Maternal stress during pregnancy causes sex-specific alterations in offspring memory performance, social interactions, indices of anxiety, and body mass.
ABSTRACT Prenatal stress (PS) impairs memory function; however, it is not clear whether PS-induced memory deficits are specific to spatial memory, or whether memory is more generally compromised by PS. Here we sought to distinguish between these possibilities by assessing spatial, recognition and contextual memory functions in PS and nonstressed (NS) rodents. We also measured anxiety-related and social behaviors to determine whether our unpredictable PS paradigm generates a behavioral phenotype comparable to previous studies. Female Sprague-Dawley rats were exposed to daily random stress during the last gestational week and behavior tested in adulthood. In males but not females, PS decreased memory for novel objects and novel spatial locations, and facilitated memory for novel object/context pairings. In the elevated zero maze, PS increased anxiety-related behavior only in females. Social behaviors also varied with sex and PS condition. Females showed more anogenital sniffing regardless of stress condition. In contrast, prenatal stress eliminated a male-biased sex difference in nonspecific bodily sniffing by decreasing sniffing in males, and increasing sniffing in females. Finally, PS males but not females gained significantly more weight across adulthood than did NS controls. In summary, these data indicate that PS differentially impacts males and females resulting in sex-specific adult behavioral and bodily phenotypes.
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ABSTRACT: Psychiatric disorders are characterized by sex differences in their prevalence, symptomatology and treatment response. Animal models have been widely employed for the investigation of the neurobiology of such disorders and the discovery of new treatments. However, mostly male animals have been used in preclinical pharmacological studies. In this review, we highlight the need for the inclusion of both males and females in experimental studies aiming at gender-oriented prevention, diagnosis and treatment of psychiatric disorders. We present behavioural findings on sex differences from animal models of depression, anxiety, post-traumatic stress disorder, substance related disorders, obsessive-compulsive disorder, schizophrenia, bipolar disorder, and autism. Moreover, when available, we include studies conducted across different stages of the estrous cycle. By inspection of the relevant literature, it is obvious that robust sex differences exist in models of all psychiatric disorders. However, many times results are conflicting and no clear conclusion regarding the direction of sex differences and the effect of the estrous cycle is drawn. Moreover, there is a lack of considerable amount of studies using psychiatric drugs in both males and females, in order to evaluate the differential response between the two sexes. Notably, while in most cases animal models successfully mimic drug response in both sexes, test parameters and treatment-sensitive behavioural indices are not always the same for male and female rodents. Thus, there is an increasing need to validate animal models for both sexes and use standard procedures across different laboratories.British Journal of Pharmacology 04/2014; · 5.07 Impact Factor
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ABSTRACT: Mothers' self-reported stroking of their infants over the first weeks of life modifies the association between prenatal depression and physiological and emotional reactivity at 7 months, consistent with animal studies of the effects of tactile stimulation. We now investigate whether the effects of maternal stroking persist to 2.5 years. Given animal and human evidence for sex differences in the effects of prenatal stress we compare associations in boys and girls.Psychological medicine. 07/2014;
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ABSTRACT: During gestation, development proceeds at a pace that is unmatched by any other stage of the life cycle. For these reasons the human fetus is particularly susceptible not only to organizing influences, but also to pathogenic disorganizing influences. Growing evidence suggests that exposure to prenatal adversity leads to neurological changes that underlie lifetime risks for mental illness. Beginning early in gestation, males and females show differential developmental trajectories and responses to stress. It is likely that sex-dependent organization of neural circuits during the fetal period influences differential vulnerability to mental health problems. We consider in this review evidence that sexually dimorphic responses to early life stress are linked to two developmental disorders: affective problems (greater female prevalence) and autism spectrum disorder (greater male prevalence). Recent prospective studies illustrating the neurodevelopmental consequences of fetal exposure to stress and stress hormones for males and females are considered here. Plausible biological mechanisms including the role of the sexually differentiated placenta are discussed.Psychoneuroendocrinology 01/2014; 49:11–25. · 5.59 Impact Factor
Maternal stress during pregnancy causes sex-specific alterations in offspring memory
performance, social interactions, indices of anxiety, and body mass
Kalynn M. Schulza,⁎, Jennifer N. Pearsona,c, Eric W. Neeleya,b, Ralph Bergera,b, Sherry Leonarda,b,c,
Catherine E. Adamsa,c, Karen E. Stevensa,c
aDepartment of Psychiatry, University of Colorado Denver, United States
bNeuroscience Program, University of Colorado Denver, United States
cVeterans Affairs Medical Research Center, United States
a b s t r a c ta r t i c l ei n f o
Received 4 October 2010
Received in revised form 18 December 2010
Accepted 10 February 2011
Medial temporal lobe
Prenatal stress (PS) impairs memory function; however, it is not clear whether PS-induced memory deficits
are specific to spatial memory, or whether memory is more generally compromised by PS. Here we sought to
distinguish between these possibilities by assessing spatial, recognition and contextual memory functions in
PS and nonstressed (NS) rodents. We also measured anxiety-related and social behaviors to determine
whether our unpredictable PS paradigm generates a behavioral phenotype comparable to previous studies.
