 |
 Previous Article | Next Article 
The Journal of Neuroscience, December 1, 1998, 18(23):10171-10179
Maternal Deprivation Effect on the Infant's Neural Stress
Markers Is Reversed by Tactile Stimulation and Feeding But Not by
Suppressing Corticosterone
Helga J.J.
van Oers1, 2,
E. Ronald
de Kloet2,
Tara
Whelan1, and
Seymour
Levine1
1 Department of Psychology, University of Delaware,
Newark, Delaware 19716-2577, and 2 Division of Medical
Pharmacology, Leiden/Amsterdam Center for Drug Research, University of
Leiden, 2300 RA Leiden, The Netherlands
 |
ABSTRACT |
After 24 hr of maternal deprivation, significant elevations in ACTH
and the naturally occurring glucocorticoid corticosterone (CORT) are
observed during the stress-hyporesponsive period. The deprived pups
also showed in the paraventricular nucleus (PVN) a marked increase of
stress-induced c-fos mRNA and a reduction of
corticotropin-releasing hormone (CRH) and glucocorticoid receptor (GR)
mRNA; in hippocampal CA1, a reduction of the mineralocorticoid receptor
(MR) and GR was observed. Here, we examined whether these changes are
reversed by (1) preventing the elevations of CORT characteristic for
the 11-d-old deprived pups by administering the synthetic
glucocorticoid dexamethasone (DEX); or (2) reinstating some aspects of
maternal behavior. The pups were either (1) left undisturbed, (2)
stroked, or (3) stroked and episodically fed by cheek cannulation. At
postnatal day 12, peripheral and neural stress markers were
measured. Nondeprived animals served as controls.
Experiment 1 demonstrates that although CORT was kept low by DEX, the
central effects on CORT receptors, CRH, and c-fos mRNA were still present, except for MR in hippocampal CA1. Experiment 2 shows that stroking alone prevented the stress-induced rise in ACTH and
c-fos mRNA and in the reduction in CRH and MR mRNA. In
pups that were fed and stroked, CORT and GR mRNA resembled nondeprived
controls. In conclusion, the changes in peripheral endocrine responses
and in the brain cannot be attributed to the effect of elevated CORT
concentrations, which are characteristic of the maternally deprived
neonate. However, reinstating some components of the dams' nurturing
behavior can reverse the effects evoked by maternal deprivation.
Key words:
maternal deprivation; stress; brain; corticosterone; dexamethasone; corticosteroid receptors
| |
INTRODUCTION |
During recent years, there has been
a growing literature that indicates that the stress-hyporesponsive
period (SHRP) is not absolute but rather is dependent on the stimulus
used and on what component is measured (Witek-Janusek, 1988 ; Widmaier,
1989; Levine et al., 1994). Different stimuli seemed to elicit
idiosyncratic patterns of ACTH release (Walker et al., 1991; Yi et al.,
1994; Kent et al., 1996, 1997). However, the response of the adrenal during development seems markedly diminished, even in the presence of
high ACTH (Rosenfeld et al., 1991, 1992).
It has also been documented that the suppression of the
hypothalamic-pituitary-adrenal (HPA) axis during the SHRP is in part under maternal regulation (Stanton et al., 1988; Suchecki et al., 1993a, 1995). Pups that are exposed to 24 hr of maternal deprivation exhibit a marked increase in "basal" level of the naturally
occurring glucocorticoid corticosterone (CORT) and an increase in
circulating levels of both ACTH and CORT in response to mild
perturbations (Stanton et al., 1988; Suchecki et al., 1993a,
1995). Maternal deprivation also alters specific components of the
neural systems that have been shown to be involved in the regulation of
the peripheral stress hormones. Maternal deprivation at postnatal day 9 (P9) and P12 results in a reduction of mineralocorticoid
receptor (MR) mRNA in the CA1 region of the hippocampus (Vazquez et
al., 1996). Further, there is a significantly greater increase in
c-fos mRNA in the paraventricular nucleus (PVN) of the
hypothalamus and a paradoxical decrease in corticotropin-releasing
hormone (CRH) mRNA after maternal deprivation (Smith et al.,
1997).
Several studies have attempted to reverse the effects of maternal
deprivation by experimentally replacing some of the elements of
maternal behavior (Cirulli et al., 1991). Suchecki et al. (1993b) indicated that when pups received tactile stimulation the effects of
maternal deprivation on the stress-induced rise in ACTH was completely
reversed. Stroking the anogenital region was capable of normalizing the
mechanisms that regulate the release of ACTH. Feeding in addition to
stroking restored elevated CORT to basal levels and reduced the
increased sensitivity of the adrenal to ACTH, which appears to be one
of the consequences of maternal deprivation. It was concluded that
different maternal behaviors regulated different components of the
developing HPA axis.
