Prenatal stress diminishes neurogenesis in the dentate gyrus of juvenile rhesus monkeys.
ABSTRACT Early life stress, including during fetal development, has been hypothesized to predispose individuals to several illnesses and psychiatric disorders later in adulthood.
To determine whether prenatal stress alters neural, hormonal, and behavioral processes in nonhuman primates, pregnant rhesus monkeys were acutely stressed on a daily basis for 25% of their 24-week gestation with an acoustical startle protocol. At 2 to 3 years of age, hippocampal volume, neurogenesis in the dentate gyrus, and cortisol levels were evaluated in the offspring generated from stressed and control pregnancies.
Prenatal stress, both early and late in pregnancy, resulted in a reduced hippocampal volume and an inhibition of neurogenesis in the dentate gyrus. These changes were associated with increased pituitary-adrenal activity, as reflected by higher cortisol levels after a dexamethasone suppression test, and also with behavioral profiles indicative of greater emotionality.
These findings indicate that the prenatal environment can alter behavior, dysregulate neuroendocrine systems, and affect the hippocampal structure of primates in a persistent manner.
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ABSTRACT: The adult central nervous system was thought to be very limited in its regenerative potential; however, the discovery that stem cell populations produce neurons in the adult brain highlights the dynamics of a previously assumed 'static' organ. The continuous generation of new neurons in the adult brain, nevertheless, leads to the question of whether neurogenesis is counterbalanced by an accompanying cell death in the same regions. The objective of this study was to stereologically analyze neurogenesis and programmed cell death in adult brain regions with known neurogenic activity. Using bromodeoxyuridine (BrdU) to identify newborn cells we find that within a few days of BrdU-labeling the adult dentate gyrus and olfactory bulb generate high numbers of newborn neurons. More importantly, dUTP-nick end labeling (TUNEL) reveals that areas of adult neurogenesis also contain high numbers of apoptotic cells. We conclude that programmed cell death may have an important regulatory function by eliminating supernumerous cells from neurogenic regions and may thus contribute to a self-renewal mechanism in the adult mammalian brain.Neuroscience Letters 10/2000; 291(1):17-20. · 2.03 Impact Factor
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ABSTRACT: To investigate whether or not aging of nonhuman primates is accompanied by a region-specific neuron loss in the hippocampal formation, we used the optical fractionator technique to obtain stereological estimates of unilateral neuron numbers of the hippocampi of eight young (0-4 years) and five aged (18-31 years) male rhesus monkeys (Macaca mulatta). Our results show a preservation of neurons (mean x 10 (3)+/-S.D.x10(3)) in the subiculum (young=588+/-124, aged=612+/-207), CA1 (young=1051+/-249, aged=1318+/-311), CA2 (young=100+/-18, aged=113+/-12), CA3 (young=478+/-125, aged=509+/-139), hilus (young=337+/-115, aged=394+/-90), and dentate gyrus (young=5550+/-1725, aged=7799+/-2087) of the hippocampal formation. These results confirm a previous stereological study in rhesus monkeys, but are in conflict with data for humans, showing age-dependent region-specific alterations in the hippocampal formation.Neurobiology of Aging 01/2003; 24(1):157-65. · 6.17 Impact Factor
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ABSTRACT: In animals, exposure to severe stress can damage the hippocampus. Recent human studies show smaller hippocampal volume in individuals with the stress-related psychiatric condition posttraumatic stress disorder (PTSD). Does this represent the neurotoxic effect of trauma, or is smaller hippocampal volume a pre-existing condition that renders the brain more vulnerable to the development of pathological stress responses? In monozygotic twins discordant for trauma exposure, we found evidence that smaller hippocampi indeed constitute a risk factor for the development of stress-related psychopathology. Disorder severity in PTSD patients who were exposed to trauma was negatively correlated with the hippocampal volume of both the patients and the patients' trauma-unexposed identical co-twin. Furthermore, severe PTSD twin pairs-both the trauma-exposed and unexposed members-had significantly smaller hippocampi than non-PTSD pairs.Nature Neuroscience 12/2002; 5(11):1242-7. · 15.25 Impact Factor
PRENATAL STRESS DIMINISHES NEUROGENESIS IN THE
DENTATE GYRUS OF JUVENILE RHESUS MONKEYS
Christopher L. Coe1, Marian Kramer2, Boldizsár Czéh2, Elizabeth Gould3,
Alison J. Reeves3, Clemens Kirschbaum4, and Eberhard Fuchs2
1 Harlow Primate Laboratory, University of Wisconsin, Madison, Wisconsin 53715, USA
