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Brain Research ~~llerin, Vol. 16, pp. 39>398, 1986. o Ankho International Inc. Printed in the U.S.A.

Effects of Prenatal Stress on
Sexually Dimorphic Asymmetries in
the Cerebral Cortex of the Male Rat
DONOVAN
Laboratory

E. FLEMING,’ RICHARD H. ANDERSON, REUBEN
EDWARD KINGHORN AND JAMES BAKAITIS

of Physiological

W. RHEES,

Psychology, Department of Psychology and the Department
Brigham Young University, Provo, UT

Received

of Zoology

26 August 1985

FLEMING, D. E., R. H. ANDERSON,

R. W. RHEES, E. KINGHORN AND J. BAKAITIS. Effects ofprenatalstress on
cortex of the male rat. BRAIN RES BULL 16(3) 395-398, 1986.-Diamond
and collaborators have reported sexual dimorphic right>left thickness asymmetries in the cerebra1 cortices of male
Long-Evans rats. In the present work we report that normal Sprague-Dawley males show a similar cortical asymmetry. On
the other hand, Sprague-Dawley males whose mothers were subjected to treatments of prenatal stress three times daily
during the third trimester of gestation showed a nonsignificant left>right pattern in the same cortical areas-a pattern
characteristic of the female cortex. These results are consistent with other findings from our laboratory wherein we have
recently shown that prenatal stress during the third trimester of gestation demasculinizes sexually dimorphic regions of the
preoptic hypothalamus in male rats. It is concluded that stress mediated changes in the prenatal environment can have a
profound effect on the developmental processes which shape the morphology of sexually dimorphic regions of the brain in
male offspring. Normal male anatomy is biased in the direction of a feminine structure. Such an anatomical picture is
consistent with demasculinized and feminized behavior patterns exhibited by male offspring of prenatally stressed dams.

sexually

dimorphic

Cortical asymmetry

crsymmetries

in the cerebral

Sexual dimorphism

Prenatal stress
-

Left-right cortical hemispheric

differences

might also influence masculine development at the cortical
level. Thus, the purpose of the present investigation was to
verify whether the masculine pattern of cortical asymmetry
reported by Diamond rt nl. for the Long-Evans strain is
present in Sprague-Dawley males, and to determine whether
prenatal stress alters this masculine pattern of cortical
asymmetry. The alteration of masculine cortical morphology
would have implications for changes in masculine sexual behavior also observed in such prenatally stressed animals
[lO,ll].

DIAMOND and collaborators [4, 5, 6, 7, 83 have reported
that there are significant left-right hemispheric differences in
cortical thickness for Long-Evans rats. More recently, she
and her co-workers have presented data which suggest that
these patterns of left-right cortical asymmetry are sexually
dimorphic [4, 7, 81. At 90 and 190 days of age Long-Evans
males show a significant right>left pattern in several cortical
areas, including Kreig’s areas 2, 3,4, 10, 17, 18, and 18a [9].
Females, on the other hand, in general show nonsignificant
left>right differences in the same areas at 90 and 185 days of
age.
We have recently demonstrated
in our laboratory that
prenatal stress during the third trimester of gestation alters
the normal morphology of the medial preoptic area of the
hypothalamus
(MPOA) in male Sprague-Dawley
rats [2].
Prental stress inhibits normal masculine development of the
sexually dimorphic nucleus of the preoptic area (SDN-POA),
and alters cellular densities within other regions of the
MPOA in a feminine direction. Prenatal stress effectively
demasculinizes
and feminizes the MPOA of the male rat
brain.
Given the profound influence of prenatal stress on the
masculine development of sexually dimorphic regions within
the hypothalamus, it appears plausible that such treatment

METHOD
Exprrimcwtcrl

Animals

and Prenatal

Stress

Treatment

Cortical thicknesses
were measured in 21 SpragueDawley male rats, 130-140 days of age at the time of sacrifice. The control animals (N= 11) were selected from the
litters of 8 non-stressed
mothers and the experimental
animals were selected from the litters of 6 prenatally stressed
mothers. The remaining male and female animals from the
two treatment groups were used in other experiments.
Mothers of both control and stressed animals were housed in
Plexiglas maternity cages with room temperature at 24”C,
water and Purina Rat Chow provided ad lib, and maintained

‘Requests for reprints should be addressed
84602.

to Donovan E. Fleming, Department of Psychology,

395

Brigham Young University,

Provo, UT

FLEMING

396

FIG. 1. A coronal section of the rat brain indicating the cortical areas measured, approximating
taken at the level of the decussation of the anterior commissure.

TABLE

ET AL.

