Behavioural Brain Research 177 (2007) 117125

Research report

Influence of gender on working and spatial memory in

the novel object recognition task in the rat
J.S. Sutcliffe, K.M. Marshall, J.C. Neill
School of Pharmacy, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK
Received 18 August 2006; received in revised form 26 October 2006; accepted 30 October 2006
Available online 22 November 2006

Abstract
Gender differences in many behavioural tasks have been observed in both humans and laboratory animals. The novel object recognition (NOR)
task is increasingly used to investigate drug effects on working memory processes, although, the influence of sexually dimorphic behaviours have
not yet been evaluated. In addition, the role of natural fluctuations in the sex steroids during the oestrous cycle has received little attention during
object recognition tasks. Therefore, the aim of the current study was to investigate the influence of gender and oestrous cycle phase on working and
spatial memory using the NOR task. Animals were tested in the NOR task and the spatial NOR task. Male and female rats completed an acquisition
trial followed by an inter-trial interval of a specified length, then a final retention trial. Vaginal cytology enabled the influence of oestrous cycle
phase to be determined in both the NOR and spatial NOR, each animal was tested during one phase of their regular oestrous cycle only. It was
found that female rats performed significantly better than male rats in the standard NOR paradigm (p < 0.05 compared to no significance (NS) at
3 h, respectively), while male rats showed improved memory in the spatial NOR paradigm compared with female rats (p < 0.05 compared to NS
at 3 h, respectively). There was no influence of phase of oestrous cycle on the NOR task, however, during the spatial NOR there was a significant
improvement in ability when oestrogen and progesterone levels have been shown to be at their lowest (i.e. p < 0.05 during oestrous compared to
NS at other stages). In conclusion, it is clear that gonadal hormones can influence components of memory and gender is an important consideration
in experimental design.
2006 Elsevier B.V. All rights reserved.

Keywords: Spatial memory; Novel object recognition; Cognition; Gender; Rat; Oestrous cycle

1. Introduction Animals typically respond to environmental changes by prefer-

A wide range of behavioural tests are available to model
human cognitive function and these tasks may be chosen for
the particular aspect of cognition under study, for example, spa-
tial and working memory or reversal learning. The novel object
recognition (NOR) task for rodents is a non-spatial, non-aversive
memory test [11] which involves the substitution of a familiar
object with a novel object in a memory retention trial. However,
the NOR task can be adapted for assessment of spatial memory,
in this task the identical objects are not replaced with a novel
object but are moved to a new position following a particular
inter-trial interval [11]. These tasks are ethologically relevant
as they rely on the animals natural exploratory behavioural
repertoire and avoid the confounding influence of reinforcement.
Corresponding author. Tel.: +44 1274 234677; fax: +44 1274 234660.
E-mail address: J.C.Neill@bradford.ac.uk (J.C. Neill).
ential exploration of novel or moved objects over those that are
familiar; it is these preferences that indicate the formation of
memories regarding the location and identity of objects. Due to
the relative simplicity and ethological relevance of this task, it is
increasingly used as a powerful experimental tool to assess drug
effects on cognition and to investigate the neural mechanisms
underlying working and spatial memory [7,20,45].
Ovarian steroid action in the brain is not limited to repro-
ductive neuroendocrinology and behaviour. Recent studies
in rodents and humans have shown that the status of both
the adrenal and gonadal axes (hypothalamicpituitaryadrenal
(HPA) and hypothalamicpituitarygonadal (HPG), respec-
tively) can influence learning and memory strategies and capac-
ities [for review see 3, 4 and 8]. Research during the past decade
confirms that gonadal steroids have the ability to influence the
structural properties of the brain regions that sub-serve learning
and memory. The majority of research has investigated the effect
of oestrogen on cognitive function and oestrogen receptors have