Pups deprived for 24 hr are exposed to at least 16 hr of elevated CORT,
which could have caused the changes in brain. To test the hypothesis
that the elevations of CORT induced the central changes after
deprivation, we eliminated the rise in CORT by the administration of
the synthetic glucocorticoid dexamethasone (DEX) at the beginning of
the deprivation period. The results of these studies failed to support
the hypothesis that CORT was responsible for the effects. Thus, a
second experiment was conducted to determine whether the central
effects of maternal deprivation could be reversed by reinstating
maternal care. Almost all of the effects of maternal deprivation were
reversed by tactile stimulation of the anogenital region. The effects
were completely reversed when the pups were stroked and fed.
| |
MATERIALS AND METHODS |
Subjects
The subjects were 12-d-old rat pups bred from Sprague Dawley
females and Long-Evans males (Simonsen, Gilroy, CA). Hybrid offspring were used because they have a lower mortality rate than purebreds. Age
was determined by checking for births every day at 9:00 A.M. and 5:00
P.M.; the date of birth was designated as P0. Litters were housed with
their dams in transparent polycarbonate cages (48.5 × 25.6 × 19.0 cm; Nalgene) with a flooring of wood shavings and a grid top.
Rat chow (Wayne Rodent Blox; Allied Mills, Chicago, IL) and tap water
were provided ad libitum. The laboratory was maintained
under constant temperature (22°C) and lighting (12 hr light/dark
cycle; lights on at 7:00 A.M.) conditions. On the day after birth (P1),
litters were culled to eight pups (four males and four females) and
placed in a clean cage. Cross-fostered pups were from litters born on
the same day and were recorded for future reference during data
analysis. From this moment on, the animals were not handled in any way,
nor were their cages cleaned, until the time of deprivation or testing
to minimize disruption of the mother-infant relationship. All
experiments were performed in accordance with protocols approved by the
Animal Care Committee of the University of Delaware.
| |
Deprivation procedure |
The mother was removed, and the litter remained in the home
cage. The home cage, containing the litter, 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 time
of testing.
| |
Sampling procedure |
Trunk blood from each pup was collected by decapitation a few
seconds after disturbing the pups and placed in precooled plastic vials
containing EDTA (60 mg/ml) kept on crushed ice. The samples were
centrifuged for 20 min at 2000 rpm at 2°C, and the plasma was
collected and placed in a marked precooled sample tube that was kept at
 20°C until radioimmunoassay procedure (RIA) for ACTH (INCSTAR kit) and CORT (ICN Biomedicals). The sensitivity of the ACTH
and CORT assays was 15 pg/ml and 0.125 µg/dl, respectively.
At the time of blood sampling, brains were collected for measurement of
mRNA expression of glucocorticoid receptor (GR), MR, and CRH. Brains
were frozen immediately in a bucket containing 2-methylbutane kept at
42°C on dry ice. The brains were stored at 80°C until time of
slicing and in situ hybridizations.
| |
Probes |
Different 35S-labeled cRNA antisense probes were
used to hybridize with complementary brain tissue MR and GR mRNA. The
antisense MR probes were transcribed from a 513 base pair (bp) rat
brain cDNA fragment, which encodes for the last 30 amino acids at the C
terminus of MR plus the adjacent highly specific 3' untranslated region
(courtesy of R. M. Evans, The Salk Institute, La Jolla, CA). The antisense GR probe was transcribed from a 500 bp cDNA fragment (courtesy of M. C. Bohn, University of Rochester,
Rochester, NY), subcloned from a 2.8 kb fragment of the rat liver GR
cDNA (courtesy of K. R. Yamamoto, University of California, San
Francisco, CA), and encoded for the N-terminal region of the GR molecule.
For CRH mRNA measurements, a synthetic oligo-encoding rat CRH (48 nucleotides: 64-111) was prepared and 3' end-labeled, using  -[35S]deoxyadenosine triphosphate (specific
activity >1000 Ci/mmol; Amersham, Arlington Heights, IL) and
terminal deoxynucleotidyltransferase (Boehringer Mannheim, Mannheim, Germany).
The c-fos probe consisted of an 860 bp fragment
containing the 3' untranslated portion of the c-DNA (courtesy of T. Curran, St. Jude Children's Research Hospital, Memphis, TN).
| |
In situ hybridization |
Serial coronal sections were cut in a cryostat through the PVN
(12 µm) and hippocampus (12 µm) according to the atlas of Paxinos and Watson (1986). Brain sections were mounted on
poly-L-lysine-coated slides and stored at 80°C until
hybridization. Tissue sections were incubated with 80 µl of
hybridization mix. The hybridization procedures described previously
for adult brain tissue (Nicot, 1996) were used with minor modifications
for the RNA probes in washing steps. Hybridization took place at 45°C
overnight. The next day, sections were rinsed three times in 2× SSC at
room temperature (RT), two times during 15 min in 2× SSC/50%
formamide at 50°C, and then a short dip at 37°C, after which the
sections were subjected to RNase A treatment (20 µg/ml at 37°C as
described previously) to remove nonspecifically bound single-stranded
probes. Then, the sections were washed three times during 15 min in 2×
SSC/50% formamide at 50°C and then in two final washes in 2× SSC at
RT. Dehydration was performed by grading ethanol series, after which the sections were air-dried and exposed for 7-14 d to Kodak X-OMAT AR
film (Eastman Kodak, Rochester, NY) for semiquantification. The control
study consisted of hybridization with the receptor-specific MR, GR, and
c-fos sense cDNA probes. In case the CRH probe was used,
control sections were exposed to an RNase A digestion (40 µg/ml for
30 min at 37°C) before hybridization.