Female Sprague–Dawley rats were exposed to daily random stress during the last gestational week and
behavior tested in adulthood. In males but not females, PS decreased memory for novel objects and novel
spatial locations, and facilitated memory for novel object/context pairings. In the elevated zero maze, PS
increased anxiety-related behavior only in females. Social behaviors also varied with sex and PS condition.
Females showed more anogenital sniffing regardless of stress condition. In contrast, prenatal stress eliminated
a male-biased sex difference in nonspecific bodily sniffing by decreasing sniffing in males, and increasing
sniffing in females. Finally, PS males but not females gained significantly more weight across adulthood than
did NS controls. In summary, these data indicate that PS differentially impacts males and females resulting in
sex-specific adult behavioral and bodily phenotypes.
© 2011 Elsevier Inc. All rights reserved.
Spatial memory ability is diminished in the offspring of dams that
were stressed during pregnancy. For example, prenatally stressed
(PS) rodents show altered performance in spatial memory-dependent
tasks such as the radial arm maze [1,2], T maze  and Morris water
maze [4–13]. Lesion studies demonstrate that spatial memory is
hippocampal based. Given that PS alters many aspects of hippocampal
morphology including glucocorticoid receptor levels, neurogenesis,
dendritic length, spine density, and overall hippocampal volume [13–
16], these stress-induced changes may contribute to the effects of PS
on spatial memory.
Although PS clearly impacts hippocampal-associated spatialmemory
function, the hippocampus is also important for recognition (visual
ability to recognize mismatched object and environmental context
pairings). For example, many studies show impairments in recognition
also depends on the integrity of the hippocampus . Thus, it is
surprising that studies of prenatal stress effects on adult memory have
only one published account on the effects of PS on recognition memory
, and no studies of the effects of PS on contextual memory. The
current study directly compares the effects of PS on recognition, spatial
and contextual memory functions in adult male and female rats. Three
variations of the novel object recognition paradigm were employed to
Physiology & Behavior 104 (2011) 340–347
⁎ Corresponding author at: Department of Psychiatry, RC-1 Room 8101 Mail Stop
8344, 12800 E. 19th Ave., Aurora CO 80045, United States. Tel.: +1 303 724 0591;
fax: +1 303 724 4425.
E-mail address: Kalynn.Schulz@UCDenver.edu (K.M. Schulz).
0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Physiology & Behavior
journal homepage: www.elsevier.com/locate/phb
test these memory types. The primary advantage of modifying the same
memory paradigm to test three different memory types is that levels of
and contextual memory trials . We also measured anxiety-related
and social behaviors to determine whether the unpredictable PS
paradigm employed here generates sex-specific behavioral alterations
comparable to previous literature reports.
2. Materials and methods
2.1. Pregnant dams
Twelve timed-pregnant Sprague–Dawley rats were ordered from
had ad libitum access to food and water. Bedding (Tekfresh, Harlan
Laboratories Inc.,Indianapolis, IN),food (2018 Teklad Global 18% Protein
were changed weekly. One day prior to parturition, the females were
provided as nesting material. Room conditions were maintained at 21 °C
NIH guidelines and all protocols were approved by the IACUC of the
University of Colorado Denver.
2.2. Prenatal stress procedure
Half of the pregnant females were randomly selected to experience
unpredictable variable stress 2–3 times daily during the last week of
gestation (prenatal days 14–21). The stressors were mild in nature and
included restraint in cylindrical restrainers (30 min), swim in water at
roomtemperature(15 min),exposuretoacoldroomat4 °C (6 h),social
received the same schedule of stressors. The remaining 6 females served
as controls and were exposed to only routine animal husbandry.
