORT levels are a function of decreased blood volume. CORT levels in the implanted DEP pups remained high during the entire testing period. Although the CORT levels were higher under both conditions of CORT administration, ACTH levels remained elevated longer (Exp III) and/or failed to return to basal values (Exp IV). In contrast, injection of CORT in the adult results in a rapid inhibition of ACTH (19, 20). The enhanced resistance to reactive CORT feedback seen in the DEP pups could occur as a function of two interacting processes. The first is best described as a central nervous system hyperdrive, which results in pronounced activation of ACTH. The second process may be related to the effects of DEP on the ontogeny of the glucocorticoid receptors.
Evidence for what we are calling a central nervous system hyperdrive comes from numerous experiments using DEP neonates (2). Consistent with the description of the SHRP, NDEP pups either do not respond or markedly reduce their responses to a variety of stimuli that elicit ACTH and CORT responses in DEP pups. Further, even under conditions that lead to an increase in ACTH in NDEP pups (i.e. injections of interleukin-1ß), this response is augmented in DEP pups (21). This suppression is not a function of a reduction in the capacity of the pituitary to release ACTH. The removal of the adrenal increased ACTH levels equivalently in NDEP and DEP animals. Further, significant elevations of ACTH were obtained during the SHRP when pups were exposed to excitatory amino acids (3). Thus, the suppression of the ACTH response is not due to any deficiency in the hypophysial component of the HPA axis. We have hypothesized that one of the consequences of the dam-pup interaction is to functionally inhibit the neural pathways that are responsible for activating the neuroendocrine cascade required to elicit an ACTH response. Accordingly, removal of the adrenal, and thus of the endogenous CORT feedback signal, may influence neural processes and override the effects of the maternal inhibition. These disinhibitory neural processes may take place in CORT-responsive extrahypothalamic circuits innervating the CRH neurons, which enhance the infant’s capacity to release ACTH. It might well be that these very same CORT-responsive circuits are affected by DEP, leading to the reduced negative feedback in the DEP pups (22).
Another reason why negative feedback does not operate with the same efficiency in maternally deprived infants after CORT replacement as in the NDEP controls may be due to differences in MR and GR in the pup’s brain. One important difference between these two receptor types is their function in negative feedback inhibition. MRs are already highly occupied by CORT at basal levels, and their function appears to be involved in the maintenance of HPA basal activity. Due to their lower affinity for CORT, GRs are mainly involved in situations where CORT levels are elevated, such as those associated with stress and the peak of the circadian rhythms (23). The acute injection of CORT provides an initial high level of CORT that rapidly dissipates over time, whereas the pellet ensures constant availability during the 3-h period. Although receptor function has not been directly determined in these studies, continuous exposure to CORT seems to be required in the neonate to completely suppress basal ACTH, and it is even more critical in suppressing ADX-elevated levels. This finding suggests that the receptors require prolonged exposure to CORT to slowly integrate the hormone signal.
During ontogeny, hippocampal MRs, mediating proactive inhibition by CORT (24), are present in the same concentrations as in adults by 8 days of age. In contrast, GR concentrations develop more gradually and are still increasing at 20 days of age, particularly in the dentate gyrus (25, 26, 27). GRs predominantly play a role in situations when CORT is high, the reactive mode. Therefore, it would be expected that negative feedback, especially reactive negative feedback, would not function as efficiently in the neonate. Moreover, 24 h of DEP results in a decrease in basal MR and stress-induced GR messenger RNA levels in the CA1 area (28), which might well be an indication for the even more reduced negative feedback observed in DEP pups. We have recently replicated the findings of Vazquez et al. (28), but DEP pups on pnd 12 also show a reduction in basal levels of GR messenger RNA in both the CA1 area and the paraventricular nucleus. Thus, DEP clearly alters the characteristics of the GRs in the neonate and thus may play an important role in the dysregulation of negative feedback in DEP pups.
In conclusion, with constant exposure to CORT, it has been shown that negative feedback is functional in the rat pup. However, there are deficiencies in both modes of negative feedback, as shown by the response to CORT injections. DEP further impairs negative feedback, particularly in the reactive mode.
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Received October 8, 1997.
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