| |
Computer-assisted imaging analysis |
Optical density was quantified with an Olympus (Paes, The
Netherlands), image analysis system equipped with a Cue
CCD camera. The film background was subtracted after shading
correction. Quantification occurred on the basis of a set of
[14C] microscales, which are comparable with
[35S] scales (Amersham). Optical density values
were converted into arbitrary units based on the respective standard
curves. Analysis of MR and GR mRNA was performed on the dorsal
hippocampus (minimum of 10 hippocampal lobi per rat). In the
hypothalamic PVN (minimum of six sections with PVN per rat), between
P1600 µm and P1200 µm posterior from bregma level GR,
c-fos and CRH mRNA levels were measured (Paxinos and
Watson, 1986).
| |
Data analysis |
Data were analyzed by ANOVA procedures (Winer, 1971),
with the level of significance set at p < 0.05. Initial ANOVA tests were performed separately for each age. Once sex
had been determined not to be a significant variable, the data were
collapsed across sexes. When appropriate, post hoc
analyses for simple main and interaction effects were analyzed by
Newman-Keuls or t test procedures. All data are expressed
as mean ± SEM, and significance was accepted at p < 0.05.
Experimental designs
Experiment 1: effects of DEX on HPA axis function
A 4 (conditions) × 5 (treatments) × 2 (gender) design was
used. The conditions were as follows: deprived and nonstressed
(DEP/NT); deprived and stressed (DEP/STRESS); nondeprived and
nonstressed (NDEP/NT); and nondeprived and stressed (NDEP/STRESS). The
treatments were as follows: nontreated (NT); DEX at onset of
deprivation (t = 0 hr) (DEX0); DEX 12 hr into
deprivation period (t = 12 hr) (DEX12); DEX at
t = 0 and 12 hr (DEX0/12); and saline at
t = 0 and 12 hr (SAL). The genders are as follows: male
(M); and female (F). Treatment and gender were intralitter variables.
DEX and SAL injections were administered at the onset of deprivation
and/or 12 hr into the deprivation time for DEP animals. NDEP animals received the injections at the equivalent time but were put back with
the mothers until the time of testing.
Testing procedure. After 24 hr of maternal deprivation or at
the equivalent time for NDEP animals (P12), half of the pups were
decapitated immediately, no stress (NS), which is within a few seconds
after disturbing the pups. The remainder of the pups was injected with
saline (0.9%, i.p.; volume, 0.1 ml/10 gm BW) and placed back in
their home cages on a heating pad set at 30-33°C for 30 min, stress (S).
Injections. A dose of 100 µg/kg body weight (BW) was used
for DEX injections. DEX was dissolved in a saline solution (0.9%, i.p.; volume, 0.1 ml/10 gm BW).
Experiment 2: effects of feeding and stroking on HPA
axis function
Feeding and stroking procedure. The cannulation
procedure and feeding apparatus have been described previously in
detail by Suchecki et al. (1993b). The pups that were stroked and fed
during the time of deprivation were cannulated 24 hr before testing
(i.e., on P11 at the onset of deprivation). After cannulation, the
pups' bladders were manually voided by gently stroking the anogenital area for 30-45 sec or until urination and/or defecation was observed. Another group of pups were only stroked at this time (STROK). All
animals, except for the NDEP group, were placed in the deprivation chamber, and the fed pups were attached to the feeding apparatus. They
were left undisturbed for the next 2 hr, and then the apparatus was
turned on and all animals were left undisturbed until testing time (24 hr after cannulation), except for two bladder-voiding sessions ~7 hr
after cannulation and 2 hr before testing in the DEP, fed and stroked
(F & S), and STROK groups. All animals were weighed at the beginning
and end of the deprivation procedure.
Testing procedure. In this experiment, a 4 (conditions) × 2 (treatments) × 2 (gender) design was used. The conditions were as
follows: nondeprived (NDEP); deprived (ISO); deprived and stroked (STROK); and deprived, stroked, and fed (F & S). The treatments were as
follows: nontreated (NT); and saline 30 min (STRESS). Gender was as
follows: male (M); and female (F). NDEP controls were left undisturbed
with their mothers until testing time (P12). The ISO, STROK, and F & S
litters were deprived for 24 hr on P11, during which time they were
either left undisturbed or manually stroked three times and stroked
plus episodically fed. After 24 hr of deprivation or at the equivalent
time for NDEP animals, half of the pups were decapitated immediately
(NT). The remainder of the pups was injected with saline (0.9%, i.p.;
volume, 0.1 ml/10 gm BW) and placed back in their home cages on a
heating pad set at 30-33°C for 30 min (STRESS).
| |
RESULTS |
Experiment 1: effects of DEX on HPA axis function
ACTH
Main effects for condition, treatment, and an interaction were
obtained by ANOVA (F(3,193) = 31.51;
p < 0.001; F(4,193) = 62.77;
p < 0.001; F(12,193) = 14.62;
p < 0.001, respectively) (Fig.
1). Deprivation induced elevations
of ACTH after stress in the NT and SAL groups (p < 0.01), as has been shown in multiple studies (Rosenfeld et al.,
1992; Suchecki et al., 1993a). Administration of DEX prevented these
stress-induced rises in ACTH. Injection at the onset of deprivation, 12 hr into the deprivation period or at both time points, did show
comparable low levels of basal and stress-induced ACTH.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 1.