All pups were born on gestation day 22. Food and water continued
tobe replacedweeklyfollowingparturition, butthebeddingandnests
were left undisturbed until weaning at 22 days of age to minimize
stress [as detailed in 24]. Cage cleanliness was closely monitored
during this time, and additional bedding was provided if necessary.
Upon weaning, weekly cage changing resumed, and animals were
housed 2 per cage with same-sex littermates. It was not feasible to
completely prevent litter effects by using only one representative pup
from each litter , however, we tried to minimize litter effects by
employing only two animals (of each sex) per litter (n=10/group;
2.4. Experimental timeline
days. On the first day animals were exposed only to the test arena,
The toys used for habituation were not used again for testing. Memory
80 days of age, and novel object–context testing (context trials)
occurred at 170 days of age. Additional testing for anxiety-related
behaviors was performed in adulthood at 180 days of age using an
elevated zero maze (San Diego Instruments, San Diego, CA). Social
interaction testing was performed last at 187 days of age. During the
object and spatial trials prenatally stressed animals appeared to be less
anxious than nonstressed controls and spent more time in the center
zone of the testing arena (data not presented here). In order to directly
test this possibility we obtained the elevated zero apparatus, which
necessarily caused a testing delay.
2.5. Behavioral testing
2.5.1. Apparatus and stimuli
Experimentaltests withthe exceptionof theelevatedzerotestwere
performed in arenas constructed of mat black expanded PVC
(70 cm×70 cm;wallheight=47.6 cm).Tofacilitatethespatiallocation
necessary spatial configuration. Testing for anxiety-related behaviors
was performed using an elevated zero maze (San Diego Instruments,
San Diego, CA). The elevated zero maze is constructed of black non-
porous plastic, elevated 30 in. (76.2 cm) off the floor, and is circular in
design (diameter of circle, 121.92 cm; widthofrunway, 20.32 cm) with
adjacent open and closed sections.
Buddies) that varied in shape, texture, and color, but were all similar in
size. Our design utilized nine total unique types of toys, and 4 identical
copies of each toy type were used interchangeably to allow for faster
cleaning between test phases. The types of objects encountered during
trials and whether toys were used as novel or familiar objects was
randomized for each animal. For object trials, the positions of objects
were fixed during exploration and recognition phases, but whether
the recognition phase was counterbalanced for each group. All objects
Specifically, each floor piece had one inverted jar lid super-glued to its
corner. The objects were attached via zip ties to the top of a plastic jar
that screwed securely into place.
All behavioral testing occurred in a dedicated behavior room
during the light phase of the light/dark cycle. Rats were transported
from their housing room into the adjacent behavior room one hour
before testing began. Experimenters were blind to group assignment
and the order in which individual animals were tested was randomized
using a random sequence generator (www.random.org).
220.127.116.11. Object and spatial trials (Figs. 1 and 2). Experimental tests
occurred over two days, but individuals experienced only one trial
type per day. Whether animals first received object or spatial trials
was counterbalanced such that half of each group received object
trials first (day 1), and spatial trials second (day two), or vice versa. To
minimize any effects time of day may have on behavior across a nine
hour testing period, subject position within the testing schedule was
also randomized. Fig. 1 illustrates the testing procedure. A single trial
consisted of 1) an exploration phase, 2) a delay phase, and 3) a
recognition phase. During the exploration phase rats were placed into
the arena and allowed to explore two identical stimuli for 5 min.
Animalsthat explored objectsfor less than 10 s during the exploration
phase were excluded from further analysis of that trial type. During
the delay phase rats were removed from the arena and placed into
their home cages for 1 h. All objects were rinsed with a 70% ethanol
solution, and then with an enzymatic solution designed to break
down biological odors (Nature's Miracle). The test arena walls and
floor panels were also washed with a non-toxic deodorizing solution
(Simply Green). During the recognition phase of object trials, one of
the previously encountered objects was replaced with an unfamiliar
novel object. During the recognition phase of spatial trials the objects
did not change but one object changed spatial locations (Fig. 2).
18.104.22.168. Novel object–context testing (Fig. 1). Novel object–context
testing determines whether animals remember encountering a specific
K.M. Schulz et al. / Physiology & Behavior 104 (2011) 340–347
object in a specific environmental context . Unique environmental
contexts were achieved by adhering different patterns of contact paper
onto the walls of each test chamber. To allow for easy visual
discrimination between environmental contexts, contact paper types
were chosen that varied significantly in pattern style and complexity.