0166-4328/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2006.10.029
118 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125
been found in several areas of the brain including the amygdala,
cerebral cortex, cerebellum and hippocampus [41,47]. Of par-
ticular interest to the current investigation is the hippocampus
which is known to play a significant role in working and spatial
memory [26] and which contains both forms of the oestrogen
receptor (ER) -ER and ER [3,4,17]. Other areas within the
brain which are fast becoming associated with mnemonic pro-
cesses include the frontal cortex and the striatum and it has
become clear that there are strong interconnections between
the hippocampus and frontal cortex [49]. In the frontal cortex
of male and female rats, each ER isoform has been shown to
display its own unique, selective distribution [32,60] the most
predominant being ER with only a small number of ER con-
taining cells [42]. Given the diverse actions of oestrogen, these
differences will undoubtedly affect cognitive processes between
the genders. Oestrogens, androgens and progestagens have been
shown to alter hippocampal electrophysiology; studies suggest
that oestrogen is excitatory whilst progesterone and its metabo-
lites are inhibitory, the function of androgens is less clear [43].
Thus, the hippocampus has been shown to display different
structural changes in response to different gonadal hormones.
Hippocampal plasticity alters across the oestrous cycle of the
rat in response to cyclic changes in oestrogen and progesterone
[3,26,5759]. During the pro-oestrous phase, when steroid levels
are at their peak [56], the number and density of CA1 pyramidal
cell spine synapses reach a maximum, in contrast, during vagi-
nal oestrous when hormones are at low levels, a 30% decline in
synaptic density and number is observed [3,58,59]. These cycli-
cal changes could induce significant behavioural changes which
may be detected using animal tests.
These mechanisms provide some biological foundation for
the complex effects of ovarian steroids on cognitive function
in various species, including humans [9,23,30,31]. What is
emerging from recent studies is that oestrogen, and perhaps
progesterone and its metabolites such as allopregnanolone, can
influence learning and memory in a task-dependent manner.
In women, oestrogen levels peak twice during the menstrual
cycle, the first peak (which is unopposed by progesterone) occurs
just prior to ovulation and the second peak is in the mid-luteal
phase when progesterone levels are also elevated. Kimura and
Hampson [23,29] have successfully demonstrated that women
are negatively affected in verbal skills and muscular coordina-
tion tasks when their oestrogen levels are high compared to the
early follicular phase (when all ovarian steroids are at a mini-
mum) when male oriented spatial tasks are favoured. In contrast,
female orientated tasks such as verbal fluency and motor skills
have been shown to be enhanced prior to ovulation when oestro-
gen action is unopposed by progesterone [28]. Clinical research
has been directed at the potential of ovarian steroids to facili-
tate the storage of new memories and to provide protection for
neuronal circuits in debilitating disease states. Although con-
troversial, some studies have shown that oestrogen supplemen-
tation/replacement may be beneficial, with respect to aspects
of cognitive function, in Alzheimers disease [1], menopausal
women [3,38,40] and schizophrenia [5,33].
Sex differences in cognition are often viewed as a product
of evolution in which women and mens roles differ, hence
their problem solving abilities became distinctive and comple-
mentary. Indeed, one evolutionary scenario suggests a female
foraging hypothesis; females should be better than males at
remembering the identity of objects [10,25] which is particu-
larly relevant to the NOR paradigm. One of the few studies that
has attempted to characterise gender differences and behavioural
responses to spatial and object novelty used adult C57BL/6J
mice [16]. It was found that males were superior to females in
remembering the identity and location of objects in an open field
after a 24 h and 7 day retention period, contradicting the female
foraging hypothesis but in agreement with the view that males
excel in spatial tasks. However, it must be noted in this instance
that the females will be in a different stage of oestrous during the
retention period due to the short oestrous cycle in rodents and this
may have had confounding effects on the findings. A more recent
study [50] used the NOR paradigm to successfully demonstrate
a positive effect of oestrogen administration on object recog-
nition in ovariectomised rats and during behavioural oestrous
(pro-oestrus) compared to other stages of the cycle in hormon-
ally intact rats. The aim of the current study was to investigate
the influence of gender and oestrous cycle phase on working
and spatial memory in the NOR paradigm. Object recognition