2 Division of Neurobiology, German Primate Center, 37077 Göttingen, Germany
3 Department of Psychology, Princeton University, Princeton, New Jersey 08544, USA
4 Department of Psychology, University of Düsseldorf, 40225 Düsseldorf, Germany
Correspondence should be addressed to
Eberhard Fuchs; Division of Neurobiology, German Primate Center
Kellnerweg 4, 37077 Göttingen, Germany
Phone: +49-551-3851 130; Fax: +49-551-3851 307; E-mail: email@example.com
Early life stress may be associated with psychopathology in humans. To determine whether
prenatal stress alters neural, hormonal and behavioral measures in nonhuman primates, preg-
nant rhesus monkeys were stressed using an acoustical startle protocol. At two-three years of
age, the offspring of stressed and control pregnancies were examined for behavior, cortisol
levels, and hippocampal anatomy. Prenatal stress, both early and late in pregnancy, resulted
in reduced hippocampal volume and an inhibition of dentate gyrus neurogenesis. These
changes were associated with higher cortisol levels, a more rapid emergence from dex-
amethasone suppression, lower levels of exploration and higher levels of motor behavior.
These findings indicate that the prenatal environment can alter behavior, dysregulate neu-
roendocrine systems and affect hippocampal structure in a persistent manner.
Keywords: neurogenesis, BrdU, hippocampus, stress, pregnancy, cortisol, infancy, monkey
Running title: Prenatal Stress and Neurogenesis
It has become increasingly evident that the antecedents of many illnesses begin in fetal life,
and further that prenatal conditions can bias us toward either health or disease in the post-
partum period. The potential vulnerability of the fetus to extrinsic factors is most apparent
when teratogenic drugs or fetal alcohol exposure cause congenital malformations in infants,
but even more moderate disturbances of the pregnant female have been found to affect fetal
physiology1,2. For example, several recent studies have confirmed that there is a significantly
increased risk for premature delivery or small-for-date infants when women experience
stressful life events during pregnancy3,4. Even in the absence of overt effects on gestation
length and fetal growth, maternal disturbance in many animals species has been shown to
influence the infant's brain chemistry, endocrine function, emotionality, and learning ability5.
These findings suggest that the fetus is actually not buffered against stressful events experi-
enced by the gravid female, and indicate a need to clarify those in utero conditions upon
which the baby relies to guide its normal development.
Our study was designed to specifically assess one sensitive and critical area of the de-
veloping brain, the hippocampus6,7. Previous research had demonstrated that exposure of the
fetal monkey to just 2 days of dexamethasone (DEX), a synthetic corticosteroid drug, one
month before term, was sufficient to cause massive atrophy of hippocampal cells at birth, and
magnetic resonance imaging showed that the hippocampal volume had still not recovered by
2 years postpartum8,9. Because stressful events activate adrenal hormones to be released,
placental transfer of maternal cortisol has often been considered to be a primary mediator of
disruptions in fetal development, although acute hypoxic episodes and altered fetal metabo-
lism are other likely pathways10,11. After birth the hippocampus becomes an important brain
center that modulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and thus
we hypothesized that monkeys from disturbed pregnancies would evince cortisol hyperactiv-
ity as seen in rats stressed early in life5,12,13. Uno et al.9 had reported that monkeys exposed to
DEX as fetuses exhibited significantly larger cortisol responses to acute stressors postpartum
. Similarly, we had found previously that infant monkeys from stressed pregnancies are
more emotionally reactive, and show immune abnormalities that are associated with altered
For the current study we took advantage of techniques that allow one to assess new
cell growth in the adult brain of vertebrates including primates19-21. We hypothesized that the
ability of certain brain regions to generate new cells during the postpartum period would be
affected by fetal experiences. Lemaire et al.22 had already documented in rats that maternal
stress during the last week of pregnancy affected neurogenesis in the offspring, but this find-
ing had not been replicated in primate infants, nor has anyone previously considered the issue
of the timing of the disturbance during pregnancy. If prenatal stress does, in fact, alter the set
point for cell growth in the hippocampus, it could give credence to many retrospective studies
in humans suggesting that learning disabilities and even mental illness might have their origin
back in fetal life23-32.