Plasma ACTH (picograms per milliliter) and
CORT (micrograms per deciliter) levels in 12-d-old pups, both under
basal condition (NT) and 30 min after a saline injection (STRESS). The
DEP group had been deprived 24 hr before testing (n = 10-12 per group). NDEP animals served as controls. The DEX0 group
had received a dexamethasone injection at the onset of deprivation, the
DEX12 group received an injection 12 hr into the deprivation period,
and the DEX0/12 group received injections at both times (equivalent
time for NDEP) of 100 µg/kg BW. SAL animals had received
saline injections at the equivalent times. *p < 0.05, significant from basal levels in DEP animals;
#p < 0.05, significant from NDEP
animals.
|
|
CORT
The CORT data also showed main effects for condition
(F(3,166) = 79.77; p < 0.001),
treatment (F(4,166) = 90.42; p < 0.001), and an interaction (F(12,166) = 25.27; p < 0.001) (Fig. 1). Deprivation-induced elevations in basal and stress values were obtained from the control groups (NT and SAL) (p < 0.001). Administration
of DEX also prevented these CORT rises, and again the different DEX
treatments showed comparable low levels of basal and stress-induced CORT.
Corticosteroid receptor mRNA expression
A downregulation in GR mRNA expression was observed in the
hippocampal CA1 area of the DEP animals compared with NDEP/NT animals (p < 0.05). DEX injection was not able to
prevent the downregulation in expression in these pups nor was DEX able
to induce effects by itself. Deprivation and/or DEX injection did not
cause changes in any of the other hippocampal regions (Fig.
2).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 2.
Basal MR and GR mRNA expression in different
hippocampal subfields in 12-d-old animals. The DEP group had been
deprived 24 hr before testing (n = 10-12 per
group). NDEP animals served as controls. The DEX animals had received a
dexamethasone injection at the onset of deprivation (or equivalent time
for NDEP) of 100 µg/kg BW. NT animals had received a saline injection
instead of a DEX injection. *p < 0.05, significant
from NDEP counterparts.
|
|
In the hippocampal CA1 area also, a downregulation in MR mRNA was
observed after deprivation (p < 0.05). DEX
injected into the DEP animals resulted in a mRNA level that was not
different from all the other groups. DEX injection was not able to
induce any effects in the NDEP pups (Fig. 2).
The PVN revealed also a decrease in GR mRNA expression after
deprivation (p < 0.05). This effect could be
affected by the DEX administration in DEP animals; DEX administration
resulted in a GR mRNA level that was not significantly different from
any of the other groups (p < 0.05) (Fig.
3).
View larger version (30K):
[in this window]
[in a new window]
|
Figure 3.
Basal CRH stress-induced (30 min after saline
injection) c-fos and basal GR mRNA expression in the PVN
of 12-d-old pups. The DEP group had been deprived 24 hr before testing
(n = 10-12 per group). NDEP animals served as
controls. The DEX animals had received a dexamethasone injection at the
onset of deprivation (or equivalent time for NDEP) of 100 µg/kg BW.
NT animals had received a saline injection instead of a DEX injection.
*p < 0.05, significant from NDEP
counterparts.
|
|
CRH mRNA expression in the PVN
All DEP animals showed a decrease in CRH mRNA expression
(p < 0.05). DEX injection at the onset of
deprivation could not reverse this downregulation. DEX injection in
NDEP animals was not able to induce any significant changes in message
level compared with NT controls (Fig. 3).
C-fos mRNA expression in the PVN
Despite the minimal endocrine response in NDEP rats, we observed a
significant increase in c-fos mRNA levels in the PVN 30 min
after the saline injection. However, stressing the DEP animals 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 (p < 0.01) (Fig. 3).
DEX injection at the onset of the deprivation period did not prevent
the stress-induced rise in c-fos mRNA levels seen after deprivation. In addition, DEX did not induce any effects in NDEP animals.
Experiment 2: effects of feeding and stroking on HPA
axis function
ACTH
ANOVA revealed main effects for condition
(F(3,96) = 13.86; p < 0.001)
and treatment (F(1,96) = 20.81;
p < 0.001) (Fig. 4). In
addition, an interaction was found between condition and treatment (F(3,96) = 17.37; p < 0.001).
As previously reported in multiple studies, 24 hr of maternal
deprivation from P11-P12 results in a significant increase in ACTH
response to a mild stimulus (saline injection) (ISO vs NDEP,
p < 0.001). Stroking the pups during the time of
separation was sufficient to maintain low levels of ACTH. The F & S
group showed comparable levels with the STROK and the NDEP groups. The
basal levels in the different condition groups did not differ.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 4.
Plasma ACTH (picograms per milliliter) and CORT
(micrograms per deciliter) levels in 12-d-old pups, both under basal
condition (NT) and 30 min after a saline injection (STRESS). Litters
were deprived for 24 hr on P11, during which time they were either left
undisturbed (ISO), stroked (STROK), or stroked and episodically fed (F
& S) (n = 10-12 per group). NDEP animals served as
controls. *p < 0.05, significant from NDEP
counterparts.
|
|
CORT
The CORT levels also showed main effects for condition
(F(3,74) = 127.87; p < 0.001)
and treatment (F(1,74) = 92.67;
p < 0.001), as well as an interaction
(F(3,74) = 20.75; p < 0.001)
(Fig. 4).