During the exploration phase, animals received 5 min exposure to two
different environmental contexts (5 min/context; Fig. 1). The presen-
tation order of environmental contexts and toys during the exploration
phase was randomized for each animal. Each environmental context
contained two toys that were identical to each other but different from
the toys encountered in the second context. Animals that explored
objects for less than 10 s during the exploration phase were excluded
from further behavioral analysis of the specific trial type. During the
arenas were cleaned as described in the spatial and object trials. During
the recognition phase animals were allowed to explore both previously
encountered toy types within one of the two previous environmental
only one of the objects was previously encountered in that particular
environmental context. Whether the environmental context used for
the recognition phase was the first or second context encountered
during the exploration phase was counterbalanced for each group.
Previous work has demonstrated that rats explore the novel object–
context pairing more than the familiar object–context pairing .
22.214.171.124. Behavioral analyses for memory tests. Previous studies have
, so our analyses were focused on the first 30 s of the recognition
terminated when the familiar object was visited. An investigation bout
was recorded by Topscan when the animal's nose was oriented within at
least 3 cm of an object. Time spent climbing or sitting on toys was not
novel or familiar object were recorded for each animal. From these
measures, the proportion of total visits to the novel toy and proportion of
total time spent investigating novel toys was calculated (novel/novel+
126.96.36.199. Elevated zero. The elevated zero is a test of anxiety similar to
the elevated plus maze, but is circular in shape allowing the rat to
continuously investigate the maze without turning around, thereby
reducing variability in the dataset. The elevated zero maze has been
validated pharmacologically with anti-anxiety drugs , and also
generates anxiety levels comparable to the elevated plus maze .
Similar to the elevated plus maze, the elevated zero maze allows indices
of anxiety to be determined based on the amount of time spent in the
open wall sections vs. the amount of time spent in the closed wall
sections. Time spent in the closed or open sections of the apparatus
took place during the light phase of the dark/light cycle over a two day
period. Animals were only tested once in the elevated zero, and whether
subjects were tested on the first or second day was counterbalanced for
Fig. 1. Schematic depicting the exploration, delay, and recognition phases of spatial, object, and context trials. For spatial and object trials, animals explored two identical objects for
5 min and were removed from the test area for 1 h. During the recognition phase, either the object location (spatial trials) or object type (object trials) was changed. For context
trials, animals sequentially explored two sets of context/object pairings for 5 min/context. The recognition phase occurred 5 min later. Even though all objects and contexts have
been previously explored and are therefore familiar, animals typically show exploratory preferences for the mismatched object/context pairing during the recognition phase. The
presentation order of environmental contexts and toys during the exploration phase was randomized for each animal, and whether the first or second context was used for the
recognition phase was counterbalanced for each group.
Fig. 2. Spatial configurations employed during spatial trials. Three unique spatial
configurations were designed to ensure the spatial arrangements of objects differed
sufficiently between explorationandrecognitionphases.Animalswere randomly assigned
to experience one of the three possible spatial configurations. Shading illustrates which
object changes locations between exploration and recognition phases of testing.
K.M. Schulz et al. / Physiology & Behavior 104 (2011) 340–347
section of the maze and allowed to investigate for a total of five minutes.
The behavior analysis system measured the duration of time spent in
both the open and closed sections and the distance traveled in the maze.
the frequency and duration of sniffing the open sections from inside the
closed sections (hind paws inside the closed section and front paws
inside the open section). From this the bout durations (duration/
frequency) of stretch-attend behavior were calculated.
188.8.131.52. Social interaction. Non-cage mates from the same stress
condition were matched according to sex and weight. Animals were
interact for a total of ten minutes. Upon completion of testing, animals
were removed from the arenas and placed back into their home cages.
The test was recorded using video cameras placed over the test arenas
sniffing of partners. Anogenital sniffing was defined as any instance in
partner's anogenital region located underneath the tail. Non-specific
bodily sniffing was defined as any instance in which the subject's nose
started, if the animal turned or otherwise lost sniffing contact with its
partner, sniffing duration was stopped.
2.6. Body weight measurements
Bodyweights were taken only in adulthood and not at earlier life
stages to minimize stress to animals during development and prior to
behavioral testing. Bodyweights were taken at three time points in
adulthood 1) following open field testing at 82 days of age, 2) following
212 and281 days of age. The age of animals range at thetime of sacrifice
because all animals underwent hippocampal electrophysiological pro-
cedures as part of a separate study. The time of sacrifice was counter-
balanced across all groups to prevent potential age biases in any one
group at sacrifice.