Observation of the monkey's behavior indicated that the ones from prenatally disturbed
pregnancies engaged in significantly lower levels of focused exploration [F(2,17) = 4.53; P <
0.05] and tended to exhibit higher levels of non-directed locomotor behavior, such as pacing
(Fig. 1). This finding is in keeping with prior reports of altered emotionality following these
types of gestational manipulations14,17,18,33.
Monkeys generated from the Early Stress and Late Stress pregnancies had significantly
higher cortisol levels than did the Controls [F(2,17) = 8.85; P < 0.05], which was a reflection
of their higher basal levels at 0900 and perhaps some influence of the the 5 min handling
required to collect blood from a conscious animal (Fig. 2a). Twelve hours after DEX ad-
ministration, both the Early Stress and Late Stress monkeys had significantly higher cortisol
levels than did the Controls [F(2,17) = 5.21; P < 0.05], indicating that their HPA axis had
emerged more rapidly from the negative feedback that DEX has on the CNS (Fig 2b).
Hippocampal neurogenesis and volume
BrdUrd-labeled cells were observed predominantly in the subgranular zone and only occa-
sionally in the hilus of the hippocampus. Quantitative analysis of the BrdUrd-labeled cells in
the dentate gyrus revealed that disturbance during either early or late pregnancy resulted in a
dramatic 32% decrease in density relative to the undisturbed Controls (Fig. 3a). The ANOVA
indicated that there was significant difference between the gestation conditions [F(2,9) =
6.85; P < 0.05] and the Tukey’s post hoc comparisons verified that monkeys from both the
Early Stress (P < 0.05) and Late Stress pregnancies (P < 0.05) showed lower neurogenesis
than did Controls. No statistically significant difference was found with regard to the timing
of the maternal stress during pregnancy. A similar pattern was observed in the hilus: both
Early and Late Stress monkeys had a lower density of BrdUrd-positive cells, by 23% and
21% respectively, but those decrements did not reach significance given the low number of
labeled cells in this hippocampal region (data not shown). In keeping with previous studies, 19
the majority of BrdUrd-labeled cells in the dentate gyrus of all animals expressed the neu-
ronal markers TuJ1 or NeuN (Fig. 4).
To characterize the hippocampal volume, cross-sectional surface areas were measured
at 5 representative levels spanning the entire rostro-caudal extent of the hippocampal forma-
tion (Fig. 3b). Comparison of these area measurements indicated that Early and Late Stress
resulted in an average 8-12% decrease across all levels [F(2, 9) = 17.03, P< .001]. Although
this might seem modest, it impacted the entire structure. Tukey’s comparisons indicated that
hippocampal volumes of both Early Stress (P < 0.001) and Late Stress monkeys (P < 0.01)
were smaller than Controls. The timing of the disturbance during pregnancy did not have a
Our findings replicate previous studies in rodents and primates indicating that manipulations
of prenatal conditions can have lasting effects on behavior, hormone activity, and brain func-
tion5,7. Specifically, in the rhesus monkey, it has been shown that disturbance of the gravid
female results in offspring with immature neuromotor reflexes at birth, greater emotionality
as infants, and lymphocyte responses that are still abnormal at 2 years of age14,16,18. By using
the dexamethasone suppression test in the current study, we have now demonstrated that the
experience of maternal stress during pregnancy causes the infants to have a different neural
set point for the HPA axis, because they emerged more quickly from the pharmacological
inhibition of adrenal cortisol induced by DEX. This result concurs with evaluations of adre-
nal function in rats after prenatal stress, and specifically with the effect of prenatal exposure
to DEX on later stress reactivity in monkey infants6,9,34. It also appears to support the hy-
pothesis the HPA hormones are involved in mediating some effects of stress on the fetus2,35.
Many studies have shown that administration of DEX or adrenocorticotrophic hormone
(ACTH) to a gravid female can mimic the effect of nonspecific psychological stressors on the
fetus36. Indeed, the deleterious consequences of transferring too much maternal cortisol to
the fetal compartment appears to be so great that the placenta has developed enzymes to me-
tabolize cortisol into less active forms, such as cortisone, and thereby protect the fetus37.