Also, the CORT levels showed a deprivation effect that has been
reported previously: 24 hr of deprivation results in elevations in
basal and stress-induced CORT levels (NDEP vs ISO, p < 0.001). To maintain low circulating levels of CORT, stroking alone of the deprived animals (ISO) was not sufficient. Feeding the pups during
the time of deprivation prevented the rise in basal and stress-induced
CORT levels (F & S < ISO; p < 0.001).
Corticosteroid receptor mRNA expression
A downregulation in GR mRNA expression was observed in the
PVN of all the DEP animals compared with NDEP controls
(p < 0.05). Stroking and feeding, although
significantly higher than the ISO and STROK groups, could not prevent
completely the GR mRNA levels from decreasing (ISO = STROK < F & S; p < 0.001) (Figs.
5,
6).
View larger version (30K):
[in this window]
[in a new window]
|
Figure 5.
Basal MR and GR mRNA expression in different
hippocampal subfields in 12-d-old animals. Litters were deprived for 24 hr on P11, during which time they were either left undisturbed (ISO),
stroked (STROK), or stroked and episodically fed (F & S)
(n = 10-12 per group). *p < 0.05, significant from NDEP counterparts.
|
|
View larger version (28K):
[in this window]
[in a new window]
|
Figure 6.
Basal CRH stress-induced (30 min after saline
injection) c-fos and basal GR mRNA expression in the PVN of 12-d-old
pups. Litters were deprived for 24 hr on P11, during which time they
were either left undisturbed (ISO), stroked (STROK), or stroked and
episodically fed (F & S) (n = 10-12 per group).
NDEP animals served as controls. *p < 0.05, significant from NDEP counterparts.
|
|
Although feeding and stroking could not completely reverse the
decrease in GR mRNA level in the PVN, these manipulations could prevent
the hippocampal decrease in GR mRNA expression in the CA1 area
(ISO < F & S; p < 0.001) (Fig. 5). Stroking
alone was able to partially reverse this decrease in the CA1 area
(ISO < STROK; p < 0.05), but the expression
level was still significantly lower than in the F & S and NDEP groups
(p < 0.05). The other hippocampal subfields
measured showed no effects.
MR mRNA expression showed a small but significant decrease after
deprivation in the CA1 area of the hippocampus
(p < 0.05) (Fig. 5). This decrease was not
observed in any of the other regions measured. Stroked animals (and
also the F & S animals) no longer revealed differences from the
NDEP rats.
CRH mRNA expression in the PVN
The downregulation in CRH mRNA (p < 0.05)
induced by 24 hr of maternal deprivation was not observed in the
animals that received either stroking alone or feeding and stroking
during the time of deprivation (Figs. 6,
7).
C-fos mRNA expression in the PVN
The marked increase in stress-induced c-fos mRNA
levels after deprivation was completely prevented by stroking the
animals during the time of deprivation. The stroked animals were not
significantly different from the NDEP pups (Figs. 6,
8).
| |
DISCUSSION |
These studies once again showed that mother-reared pups showed
little or no change in the endocrine parameters in response to the
injection of isotonic saline, whereas pups displayed significant elevations of ACTH and CORT after 24 hr of maternal deprivation after
mild stress (Stanton et al., 1988; Rosenfeld et al., 1992; Suchecki et
al., 1993a). The studies also showed that there are consistent changes
in several components of the developing HPA axis. In the experiments
reported here, the deprived pups exhibited the following changes: a
marked stress-induced increase in c-fos mRNA, a reduction of
CRH and GR mRNA in the PVN, and a reduction in GR and MR mRNA in the
CA1 region of the hippocampus. These effects of maternal deprivation
are very robust and have now been demonstrated in various studies
(Vazquez et al., 1996; Smith et al., 1997). What is apparent from the
current studies is that the changes that occur in the brain are not a
function of the elevations of peripheral circulating hormones, but
rather the disinhibition of the peripheral hormone response is more
likely a function of the changes in the brain.
The objective of the current studies was to determine whether the
effects of maternal deprivation were reversible. Our original hypothesis was that the deprivation-induced changes in the brain were a
consequence of the high circulating basal levels of CORT. It was
thought that CORT would act on the developing CNS to transiently and permanently alter those structures that have been shown to be
involved in the regulation of the HPA axis. Numerous studies have
reported that exposure to high levels of CORT have detrimental effects
on the developing brain (Bohn, 1984). To test the hypothesis, we
administered a single injection of DEX at the onset of the deprivation
period. This procedure effectively blocked the basal and stress
elevations of CORT in deprived pups. However, in the absence of CORT
elevations, all neural markers, except for MR, in hippocampal CA1
remained significantly different from mother-reared pups. Recently,
Vazquez et al. (1996) also reported a reduction of MR mRNA after
deprivation, which was attributed to the deprivation-induced elevations
of CORT. It is therefore tempting to speculate that MR, which is
normalized in DEX-treated deprived pups, is one aspect of the
developing brain that is CORT-sensitive. However, the results of the
stroking and feeding experiment failed to provide evidence that
elevations of CORT are solely responsible for the downregulation of MR
mRNA. Thus, the hypothesis was not supported that the mechanism responsible for the brain changes seen in deprived pups was solely dependent on the persistent elevation of CORT.