2.7. Statistical analysis
2.7.1. Sample sizes
Animals that spent b10 s total toy investigation during the
exploration phase of memory tests were excluded from further analysis
for spatial trials, 4 animals for object trials, and 2 animals for context
trials. The elevated zero data for 5 animals were lost due to a temporary
technical problem with the video recording.
a paired t-test. Using one-factor ANOVA we assessed the effects of PS on
litter size and the effects of PS on the number of males or females in
litters. Memory tests were analyzed by 3-factor mixed ANOVA treating
sex and stress condition as between subject variables, and toy type
(familiar or novel) as a within subject variable. One sample t-tests were
also employed to assess whether rats discriminated between novel and
familiar objects by comparing the proportion of novel visits and
proportion of time investigating the novel object with what would be
expected by chance (i. e. a ratio of ~0.50). Elevated zero and social
interaction data were analyzed using a two-factor ANOVA treating sex
and stress condition as independent variables (male vs. female, PS vs.
NS). Body weight data were collected over time and therefore analyzed
by 3-factor repeated measures ANOVA. Significant interactions were
probed using Fischer's PLSD. All statistical analyses were conducted
using Statview Version 7. Differences were considered significant when
3.1. Litter size and sex ratio
No differences in litter size were found between PS and NS litters
(PS=13.00±0.47 SEM; NS=13.87±0.47 SEM). Although litters were
male-biased overall [t(1,11)=6.17, pb0.001], prenatal stress had no
influence on the litter sex ratios (PS males=7.83±0.48 SEM; NS
males=8.17±0.48 SEM; and PS females=5.17±0.48 SEM; NS
3.2. Memory tests
3.2.1. Latency to novel spatial locations, object types, and
The latency to visit the novel or familiar object varied by sex and
stress condition for both spatial [Fig. 3A; F(1,28)=5.92, pb0.05] and
object trials [Fig. 3B; F(1,31)=4.03, p=0.05]. NS males were theonly
Fig. 3. Effectsofprenatalstressonlatenciestoinvestigatenovellocations,objecttypes,and
to investigate novel over familiar locations (A) and object types (B), suggesting that
prenatal stress (PS) reduces memory for spatial locations in males. In contrast, PS showed
significantly faster latencies to investigate novel object/context pairings over familiar
object/context pairings, suggesting PS enhances memory for contextual memory in males
in PS and NS females. NS females: spatial n=9; object n=8; context n=9. PS females:
spatial n=8; object n=10; context n=10. NS males: spatial n=6; object n=9; context
n=9.PSmales: spatial n=9;objectn=8;context n=9.Dataexpressed asmean±SEM.
Asterisk (*) indicates a significant difference (pb0.05) between groups.
K.M. Schulz et al. / Physiology & Behavior 104 (2011) 340–347
the familiar objects [spatial, F(1,28)=5.36, pb0.05; object, F(1,31)=
4.53, pb0.05]. For context trials a trend toward an interaction between
stress condition and toy type (novel or familiar object/context pairing)
was detected [Fig. 3C; F(1,33)=3.81, p=0.05]. Surprisingly, stressed
animals showed significantly faster latencies to the novel than to the
familiar object/context pairings [F(1,33)=10.98, pb0.01]. However,
when stressed males and females were analyzed separately, only the
stressed males showed significantly faster latencies to investigate the
novel object/context pairings over the familiar object/context pairings
[males, F(1,33)=9.52, pb0.01; females F(1,33)=2.56, pN0.05].
3.2.2. Proportion of visits and time spent investigating novel locations,
object types, and object/context pairings
For spatial trials, sex and condition interacted to influence the
proportionof visitstothenovellocation(Fig.4A),and theproportionof
time (Fig. 4B) investigating the novel object locations [% novel visits, F
(1,27)=7.44, pb0.01;% novel duration,[F(1,27)=5.12, pb0.05].Stress
significantly reduced the proportion of visits and time investigating
novel locations in males [% novel visits, F(1,27)=9.92, pb0.01;% novel
duration, F(1,27)=4.00, pb0.05]. No differences were found between
displayed above chance preferences for the novel spatial locations
[Fig. 4A, % novel visits, t=3.11, pb0.05; Fig. 4B, % novel duration,
t=2.43, pb0.05]. No significant effects of sex or stress condition were
found for object or context trials. However, one group t-tests indicated
novel objects [Fig. 4C, % novel visits, t=1.90, pb0.05]. No other groups
trials (Fig. 4E and F).