Nevertheless, subsequent to maternal stress, hormone alterations as well other physiological
changes, including reduced placental blood flow and oxygen availability, appear to be able to
overide this placental buffer and derail fetal development.
There is now a substantial literature indicating that fetal exposure to DEX can have
adverse effects on brain development, which must be weighed against its benefits when used
in the Neonatal Intensive Care Unit to take advantage of its ability to promote rapid matura-
tion of the baby's lungs38. Fetal monkeys administered DEX at doses 5-10 times higher than
used with premature human babies exhibited marked atrophy of hippocampal cells at birth,
and their hippocampi failed to recover to normal size even at 2 years postpartum9. The brain
alterations in the current study are perhaps even more remarkable because a similar hippo-
campal vulnerability was found after psychological disturbance of a relatively moderate na-
ture. It should be emphasized that the disturbance manipulations lasted only 10 minutes per
day, and spanned only 25% of pregnancy. Yet, they were sufficient to alter a fundamental
and important aspect of brain function, the regenerative capacity to grow new cells. In a
previous study of other infant monkeys, we reported that the same type of pregnancy ma-
nipulation also had effects on the size and shape of the corpus callosum39. To this effect on
interhemispheric connections, we now add a reduced set point for neurogenesis and a de-
creased hippocampal volume, which was manifest in both male and female offspring, and
seen equally after stress early and late in pregnancy.
The lack of an effect of gestational timing was somewhat surprising given that there
are very important differences in the neuronal growth within the fetal brain corresponding to
the two periods we selected for the gestational manipulations40,41. Thus, it would suggest that
the lingering effect postpartum was due to an sustained shift in the developmental trajectory
after birth, rather than just mediated by a single, punctate insult before term. Given that we
have evidence of different behavioral responses in the prenatally disturbed infants, alterations
in stress reactivity, and heightened HPA activity, the brain changes probably reflect multiple
influences. It has already been shown that adrenal hormone feedback on the hippocampus can
affect the ongoing rate of neurogenesis in the adult organism42.Moreover, in rodents there
have been several elegant studies demonstrating mother-infant interactions play an important
role in extending the effects of early rearing manipulations12.
Now that evidence for an effect of prenatal disturbance on neurogenesis and hippo-
campal anatomy has been extended from the rodent22 to the nonhuman primate, there is a
compelling need to consider the possible implications for children. Hopefully we benefit
some from the fact that the human brain is more dependent on growth during the postpartum
period; the brain of the neonatal monkey is 60% of adult size, whereas ours is only 24% of
adult size at birth. Thus, there may be more capacity for recovery in children, and a greater
reliance on the rearing environment for creating the right milieu needed for healthy develop-
ment. It is already known, for example, that learning can potentiate neurogenesis in the hip-
pocampus and there can be additional neurogenesis stimulated during the recovery process
after brain damage43,44. However, as a societal concern, we should strive for an optimal
pregnancy environment, followed by a good rearing environment, in order to prevent irre-
versible changes in the set point for neurogenesis, which is currently impossible to correct in
Subjects and housing. Rhesus monkey infants (Macaca mulatta) were generated from 3
types of pregnancies, including a control and two prenatal disturbance conditions. Initially,
20 healthy male and female offspring were selected for the hormone and behavior assess-
ments when they were 2 - 2.5 yr of age, prior to puberty. Then, a smaller subset of 12 were
chosen for the neuroanatomical evaluation because it required euthanasia, including two
males and two females from each rearing condition.
The mothers were laboratory-reared, multiparous females in a long-established
breeding colony. They were time-mated with one adult male during the 4 days around ovu-
lation in order to verify the date of conception for the pregnancy manipulations. The gravid
females were maintained in undisturbed and standardized housing conditions, except on the
acute disturbance days (see below). After birth, the infants were reared normally by the
mother through 7 months of age, and then transferred into small social groups comprised of
4-8 animals from both types of pregnancies. To facilitate the current blood and behavioral
evaluations, they were re-housed as pairs. The husbandry conditions were in accordance with
NIH guidelines for the proper care and treatment of laboratory animals. Commercial monkey
chow was provided daily, fruit was given 3 times per week, and water was available ad libi-
tum. The light:dark schedule was 16:8, with lights on at 0600; ambient room temperature was
maintained at 21 oC. All experimental procedures were approved by the Institutional Animal
Care and Use Committee.