The purpose of DEX administration was to block the secretion of
endogenous CORT in deprived pups. There were no indications that DEX as
used in these experiments had any effects on gene expression in any of
the transcripts measured in nondeprived pups. There is some debate as
to whether DEX crosses the blood-brain barrier. It has been reported
that DEX poorly penetrates the blood-brain barrier because of
the P-glycoproteins in the blood-brain barrier (Schinkel et al., 1996;
De Kloet, 1997; Meijer et al., 1998). It is likely that the feedback
action of CORT was at the level of the pituitary and that the brain of
both the nondeprived and deprived pups were minimally exposed to DEX.
Manipulations, which were intended to mimic some of the critical
aspects of maternal behavior, were capable of reversing most of the
effects of maternal deprivation. The deprived pups that were stroked
and fed during the deprivation period closely resembled nondeprived
pups. This was the case for the endocrine responses, as well as for
those changes observed in brain. The stroked and fed pups failed to
show elevations in basal and stress-induced ACTH and CORT and further
failed to elicit a c-fos response in PVN. CRH and GR mRNA
were also normalized. Stroking the anogenital region to stimulate
urination and defecation reversed the stress-induced ACTH response,
although the basal and stress-related elevations of CORT were similar
to totally deprived neonates. The c-fos and CRH changes that
occur with deprivation were reversed, as well as the downregulation of
MR mRNA. However, the deprivation-induced downregulation of GR mRNA in
the hippocampus and PVN were still present in stroked pups.
It has been proposed that there are two distinct components of maternal
behavior that regulate different elements of the HPA axis in the
neonate: (1) feeding, which reduces the sensitivity of the adrenal to
ACTH; and (2) stimulation of the anogenital region, which is involved
in the inhibition of ACTH (Suchecki et al., 1993b). It appears that a
similar dual regulatory process also occurs in the brain. Thus,
stroking alone is sufficient in eliminating the effects of maternal
deprivation on CRH and c-fos that occur in the PVN and
hippocampal MR mRNA, whereas the effects on GR mRNA in both hippocampus
and PVN require the addition of feeding. It is difficult to determine
to what extent feeding and stroking interact, because anogenital
stimulation is required to stimulate eliminative functions during this
period of development and is an integral component of the nurturing
activities of the dam. What is clear is that when both components of
the dam's behavior are reinstated the pup is more like a mother-reared
animal and is stress-hyporesponsive. It should be noted that the degree
of stimulation provided to the neonate in these studies is indeed minimal: a total of 135 sec divided into three 45 sec bouts of anogenital stroking. Yet this is sufficient to reverse many of the
effects of maternal deprivation. That somatosensory experiences influence some aspects of cortical development is discussed and demonstrated in reviews by Greenough (1990) and Meszenich (1990). The
process by which maternal tactile stimulation can alter cortical and
subcortical development is unexplored. We have investigated only a few,
albeit key parameters, of the numerous elements that make up the
neuroendocrine regulatory mechanisms of the HPA system. It is difficult
to determine at this time how many other aspects of the developing
brain are dependent on maternally initiated tactile stimulation.
In many ways, the deprived neonate is similar to the adult that is
stress-responsive. ACTH and CORT are elevated, and there is a marked
increase in c-fos mRNA in the PVN (Smith et al., 1997), which has been used as a marker of neuronal activity and has been validated as a marker of increased neuronal activity after exposure to
a variety of stressful events. However, a significant difference is
that in the mature animal ACTH elevations are generally preceded by the
release of CRH from the nerve terminals of the median eminence, which
act on the pituitary corticotrophs. This is followed by a compensatory
increase in CRH mRNA in the PVN (Lightman and Harbuz, 1993; Yi
et al., 1994). In the deprived pup, a downregulation in CRH mRNA seems
to be characteristic after 24 hr of maternal deprivation. This suggests
that perhaps earlier during the deprivation period CRH had been
released and that the neonate does not have the capacity to compensate
either by decreasing CRH mRNA transcription or increasing CRH
degradation. In general, changes in CRH mRNA have been difficult to
elicit in the mother-reared neonates. In one instance, 9-d-old pups did
show an increase in CRH transcription in response to severe cold (Yi et
al., 1994). However, it has also been reported by this same group that
CRH mRNA is decreased in response to a chronic stress and disturbed
mother-infant interactions and more recently that CRH mRNA is reduced
after 10 d of brief disturbances of mother-infant interactions
(handling) (Eghbal-Ahmadi et al., 1997).
In conclusion, we find that 24 hr of maternal deprivation is associated
with changes in the endocrine and neuroendocrine response to stress in
the 12-d-old rat pup. The changes in the peripheral endocrine responses
and in the brain cannot be attributed to the effects of elevated CORT
levels, which are characteristic of maternally deprived neonates.
However, reinstating some of the components of the dam's nurturing
behavior can reverse the effects of maternal deprivation. Stroking and
feeding the pup can reverse almost all of the effects created by
maternal deprivation. Stroking alone also reverses some of the effects
of maternal deprivation. Thus, the increased ACTH response and the
increased c-fos and decreased CRH mRNA are returned to
normal, whereas the elevation of CORT levels and the downregulation of
the GR mRNA are not affected by tactile stimulation.
| |
FOOTNOTES |
Received May 18, 1998; revised Sept. 14, 1998; accepted Sept. 16, 1998.
This work was supported by National Institutes of Mental Health Grant
MH-45006 (to S.L.) and NATO Collaboration Research Grant CRG
97.0477.