3.3. Elevated zero
The bout durations (duration/frequency) of stretch-attend risk-
arms) varied depending on sex and condition [Fig. 5A; Interaction: F
(1,31)=4.01, p=0.05]. When we compared the behavior of males and
females separately, each stretch-attendbout wassignificantly longer for
PSfemales thanNS females [Fig. 5A;F(1,31)=4.28, pb0.05],suggesting
increased anxiety. In contrast, no significantdifferences between PS and
NS males were found in stretch-attend bout durations. The duration of
time spent in the open walls [Fig. 5B; F(1,31)=5.00, pb0.05] and
distance traveled [Fig. 5C; F(1,31)=3.95, p=0.05] in the open arms of
the elevated zero followed a similar pattern of interaction. Specifically,
PSfemales spentsignificantly less time [5B; F(1,31)=6.89, pb0.05] and
traveled shorter distances [5C; F(1,31)=4.40, pb0.05] in the open arm
than NS females, whereas these behaviors were unaffected by PS in
3.4. Social interaction
overall [Fig. 6A and B; F(1,36)=12.22, pb0.002]. No effects of stress
condition or interactions between stress condition and sex were found,
but anogenital sniffing significantly decreased across the 10 min test [F
(1,144)=7.72, pb0.0001]. In contrast to female-biased anogenital
sniffing, males engaged in more nonspecific sniffing than females
[Fig. 6C and D; F(1,36)=3.84, p=0.05]. Sex also interacted with stress
condition [Interaction: F(1,36)=4.32, pb0.05]. Post-hoc analyses
indicated that NS males sniffed partners more thannonstressed females
(Fig. 6C), whereas PS males and females did not differ in nonspecific
found for nonspecific sniffing, such that sniffing generally decreased
across the 10 min test [Fig. 6; F(1,144)=11.80, pb0.0001].
3.5. Body weight
Sex and condition interacted to influence body weight [Fig. 7;
F(1,36)=6.20, pb0.02]. Specifically, stress significantly increased
body weight in males [F(1,36)=11.30, pb0.004] but not females
[F(1,36)=0.004 p=0.96]. Agealsosignificantlyinfluencedbodyweight
[F(1,72)=465.6, pb0.0001], and age and sex significantly interacted to
affect body weight [F(1,72)=97.58 pb0.0001]. Post-hoc analyses
indicated significant weight gains at each age tested, and for both sexes
(82 days, 168 days, and the final weights ranging from 212 to 281 days).
However, males displayed proportionally greater gains than females
between 168 days of age and the final weight [males: 168 vs. final
weight=pb0.0001; females: 168 vs. final weight=pb0.02].
Fig. 4. Effects of prenatal stress on novelty preferences during spatial, object, and context trials. For spatial trials prenatal stress (PS) significantly reduced the % of visits (A) and %
time investigating (B) novel locations in males but not females. PS did not significantly affect the % of visits or % time investigating objects during object or context trials, however,
nonstressed males displayed above chance preferences for novelty during object trials (†). NS females: spatial n=9; object n=8; context n=9. PS females: spatial n=8; object
n=10; context n=10. NS males: spatial n=6; object n=9; context n=9. PS males: spatial n=9; object n=8; context n=9. Data expressed as mean±SEM. Asterisk (*) indicates
a significant difference (pb0.05) between groups. Cross (†) indicates an above chance preference for novelty (N50%) in that group.
K.M. Schulz et al. / Physiology & Behavior 104 (2011) 340–347
hippocampal-associated memory function in rats. Three types of
memory were tested using spatial, recognition, and contextual variants
of the novel object recognition task . PS impaired memory for spatial
locations and novel object recognition in males, but modestly facilitated
memory for object/context associations in males. In contrast, PS did not
affect female performance on these measures. These findings suggest
that PS impacts hippocampal-associated forms of memory, but this
relationship depends on the sex of animals and the specific type of
memory being tested.