should be deceased as the inter-trial interval increases for both
genders as essentially, the task gets harder. Females are expected
to display novel object recognition over a longer inter-trial inter-
val than males due to the presence of ovarian steroids and their
effects on neurotransmitter systems, however, ovarian steroids
have been proven to be detrimental to spatial memory tasks so,
in the spatial NOR task it is assumed males will excel. It is
further hypothesised that females will demonstrate variable per-
formance in spatial and working memory tasks, which may be
related to changes in the hippocampal structure in response to
natural cyclic hormonal fluctuations.
2. Methods
2.1. Subjects
All experiments were carried out using mature male and female experimen-
tally nave, hooded Lister (hL) rats (Harlan, Bicester, UK) weighing 373 17 g
and 234 17 g, respectively at the onset of the studies. Animals were housed
in groups of five (in cages measuring 38 cm 59 cm 24 cm) with cardboard
tubes (Datesand Ltd, Manchester, UK) for environmental enrichment as stan-
dard practice. Animals were maintained under standard laboratory conditions
on a 12 h light:12 h dark cycle (lights on at 07:00 h) in a temperature controlled
room at 21 2 C, with humidity of 4050%. All experimental procedures were
carried out in the light phase. Rats had free access to food (standard laboratory
chow, Special Diet Services, Essex, UK) and water. Experiments were carried
out in accordance with the Animals Scientific Procedures Act (1986) and were
approved by the University of Bradford Ethical Review Process.
2.2. Apparatus
The novel object arena used in the current study consisted of an open box
(52 cm wide by 40 cm high by 52 cm long) with black Perspex sides, a white
Perspex base and dividing black grid lines for locomotor activity (LMA) assess-
ment [11]. No additional illumination was placed on the box and the box was
placed in the experimental room in such a position where no shadows fell. The
objects to be discriminated were available in quadruplicate, held no natural sig-
nificance to the rats and had never been associated with a reinforcer. They were
made of a biologically inert substance (glass, plastic, metal, ceramic or wood)
 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125
and were chosen to enable ease of cleaning (10% alcohol) between subjects in
an attempt to remove olfactory cues. For each subject, the role and position of
the objects was counterbalanced and randomly changed to reduce any effects of
object and place preference. Each animal only completed one test in the novel
or spatial object task and was not used for further tests to ensure that all animals
were experimentally nave.
2.3. Experimental procedure
Animals were handled daily for 1 week and habituated to the test box for
3 days prior to the initial test for a period of 30 min each time, as it has been
suggested that when a rat is initially placed in an open environment it will take
longer to explore a novel stimulus or the middle of the arena [39]. On the day of
testing, the animals were given an additional 3 min re-habituation period prior
to commencing the test. The NOR and spatial NOR tests are divided into three
phases with two trials, the acquisition trial, the retention trial and an inter-trial
interval of varying times.
Acquisition trial: in this first trial the animals explored two identical objects
(A1 and A2) for a period of 3 min.
Inter-trial interval (ITI): the animals were returned to the home cage for 3 min,
30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 24 h or 48 h.
Retention trial: in this second trial, the animals explored a familiar object
(A) that is a duplicate of those objects from the acquisition trial (to minimise
olfactory cues) and a novel object (B) for a further 3 min. In the case of the
spatial NOR task copies of the same objects from the acquisition trial were
presented to the animal, however, one object occupied a novel position within
the arena.
Object exploration is defined by animals licking, sniffing or touching the
object whilst sniffing but not leaning against, turning round, standing or sitting
on the object. Objects were of sufficient weight and were secured to the floor
of the arena to ensure that they could not be knocked over or moved around by
the animal. The acquisition and retention trials were video taped for subsequent
scoring of object exploration by an individual blind to the experimental con-
dition. The exploration times (s) of all objects was recorded via stopwatch for
subsequent statistical analysis. LMA was assessed by counting the number of
times the animal crossed the black grid lines during the acquisition and retention
trial, the line was only considered crossed when the base of the rats tail had
passed over the line.
2.3.1. Experiment 1: effect of phase of oestrous cycle in the NOR and
spatial NOR task
Vaginal smears were taken daily between 09:00 h and 11:00 h. This involved