Correspondence should be addressed to Dr. S. Levine, Department of
Psychology, University of Delaware, Newark, DE 19716-2577.
| |
REFERENCES |
-
Bohn MC
(1984)
Glucocorticoid induced teratologies of the nervous system.
In: Neurobehavioral teratology (Yanai J,
ed), pp 365-387. Amsterdam: Elsevier Science.
-
Cirulli F,
Gottlieb SL,
Rosenfeld P,
Levine S
(1991)
Maternal factors regulate stress responsiveness in the neonatal rat.
Psychobiology
20:143-152.
-
De Kloet ER
(1997)
Why dexamethasone poorly penetrates in brain.
Stress
2:13-21.[Medline]
-
Eghbal-Ahmadi M, Avishai-Eliner CG, Hatalski AG, Snodgrass SR, Baram
TZ (1997) The corticotropin releasing factor receptor CRF2 is
down-regulated in maternally deprived neonatal rats. 23rd Annual
Meeting of the Society for Neuroscience 23:990.
-
Greenough W
(1990)
Brain storage of information from cutaneous and other modulations in development and adulthood.
In: Touch: the foundation of experience (Katheryn ED,
Barnard E,
Berry Brazelton T,
eds), pp 97-128. Madison, CT: International Universities.
-
Kent S,
Kernahan SD,
Levine S
(1996)
Effects of excitatory amino acids on the hypothalamic-pituitary-adrenal axis of the neonatal rat.
Dev Brain Res
94:1-13[Medline].
-
Kent S,
Tom C,
Levine S
(1997)
Pituitary-adrenal, feeding, and immune responses to interleukin-1
 in the neonatal rat: interaction with maternal deprivation.
Stress
1:213-231.[Medline] -
Levine S,
Berkenbosch F,
Suchecki D,
Tilders FJH
(1994)
Pituitary-adrenal and interleukin-6 responses to recombinant interleukin-1 in neonatal rats.
Psychoneuroendocrinology
19:143-153[Medline].
-
Lightman SL,
Harbuz MS
(1993)
Expression of corticotropin-releasing factor mRNA in response to stress.
In: Corticotropin releasing factor, pp 173-198. Chicester, UK: Wiley.
-
Meijer OC,
De Lange ECM,
Breimer DD,
De Boer AG,
De Kloet ER
(1998)
Penetration of dexamethasone into brain glucocorticoid targets is enhanced in mdr1a P-glycoprotein knock-out mice.
Endocrinology
139:1789-1793[Abstract/Free Full Text].
-
Meszenich M
(1990)
Development and maintenance of corticalsomatosensory representation: functional "maps" and neuroanatomical repertoires.
In: Touch: the foundation of experience (Katheryn ED,
Barnard E,
Berry Brazelton T,
eds), pp 47-72. Madison, CT: International Universities.
-
Nicot A,
Rostène W,
Bérod A
(1995)
Differential neurotensin receptor mRNA expression in dopaminergic cell groups of the rat di- and mesencephalon.
J Neurosci Res
40:667-674[ISI][Medline].
-
Paxinos G,
Watson C
(1986)
In: The rat brain in stereotaxic coordinates. San Diego: Harcourt Brace Jovanovich.
-
Rosenfeld P,
Gutierrez YA,
Martin AM,
Mallett HA,
Alleva E,
Levine S
(1991)
Maternal regulation of the adrenocortical response in pre-weanling rats.
Physiol Behav
50:661-671[Medline].
-
Rosenfeld P,
Suchecki S,
Levine S
(1992)
Multifactorial regulation of the hypothalamic-pituitary-adrenal axis during development.
Neurosci Biobehav Rev
16:553-568[ISI][Medline].
-
Schinkel AH,
Wagenaar E,
Van Deemster L,
Mol CAAM,
Borst P
(1996)
Absence of mdr1a-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxine and cyclosporin A.
J Clin Invest
96:1698-1705.
-
Smith MA,
Kim S-Y,
Van Oers HJJ,
Levine S
(1997)
Maternal deprivation and stress induce immediate early genes in the infant rat brain.
Endocrinology
138:4622-4628[Abstract/Free Full Text].
-
Stanton ME,
Gutierrez YR,
Levine S
(1988)
Maternal deprivation potentiates pituitary-adrenal stress responses in infant rats.
Behav Neurosci
102:692-700[ISI][Medline].
-
Suchecki D,
Mozaffarian D,
Gross G,
Rosenfeld P,
Levine S
(1993a)
Effects of maternal deprivation on the ACTH stress response in the infant rat.
Neuroendocrinology
57:204-212[ISI][Medline].
-
Suchecki D,
Rosenfeld P,
Levine S
(1993b)
Maternal regulation of the hypothalamic-pituitary-adrenal axis in the infant rat: the roles of feeding and stroking.
Dev Brain Res
75:185-192[Medline].
-
Suchecki D,
Nelson DY,
Van Oers H,
Levine S
(1995)
Activation and inhibition of the hypothalamic-pituitary-adrenal axis of the neonatal rat: effects of maternal deprivation.
Psychoneuroendocrinology
20:169-182[ISI][Medline].
-
Vazquez DM,
Van Oers HJJ,
Levine S,
Akil H
(1996)
Regulation of glucocorticoid and mineralocorticoid receptor mRNAs in the hippocampus of the maternally deprived infant rat.