Our findings corroborate and extend previous work investigating the
effects of stress during gestation on the memory performance of adult
performance in males [2,4,5,7,10–13,23]. However, to our knowledge,
this is the first demonstration of prenatal stress-induced spatial memory
deficits using the novel object spatial location paradigm. The strength of
using variants of the novel object recognition paradigm is that spatial,
recognition, and contextual memory functions can be compared while
controlling for the amount of training, handling, and the levels of stress
PS-induced spatial memory deficits using the Morris water maze [4–13],
findings by also testing recognition and contextual memory functions in
the same animals. Another report found no effect of PS on recognition
memory . In this study rats were acclimated to the behavioral arena
Fig. 5. Investigatory behavior in the elevated zero maze. (A) Prenatally stressed (PS)
females displayed longer bout durations of stretch-attend (risk assessment) than did
nonstressed (NS) females. No differences in stretch-attend bout durations were
observed between PS and NS males. (B) PS females spent less time in the open walls of
the elevated zero than NS females, whereas no differences were observed between PS
and NS males. (C) PS females traveled less in the open walls than did NS females,
whereas no difference in distance traveled in open arms was found between PS and NS
males. NS females, n=9; stressed PS females, n=9; NS males, n=9; PS males, n=8.
Data expressed as mean±SEM. Asterisk (*) indicates a significant difference (pb0. 05)
Fig. 6. Anogenital and nonspecific bodily sniffing of same-sex and -stress condition
partners across a 10 min test. (A and B) Females engaged in more anogenital sniffing of
partners than males, regardless of stress condition (histograms illustrate main effect of
sex; prenatal stress = PS; nonstressed = NS). (C) Males engaged in more nonspecific
sniffing of partners than did females, but only in the NS condition (histogram illustrates
main effect of sex). (D) No sex differences in nonspecific sniffing were observed in
prenatally stressed animals. Both anogenital and nonspecific sniffing decreased
significantly across the 10-min test. NS females, n=10; PS females, n=10; NS males,
n=10; PS males, n=10. Data expressed as mean±SEM. Asterisk (*) indicates a
significant difference (pb0.05) between groups.
Fig. 7. Effects of prenatal stress on body weight across adulthood. (A) Prenatal stress (PS)
significantly increased the body weight of males but not females, and PSmales gained the
most weight across time. (B) Photo of representative PS (top) and nonstressed (NS,
bottom) malesfrom thisexperiment. Note thatthe bodyweights of PS and NS femalesare
completely overlapping,so the symbols for the NS female groupswereshifted to the right
to allow forvisualinspection.NS females, n=10;PSfemales,n=10;NSmales,n=10;PS
(pb0.05) between groups.
K.M. Schulz et al. / Physiology & Behavior 104 (2011) 340–347
and received 4 novel object testing sessions before data collection
occurred. In contrast, our rats were acclimated to the behavioral arena
and were not repeatedly tested in any given trial-type (object, spatial, or
context). Thus, it is possible that testing experiences mitigate the initial
negative effects of PS on recognition memory. Additional experiments
manipulating both stress and testing experience will help determine
whether these factors interact to influence recognition memory.
PS also impacted contextual memory function. Surprisingly, prena-
to investigate novelobject/context pairings, suggesting that PS improves
latencies to investigate novel object/context pairings, the proportion of
visits and time spent with the novel object/context did not significantly
differ between PS and NS males. Thus, while these initial findings are
intriguing, experimental replication will help to determine the extent to
which PS affects contextual memory.
Prenatal stress only affected memory performance in males.
However, even NS females did not show a preference for novel objects.
The reason for this is not clear, especially since females showed definite
novelty preferences in our pilot experiments (data not presented here).
One possibility is that the female subjects were in the low-estrogen and
-progesterone estrous cycle phase diestrus when memory performance
is decreased relative to proestrus and estrus phases [31–33]. This
possibility is only speculative given that we did not track estrous cycle
phase. While most studies investigating the effects of prenatal stress on
behavior have not controlled for estrous cycle phase [but see 15,34,35],
our future experiments will account for potential estrous cycle phase
effects on memory performance.
Given that spatial, recognition, and contextual memory functions are
recognition memory functions while facilitating contextual memory. An
interesting possibility is that different subcomponents of the hippocam-
pus are important for different hippocampal-associated memory types.