the insertion of 0.2 ml of 0.9% (w/v) saline solution into the vagina by means of
a thin plastic pipette. Histological examination of the vaginal lavage was carried
out by light microscopy [12,15,52]. All animals used in the study displayed a
regular 4-day cycle for at least 2 weeks prior to starting the experiment. Each
sample was classified as being in one of the following phases: pro-oestrus,
oestrus, di-oestrus (early) or di-oestrus (late) and animals (n = 24) were tested
in the NOR and the spatial NOR task. Results were grouped according to stage
of the cycle (n = 6 per stage). Acquisition and retention trials were conducted as
previously described but with a 1 h inter-trial interval (since object recognition
in both NOR and the spatial NOR is maintained by females at this time point,
experiments 2 and 3).
2.3.2. Experiment 2: gender effects in the NOR task
A total of 54 male and 54 female animals were tested following the procedure
described in Section 2.3. Two identical objects were presented in the 3 min
acquisition trial followed by an ITI of 3 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 24 h
or 48 h, and finally the 3 min retention trial where both objects were replaced by
a duplicate familiar object and a novel object, six animals of each gender were
tested per inter-trial interval.
2.3.3. Experiment 3: gender effects in the spatial NOR task
This experiment followed the same procedure as the NOR task (described
in Section 2.3) with a 3 min acquisition trial followed by an ITI of 3 min to 48 h
119
before the 3 min retention trial. During this experiment, 54 new male and 54
new female animals were used. The objects presented in the retention trial were
identical copies of those investigated during the acquisition trial; only one of
the objects occupied a new position in the arena with respect to the previous
trial (moved object). It must be noted that in each trial for each experiment 13,
new, experimentally nave animals were used. That is so experimentally nave
animals were assessed at one of the inter-trial intervals only, or, in the case of
females, were tested during one phase of the oestrous cycle only.
2.4. Statistical analysis
Differences in object exploration for both trials for all group means were
tested for significance initially by two-way ANOVA using objects and ITI as
set variables. A paired t-test was used to evaluate any statistical difference
between exploration for each object, in each trial, for each experimental con-
dition. One-way ANOVA followed by post hoc Dunnetts t-test was used to
determine discrimination index (DI) and LMA differences for each ITI, gender
and stage of the oestrous cycle in each experiment.
2.4.1. Discrimination index (DI)
This value is the time spent exploring the novel/moved object minus the time
spent exploring the familiar/stationary object, divided by the total time spent on
all object exploration in the retention trial. A value below zero describes sub-
jects exploring the familiar/stationary object more than the novel/moved object.
A value above zero describes animals exploring the novel/moved object more
than the familiar/stationary object. Use of the DI allows gender comparisons
for each inter-trial interval tested and for the discrimination between objects
following different inter-trial intervals. Statistical evaluations were made using
SPSS for Windows (v13.0). Results were considered significant when p < 0.05
and confidence intervals were set at 95%.
3. Results
3.1. Experiment 1: effect of phase of oestrous cycle in the
NOR and spatial NOR task
3.1.1. Working memory
Overall two-way ANOVA revealed no significant effect of
objects during the acquisition trial (F1,3 = 2.32, p = 0.14) or
object versus stage of cycle (F3,23 = 0.79, p = 0.51) for natu-
rally cycling female rats during the acquisition phase of the
NOR paradigm (data not shown). During the retention trial,
there was a significant difference in novel object exploration
(F1,3 = 27.16, p < 0.001) but no effect of stage of the oestrous
cycle (F3,23 = 0.29, p = 0.83). Paired samples t-test showed that
female rats recognised the novel object following a retention
period of one hour at all stages of the oestrous cycle (i.e. the rats
spent significantly more time exploring the novel compared to
familiar object, p < 0.050.01, Fig. 1). Exploration time of the
novel object at pro-oestrus and oestrous was higher compared
with early and late di-oestrus, although no significant effect was
observed following one-way ANOVA (F3,23 = 1.11, p = 0.37).
A one-way ANOVA revealed no significant effect of stage of
oestrous cycle on DI values (F3,23 = 0.05, p = 0.98) for object
recognition as assessed by the NOR paradigm (Table 1). There
were no significant differences in LMA during each stage of the
oestrous cycle (data not shown, F3,23 = 2.03, p = 0.121).
3.1.2. Spatial memory
Overall two-way ANOVA revealed no significant effect of
objects during the acquisition trial (F1,3 = 3.00, p = 0.10) or
120 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125