Brain Res
731:79-90[ISI][Medline].
-
Walker CD,
Scribner K,
Cascio CS,
Dallman MF
(1991)
The pituitary-adrenocortical system of neonatal rat is responsive to stress throughout development in a time-dependent and stressor-specific fashion.
Endocrinology
128:1385-1395[Abstract].
-
Widmaier EP
(1989)
Development in rats of the brain-pituitary- adrenal response to hypoglycemia in vivo and in vitro.
Am J Physiol
257:E757-E763[Abstract/Free Full Text].
-
Winer BJ
(1971)
Analysis of covariance.
In: Statistical principles in experimental design (Garmezy N,
Solomon RL,
Jones LV,
Stevenson HW,
eds), pp 752-812. New York: McGraw-Hill.
-
Witek-Janusek L
(1988)
Pituitary-adrenal response to bacterial endotoxin in developing rats.
Am J Physiol
225:E525-E530.
-
Yi S-J,
Baram TZ
(1994)
Corticotropin-releasing hormone mediates the response to cold stress in the neonatal rat without compensatory enhancement of the peptide's gene expression.
Endocrinology
6:2364-2368.
Copyright © 1998 Society for Neuroscience 0270-6474/98/182310171-09$05.00/0
This article has been cited by other articles:
|
|
|
|
 
S J Henare, D J Mellor, R G Lentle, and P J Moughan
An appraisal of the strengths and weaknesses of newborn and juvenile rat models for researching gastrointestinal development
Lab Anim,
July 1, 2008;
42(3):
231 - 245.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V Schmidt, C. Liebl, V. Sterlemann, K. Ganea, J. Hartmann, D. Harbich, S. Alam, and M. B Muller
Neuropeptide Y mediates the initial hypothalamic-pituitary-adrenal response to maternal separation in the neonatal mouse
J. Endocrinol.,
May 1, 2008;
197(2):
421 - 427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Moriceau, D. A. Wilson, S. Levine, and R. M. Sullivan
Dual circuitry for odor-shock conditioning during infancy: corticosterone switches between fear and attraction via amygdala.
J. Neurosci.,
June 21, 2006;
26(25):
6737 - 6748.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Fenoglio, Y. Chen, and T. Z. Baram
Neuroplasticity of the Hypothalamic-Pituitary-Adrenal Axis Early in Life Requires Recurrent Recruitment of Stress-Regulating Brain Regions
J. Neurosci.,
March 1, 2006;
26(9):
2434 - 2442.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Schmidt, S. Levine, M. S. Oitzl, M. van der Mark, M. B. Muller, F. Holsboer, and E. R. de Kloet
Glucocorticoid Receptor Blockade Disinhibits Pituitary-Adrenal Activity during the Stress Hyporesponsive Period of the Mouse
Endocrinology,
March 1, 2005;
146(3):
1458 - 1464.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. van voorhees and A. Scarpa
The Effects of Child Maltreatment on the Hypothalamic-Pituitary-Adrenal Axis
Trauma Violence Abuse,
October 1, 2004;
5(4):
333 - 352.
[Abstract]
[PDF]
|
|
|
|
|
|
|
I. S. Hairston, C. Peyron, D. P. Denning, N. F. Ruby, J. Flores, R. M. Sapolsky, H. C. Heller, and B. F. O'Hara
Sleep Deprivation Effects on Growth Factor Expression in Neonatal Rats: A Potential Role for BDNF in the Mediation of Delta Power
J Neurophysiol,
April 1, 2004;
91(4):
1586 - 1595.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Pryce, D. Ruedi-Bettschen, A. C. Dettling, and J. Feldon
Early Life Stress: Long-Term Physiological Impact in Rodents and Primates
Physiology,
August 1, 2002;
17(4):
150 - 155.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. K. Okimoto, A. Blaus, M. Schmidt, M. K. Gordon, G. W. Dent, and S. Levine
Differential Expression of c-fos and Tyrosine Hydroxylase mRNA in the Adrenal Gland of the Infant Rat: Evidence for an Adrenal Hyporesponsive Period
Endocrinology,
May 1, 2002;
143(5):
1717 - 1725.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. W. Groer, J. Hill, J. E. Wilkinson, and A. Stuart
Effects of Separation and Separation with Supplemental Stroking in BALB/c Infant Mice
Biol Res Nurs,
January 1, 2002;
3(3):
119 - 131.
[Abstract]
[PDF]
|
|
|
|
|
|
|
M. F. Dallman
Editorial: Moments in Time--The Neonatal Rat Hypothalamo-Pituitary-Adrenal Axis
Endocrinology,
May 1, 2000;
141(5):
1590 - 1592.
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. W. Dent, M. A. Smith, and S. Levine
Rapid Induction of Corticotropin-Releasing Hormone Gene Transcription in the Paraventricular Nucleus of the Developing Rat
Endocrinology,
May 1, 2000;
141(5):
1593 - 1598.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Eghbal-Ahmadi, S. Avishai-Eliner, C. G. Hatalski, and T. Z. Baram
Differential Regulation of the Expression of Corticotropin-Releasing Factor Receptor Type 2 (CRF2) in Hypothalamus and Amygdala of the Immature Rat by Sensory Input and Food Intake
J. Neurosci.,
May 15, 1999;
19(10):
3982 - 3991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