For example, studies have found that adult adrenalectomy results in a
selective loss of cells in the dentate gyrus [36,37]. When spatial,
recognition, and contextual memory functions were assessed following
gyrus development differently than other hippocampal subregions,
leading to dissociations in hippocampal-associated memory functions
While the effects of PS on memory function were male-biased, PS
preferentially affected the anxiety-related behaviors of females. Speci-
fically, PS decreased the time females spent in the open arms of the
elevatedzeromaze,andsignificantlyincreased female boutdurationsof
stretch-attend behavior (i. e. risk assessment), suggesting that PS
females were more anxious than controls. These data are in agreement
with previous studies demonstrating PS preferentially increases female
anxiety-related behavior in the elevated plus maze. Richardson et al.,
theopen arm of the elevated plus maze. Similarly, PS female Wistar
rats showed increased anxiety-related behavior when tested in the
females supports previous research, and validates the elevated zero for
use in PS animals. It should be noted, however, that other studies
employing the elevated plus maze have found anxiety increases in both
male and female PS rats [39,40]. These differences between studies may
reflect differences in the implementation of the conditions surrounding
theelevated plus mazetestsuch as lighting conditionsduringtesting,or
possibly differences in maze configuration (circle vs. plus).
Social interactions were affected by sex and stress condition in a
behavior-specific manner. Females engaged in more anogenital sniffing
more nonspecific bodily sniffing than females. However, bodily sniffing
was only male-biased in NS animals. PS eliminated the male-biased sex
difference by decreasing nonspecific bodily sniffing in males, and
increasing bodily sniffing in females. Thus, while previous studies have
rats [41,42], here we extend those findings by demonstrating that PS
increases female social investigation.Interestingly, PS females displayed
increased anxiety-related behavior in the elevated zero yet displayed
increased bodily sniffing of partners, suggesting that the effects of PS on
anxiety-related behavior do not extend to social interactions. Likewise,
nonspecific bodily sniffing in PS males is not simply attributable to
and social interactions suggest that altered social behaviors are not
secondary to PS-induced changes in the neural circuitry regulating
anxiety, but rather, PS may independently organize neural circuits
underlying the motivation to engage in social interactions.
Exposure to gestational stress also influenced body mass in a sex-
specific manner. Specifically, PS significantly increased the bodyweight
fact, PS males were on average 100 g larger than NS males. These data
and female rats . Although PS did not increase female body weights
in the fat content of the post-weaning diet. Tamashiro et al.  fed
animals a high fat diet at weaning (60% kcal from fat), whereas our
animals were fed a standard rodent diet at weaning (17% of kcal from
fat). The species of rodent is another important factor mediating the
effects of gestational stress on bodyweight. Stress experienced early in
gestation decreases bodyweight and adiposity in male and female
C57Bl/6:129 mice . Taken together, these studies highlight the
complexity of the relationships between gestational stress, species, sex,
Animal models of human disorders linked to the prenatal period
are necessary for the development of appropriate preventions and
treatments for conditions such as schizophrenia, anxiety, depression,
autism, and obesity. These disorders are also highly gender-specific in
the incidence and/or expression of symptoms. Although PS in rodents
generally induces behavioral and bodily phenotypes analogous to
those found in these human conditions, determining the nature and
direction of stress-induced sex differences has proved difficult. The
rodent species, strain, timing of stress and testing environment have
all been linked to the sex-specific expression of behavior, but the
direction ofsex-specific changesvariesbystudy [45,46]. Inthecurrent
study, PS induced female anxiety-related behavior in the elevated
zero maze. This finding maps nicely onto female-biased disorders
such as depression and anxiety in humans.In addition, the PS-induced
social deficits in male rats may be analogous to the male-biased social
withdrawal experienced in conditions such as schizophrenia and
autism [47–50]. In contrast, the stress-induced and male-specific
increases in bodyweight did not recapitulate the female-biased
incidence of obesity in the general population . However, whether
PS affects obesity in a gender-dependent fashion in humans is not yet
known. Future studies focused on the mechanisms by which PS
induces these bodily and behavioral phenotypes may help elucidate
themechanisms bywhichPSincreasessusceptibilityto disorderswith
etiological links to the prenatal period.
This work was supported by T32 MH15442-30, MH073725 to KES,
MH081177, DA09457 and Veterans Affairs Merit Review to SL.
We thank Drs. Kristin Wildeboer-Andrud, Randy Ross, Heather
Richardson, and Kaliris Salas-Ramirez for the critical feedback on the
early drafts of this manuscript. We also thank Joan Yonchek for the
valuabletechnicalassistance, andDr. Michael Hallfor constructing the
testing chambers for memory testing.
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