Prenatal Manipulations. Juvenile monkeys from undisturbed, normal pregnancies (Control)
were compared to ones that had been disturbed for 6 weeks during the 24-week pregnancy,
either early or late in gestation. The Early Stress period began on Day 50 post-conception
and lasted until Day 92, whereas Late Stress spanned Days 105-147 (these periods corre-
spond to 2 distinct stages of cell growth and synaptogenesis in the fetal monkey brain40. The
pregnant female was acutely disturbed 5 days per week, M-F, by being moved to a darkened
test room between 1430 and 1600. While located there for 10 min in a small transport cage,
she was intermittently aroused with an acoustical startle protocol (three 1-sec broadcasts of a
horn, randomly at 1-4 min intervals). Prior research using the same paradigm demonstrated
that it significantly elevates maternal cortisol above the normal level for pregnant monkeys,
and can affect the infant's behavioral reactivity and immunity postpartum14-16. Both before
and after the 6-week manipulations, the females lived undisturbed in their home cages until
the natural birth of their infants.
Behavioral characterization. When the offspring were 2.0-2.5 yr of age, each monkey's
behavior was observed and recorded on a lap top computer across 12 5-min sessions. The
mean duration of motoric activity and exploratory behavior was used as an index of emotion-
Hypothalamo-pituitary-adrenal hormone axis. To characterize adrenal hormone secretion,
plasma cortisol levels were determined under 2 conditions: 1) baseline at 0900-1000, which
also incorporated a measure of acute reactivity because it took approximately 5 minutes to
collect blood from the awake monkey, and 2) 12 hr after overnight dexamethasone treatment
at 0900 on the following morning (DEX, dexamethasone sodium phosphate, 0.25 mg/kg,
i.m.). The latter condition should normally inhibit HPA activity, and thus is a measure of the
negative feedback sensitivity of the CNS to corticosteroid hormones. Cortisol was deter-
mined by enzyme linked immunoabsorbent assay (ELISA).
Bromodeoxyuridine injection and immunocytochemistry. At 2.5 - 3.0 yr of age, 12 of the
monkeys were anesthetized with ketamine (15 mg/kg, i.m.) and administered 5-bromo-2’-
deoxyuridine (BrdUrd, 75mg/kg, i.p.) on three consecutive days. Three weeks following the
BrdUrd injections, the animals were perfused under ketamine (20 mg/kg) and deep nembutal
anesthesia (60 mg/kg) with 4.0% paraformaldehyde in 0.1 M phosphate buffer. The 21-day
survival time was chosen to allow for the determination of the phenotype of any newly gen-
erated cells. An oscillating tissue slicer was used to collect 40-µm coronal sections through
the entire hippocampal formation. For quantification of BrdUrd-positive cells, the tissue was
processed immunohistochemically with the peroxidase methods according to our standard
protocol19. In brief, the tissue was first treated by heating in 0.1 M citric acid buffer (pH 6.0),
followed by incubation in hydrogen peroxide, trypsin, and 2 M HCl, and then blocked in
normal horse serum and incubated in mouse monoclonal antibody to BrdUrd (Novocastra,
Newcastle, U.K.; 1:250). These sections were processed using a Vectastain Elite ABC kit
(Vector Laboratories, Burlingame, CA), followed by a brown diaminobenzidine reaction, and
finally counterstained for Nissl by using cresyl violet.
For fluorescence immunocytochemistry, the sections were denatured in 2N HCl for 30
min, rinsed, and incubated overnight at 4 °C in rat monoclonal antibody against BrdUrd
(1:200 + 0.5% Tween-20; Accurate, Westbury, NY). The sections were rinsed and incubated
in biotinylated rat secondary antisera for 60 min (1:200; Chemicon, Temecula, CA), rinsed,
and incubated in streptavidin Alexa-568 (1:500; Molecular Probes, Eugene, OR). Then,
rinsed and incubated in either mouse anti-TuJ1 (1:500; a gift from Dr. Anthony Frankfurter)
or mouse anti-NeuN (1:200, Chemicon) for two days at 4 °C. After several more rinses, sec-
tions were incubated in the appropriate secondary antisera conjugated to Alexa-488 for 30
min (1:500; Molecular Probes). As the final step, rinsed sections were mounted and dried,
then preserved in 25% glycerol in TBS under a cover slip . Tissue stained for fluorescence
was viewed with a BX-60 fluorescent microscope Olympus (New Hyde Park, NY) and also
with a confocal scanning laser microscope (Zeiss Axiovert 510 LSM; lasers: Argon 458/488,
HeNe 543, UV 351/364) for verification of double labeling. Optical stacks of 1 um sections
revealing double-labeled cells were obtained, and the images rotated in orthogonal planes to
verify double labeling.