Kreig’s areas 4, 3, and 2. This section was

1

MEAN LEFT-RIGHT CORTICAL THICKNESS VALUES FOR CONTROL AND STRESSED MALES*

Kreig’s Areas?
4

3

L
Control Animals (N= 11)
Mean Thickness
% Difference
Significance (p <)
Stressed Animals (N= 10)
Mean Thickness
% Difference
Significance @<)

1.45 It 0.013

R

L

1.51 * 0.013

1.53 + 0.020

4
NS
1.57 * 0.020

2
R

L

1.61 + 0.006

1.61 + 0.027

5
0.01
1.54 & 0.033

2
NS

*Values in millimeters + standard error of the mean.
tApproximations to Kreig’s areas.

1.67 -t 0.027

1.68 2 0.013
4
0.01

1.62 f 0.033
3
NS

R

1.65 + 0.027

1.65 ? 0.033
0
NS

CORTICAL

ASYMMETRY

AND PRENATAL

STRESS

on a reversed 12:12 hour dark/fight cycle (lights were off
from 0800 hours to 2000 hours). In a procedure described
elsewhere [lo], mothers of prenatally stressed males were
subjected to treatments of heat/fight/restraint
stress during
the third trimester of gestation (day 14 of pregnancy to parturition, plug day=day 0). This stress consisted of restraint
three times daily for 45 mitiute periods in 13x6~ 8 cm
semicircular transparent
Plexiglas chambers which were
placed under the illumination of two 150 watt floodlamps
(2150 1m/m2). During a 45 minute session the temperature of
the chamber reached about 31°C. The stress treatment
produced urination and defecation in all animals. The
mothers of control animals remained in the maternity cages
through pregnancy, pa~urition,
and weaning of the offspring.
At day 2.5 postpartum, control and stressed offspring were
sexed and weaned; males were separated and housed singly.
At the time of weaning the identification of both prenatally
stressed and control male offspring were coded so that all
further procedures were performed without knowledge of
treatment group.
Histology

und Cortictrl

Metmrrernmts

At 130-140 days postpartum,
all animals
were
anesthetized with ketamine and perfused through the left
ventricle of the heart under gravity, first with normal saline,
followed by 1% formalin. Equal perfusion pressures were
maintained for all animals. Brains were removed from the
cranium, fixed, embedded in paraffin, and sectioned serially
at 40 /* in the DeCroot plane [3] and stained with thionin.
For each animal, 8 to 12 consecutive anterior to posterior
coronal sections were used for analysis. The initial section
was taken at the level where the anterior commissure crosses
the midline. The cortex at this level encompasses Kreig’s
area 4, which is part of the somatic motor cortex, and areas 3
and 2, which are somatonsensory
regions [9]. Outlines of
brain sections were traced from a microslide-projected
image, using a Leitz microprojector at a magnification of 150x.
Corticai thickness was measured with a millimeter rule directly from the tracings. The measurements extended vertically from the dorsal aspect of layer II to the ventral aspect
of layer VI. To provide a reliable base for measurement, a
template was devised which when laid along the tracing of
the corpus calfosum yielded a series of vertical ordinates that
was consistent from tracing to tracing. In keeping with the
procedures used by Diamond rf rrl. [5,6], layer I was not
measured. Thickness measurements
were taken from the
tracing at 2.5 cm intervals laterally along the cortex, beginning immediately lateral to the medial elevation of the corpus
callosum. These 2.5 cm intervals encompassed
approximately 167 F of brain tissue. Twelve measurements were
made along each cerebrai hemisphere. In the case where an
artifact in the section impeded a clear measurement of cortical thickness, when possible an estimate was made; if a reasonable estimate
could not be made, the section was discarded. A coronal section of the cortical area examined for
thickness with an accompanying depiction of the measuring
sites is displayed in Fig. 1.
Cortical thicknesses for areas 4, 3, and 2 were computed
bilaterally for each animal by averaging measurements taken
within each area (see Fig. I). These values were again averaged to yield mean area thicknesses, bilaterally, for each of
the stressed and control groups. Hemispheric differences in
thickness were then tested for significance using Student’s
t-test for matched pairs.

Control

Stress

FIG. 2. Left and right cortical thickness differences in stressed and
control male rats at 130-140 days of age. Beginning at the midline,
moving left to right, the measurements approximate Kreig’s areas 4,
3, and 2. For stressed rats, N= 10; for control rats, N= 11.

RESULTS

Table 1 and Fig. 2 display left and right hemispheric
thickness differences for control and prenatally stressed
male Sprague-Dawley rats. From Fig. 2 it is clear that cortical thickness differences were noted in control animals in
Kreig’s areas 2,3, and 4. In area 2, t(10)=3.4,p<0.01,
and area
3, r(I0)=3.2, p<O.Ol, the right hemisphere was significantly
thicker than the left; in area 4, the right side was also thicker,
although not significantly so, t(10)=2.1, ~~0.06. The right
side was thicker than the left on 27 of 33 comparisons. The
greatest degree of asymmetry noted was a 5% difference in
area 3.
In contrast to control animals, prenatally stressed males
showed no significant differences in cortical thickness between hemispheres. In area 4, t(9)= 1.9, p<O. 16, and in area
3, t(9)=1.3,
~~0.2, the left side was thicker than the right,
but differences did not achieve statistical significance. In
area 2, the hemisphe~c thicknesses were also found to be
nonsigni~~ant, t(9)=0.12,
p<O.O6. The greatest difference in
thickness observed in prenatally stressed animals was 3% in
area 3.
DISCUSSION