BrdUrd quantification. Every 15th section (from an average 20 sections per animal) was
examined throughout the antero-posterior extent of the left hippocampal formation. To ensure
objectivity, slides were coded before quantitative analysis, and the code was not broken until
the analysis was completed. All BrdUrd-labeled cells were counted under 400x magnification
regardless of size or shape. The number of BrdUrd-positive cells was assessed in the granule
cell layer (gcl), together with the subgranular zone (sgz, defined as a 2-cell body wide zone
along the border of the granule cell layer) and separately in the hilus. Values are expressed as
densities (number of BrdUrd-labeled cells / 1 mm3 of the granule cell layer).
Hippocampal volume. Cross-sectional surface area measurements of the hippocampus
were made at 5 representative levels spanning the entire rostro-caudal extent of the hippo-
campal formation: at -11, -14, -16, -18, and -21 mm in distance from Bregma45. Briefly, the
hippocampal area (hippocampus proper and dentate gyrus) was outlined using the Neurolu-
cida system (Microbrightfield, Colchester, VT), then the surface areas were calculated. Area
measurements are reported as mm2.
Data analysis. The results are presented as the mean ± SEM. Prenatal treatment effects
were assessed with either one- or two-way analyses of variance (ANOVA), followed by the
Tukey’s or Newman-Keuls post hoc analysis for examination of group differences.
Marian Kramer died unexpectedly as he was finishing these experiments; the manuscript was
written posthumously by his colleagues who mourn the loss of this talented, young scinetist.
M.K. had been the recipient of a fellowship of the Studienstiftung des Deutschen Volkes and
supported by a grant from the G.A. Lienert-Siftung für die Nachwuchsförderung in Biopsy-
chologischer Methodik. Additional research support was provided to E.F. by the Deutsche
Forschungsgemeinschaft (Fu174 /16-1), and a National Institutes of Health grant to C.L.C.
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Exploratory behavior (a) and locomotor activity (b) per 5-min observation session. Time
spent in focused environmental exploration was significantly decreased in both the Early and
Late Stress monkeys as compared to Controls (one-way ANOVA followed by Newman-
Keuls multiple comparison test). Nondirected locomotor activity was exhibited 22% more
often by prenatally disturbed monkeys, a tendency that did not reach significance with this
small sample size. * p < 0.05 significantly different from Controls.
Basal cortisol (a) and levels following an overnight Dexamethasone Suppression Test (DST)
(b). Animals generated from Early and Late Stress pregnancies had significantly elevated
cortisol levels both at baseline and following the DST. Horizontal dashed line indicates the
maximum level of plasma cortisol (5µg/dL) considered abnormal (e.g., failure to stay sup-
pressed). Results are presented as mean ± SEM (µg cortisol / dL). For statistical comparisons
one-way ANOVA was used followed by Tukey`s multiple comparison test. * p < 0.05 sig-
nificantly different from the Controls.
Adult neurogenesis in the hippocampus was quantified using BrdUrd immunohistochemistry
(a). Cell proliferation in the dentate gyrus was significantly lower in monkeys from both
Early and Late Stres pregnancies. Results are the mean density ± SEM (number of BrdUrd-
positive cells / 1 mm3 of the granule cell layer). * p < 0.05 significantly different from un-
treated Controls. Hippocampal volume measurements (b). Five representative sections were
analyzed and indicated that the cross-sectional surface area was significantly smaller in both
Early (p < 0.001) and Late Stress monkeys (p < 0.01). For comparisons one-way ANOVA
was used followed by Tukey`s multiple comparison test.
Confocal images of new neurons in dentate gyrus of the prenataly stressed subadult ma-
caques. Granule cells labeled NeuN (green) and a newly generated neuron labeled with