As might be expected, the differences that normally exist
in cortical thicknesses between cerebral hemispheres of male
Long-Evans rats also occur in the male Sprague-Dawley
animal. In addition, this sexually dimorphic feature of the rat
cortex is sensitive to prenatal stress in a manner not unlike
the sexually dimorphic nucleus of the hypothalamic preoptic

FLEMING

398

area. It has been demonstrated in our laboratory [2] that
stress applied to pregnant dams significantly reduces the
volume of the SDN-POA in male offspring as compared with
non-stressed counterparts. In the present investigation, male
offspring of prenatally stressed dams did not display the
normal right>left hemispheric difference in cortical thickness noted in control animals. On the contrary, the differences in cortical thickness between hemispheres were not
statistically reliable.
Examination of Table 1 indicates that, in comparison with
control animals, the thickness of the right hemisphere of
experimental animals was not reduced, but rather, the thickness of the left hemisphere increased. Regarding this observation, in a recent summary of the work of her laboratory,
Diamond [4] indicated that male gonadectomy at postnatal
day 1 reduces the normal male right>left hemisphere differences in cortical thickness, a pattern similar to that of the
female. The female brain normally has a thicker left cortex
than the male. With ovariectomy on day 1, however, the
female pattern actually reverses, in that the animals develop
a male pattern by 90 days of age with a significantly thicker
right cortex. These results would indicate a role for ovarian

ET AL.

hormones in the maintenance
of a symmetrical cortical
thickness.
In light of these observations it would appear that prenatal stress interferes with the normal mascutine organization of the cerebral cortex and produces a neural pattern
approaching that seen in female rats. This is consistent with
our observations of the effects of prenatal stress on the sexually dimorphic nucleus of the preoptic area published
elsewhere [1,2]. A feminized pattern of cortical thickness
following prenatal stress is also consistent with our behavioral investigations which have shown that prenatal stress
both demasculinizes
and feminizes the sexual behavior of
adult male rats [9,10].

ACKNOWLEDGEMENTS

We wish to express our appreciation to Robert E. Seegmiller for
expert photographic assistance. Thanks also to Cathy Paulson for
technical assistance. This work was supported in part by funding
provided by the College of Family, Home, and Social Sciences and
by the Department of Zoology, Brigham Young University.

REFERENCES
1. Anderson, D. K., R. W. Rhees and D. E. Fleming. Effects of

2.

3.
4.

5.

prenatal stress on differentiation of the sexually dimorphic nucleus of the preoptic area (SDN-PGA) of the rat brain. Brain RPS
332: 113-118, 1985.
Anderson, R. H., D. E. Fleming, R. W. Rhees and E. Kinghorn.
Relationships between sexual activity, plasma testosterone, and
the volume of the sexually dimorphic nucleus (SDN-POA) in
prenatally stressed and non-stressed male rats. Bruin Res, in
press.
DeGroot. J. The rat forebrain in sterotaxic coordinates. Verh K
Ned Akod Wet 52: I-40, 1959.
Diamond, M. C. Age, sex, and environmental influences. In:
Cerehrul Dominance: The Biological Foundations,
edited by N.
Geschwind and A. M. Galaburda. Cambridge: Harvard, 1984.
Diamond, M. C., R. E. Johnson, D. Young and S. S. Singh.
Age-related morphologic differences in the rat cerebral cortex
and hippocampus: male-female: right-left. Ewp Neural 81: l-13,
1983.

6. Diamond, M. C., G. A. Dowling and R. E. Johnson. Morphologic cerebral cortical asymmetry in male and female rats.
Exp Neural 71: 261-268, 1981.
7. Diamond, M. C., M. R. Rosenzweig, E. L. Bennett, B. Lindner
and L. Lyon. Effects of environmental enrichment and impoverishment on rat cerebral cortex. J Neurohiol3: 47-64, 1972.
8. Diamond, M. C., D. Krech and M. R. Rosenzweig. The effects
of an enriched environment on the histology of the rat cerebral
cortex. J Camp Neural 123: 11 I-120, 1964.
9. Kreig, W. J. S. Connections of the cerebral cortex. I. The albino
rat. A. Topography of the cortical areas. J Camp Nrlfrol 84:
221-275, 1946.
IO. Rhees, R. W. and D. E. Fleming. Effects of malnutrition, maternal stress, or ACTH injections during pregnancy on sexual
behavior of male offspring. Physiol Behn~~27: 87%882, 1981.
11. Ward, I. L. Prenatal stress feminizes and demasculinizes the
behavior of males. Scic~?c,c,175: 82-84, 1972.


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