Fig. 2. Exploration times of mature female hL rats (n = 6 per group) during dif-
ferent stages of the oestrous cycle in the retention trial of the adapted NOR task
Fig. 1. Exploration times of mature female hL rats (n = 6 per group) during
for spatial working memory, where two familiar objects are presented to the
different stages of the oestrous cycle in the retention trial of the NOR task, where
subjects, one occupying a novel position with respect to the previous acquisition
one familiar and one novel object are presented to the subjects. D = di-oestrus
trial. D = di-oestrus (early) and (late), P = pro-oestrus and O = oestrus. Signif-
(early) and (late), P = pro-oestrus and O = oestrus. Significant discrimination
icant moved object preference was observed following Students paired t-test
with preference for the novel object was observed following paired samples t-
conducted for each group during vaginal oestrous (* p < 0.05 moved object com-
test conducted for each group (* p < 0.05, ** p < 0.001 novel compared to familiar
pared to stationary object exploration, data are expressed as the mean S.E.M.).
object exploration, data are expressed as the mean S.E.M.).

During the retention trial, when a novel object replaced one of

object versus stage of the cycle (F3,23 = 0.42, p = 0.73) in cycling
female rats during the acquisition phase of the adapted NOR
paradigm (data not shown). During the retention trial, there was
a significant difference in novel object exploration (F1,3 = 11.13,
p < 0.05) but the phase of the cycle had no significant effect
(F3,23 = 0.77, p = 0.38). Paired samples t-test indicated a sig-
nificant discrimination for the moved object during oestrus
(p < 0.05) but not at any other stage of the cycle (Fig. 2). A
one-way ANOVA revealed no significant effect of stage of the
oestrous cycle on DI values (F3,23 = 0.67, p = 0.58) for spatial
memory as assessed by the adapted NOR paradigm (Table 1).
There were no significant differences in LMA during each stage
of the oestrous cycle (data not shown, F3,23 = 2.61, p = 0.075).
3.2. Experiment 2: gender effects in the NOR task
Overall, a two-way ANOVA revealed no significant effect of
gender on exploration of the identical objects in the acquisition
trial (F1,8 = 0.63, p = 0.43 and F1,8 = 1.77, p = 0.19) and no effect
of object exploration with regard to ITI (F8,45 = 0.44, p = 0.89
and F8,45 = 0.96, p = 0.48). When each group was analysed sep-
arately by paired samples t-test there were no significant differ-
ences in the exploration of two identical objects presented in the
acquisition trial regardless of gender or ITI (data not shown).
the familiar objects, a two-way ANOVA indicated a significant
discrimination between objects for both males (F1,8 = 21.59,
p < 0.001) and females (F1,8 = 50.95, p < 0.001). There was
also a significant effect of ITI for both male and female rats
(F8,45 = 6.64, p < 0.001 and F8,45 = 4.67, p < 0.001, respectively).
Paired samples t-test showed that female rats were able to dis-
criminate the familiar from the novel object following ITIs of
3 min, 0.5 h, 1 h, 2 h and 3 h (p < 0.050.01). This is shown in
Fig. 3a as a significant increase in time spent exploring the novel
versus the familiar object. In marked contrast, the only statisti-
cally significant discrimination between the novel and familiar
object in males occurred at 3 min and 30 min (p < 0.01 and 0.001,
respectively, Fig. 3b).
A one-way ANOVA revealed no significant differences in
DI (Table 2) when comparing the different ITIs for female
object exploration (F8,53 = 1.50, p = 0.19), however, there was
a significant effect of length of ITI on male exploration
(F8,53 = 7.20, p < 0.001). Post hoc Dunnetts t-test showed a sig-
nificant increase in DI following ITIs of 3 (p < 0.05) and 30 min
Table 2
Discrimination index values obtained during the retention period of the NOR
task for both male and female groups (n = 6 per group per gender)
Inter-trial interval (h) Females Males

0.05 0.19 0.26 0.33 0.08*

0.5 0.39 0.05 0.50 0.08**
Table 1 1 0.21 0.07 0.03 0.07
Discrimination index values observed during each stage of the oestrous cycle in 2 0.30 0.08a 0.05 0.06
the NOR task for working and spatial memory (n = 6 per group) 3 0.32 0.14a 0.08 0.09
DI values D (early) D (late) P O 4 0.01 0.04 0.03 0.02
for 5 0.01 0.05 0.05 0.06
24 0.18 0.06 0.11 0.08
Working 0.46 0.24 0.39 0.21 0.43 0.47 0.45 0.31 48 0.02 0.09 0.01 0.04
memory
Spatial 0.12 0.10 0.02 0.07 0.003 0.12 0.14 0.08 Data are expressed as the mean S.E.M. and were analysed using one-way
memory ANOVA. * p < 0.05 and ** p < 0.01 at that ITI compared to the 48 h ITI for that
gender.
D = di-oestrus (early) and (late), P = pro-oestrus and O = oestrus. Data are a p < 0.05 significant difference in DI between males and females at that ITI

expressed as the mean S.E.M. (n = 6 per group). following one-way ANOVA.

 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125
Fig. 3. (a) Exploration times of mature female hL rats during the retention trial
in the NOR task, where one familiar and one novel object are presented to
the subjects. * p < 0.05 and ** p < 0.01 represent significant differences between
the time spent exploring the familiar and the novel object and (b) exploration
times of mature male hL rats during the retention trial of the novel object
task, where one identical and one novel object are presented to the subjects.
** p < 0.01, *** p < 0.001 represent significant differences between the time spent
exploring the familiar compared to the novel object. Data are expressed as the
mean S.E.M. and were analysed at each time point using paired samples t-
tests (n = 6).
(p < 0.001) when compared to the 48 h ITI. One-way ANOVA
shows a significant decrease in novel object recognition assessed
by the DI value for male animals when compared to females
at ITIs of 2 h (F1,10 = 7.35, p < 0.05) and 3 h (F1,10 = 13.48,
p < 0.05, Table 2). One-way ANOVA indicated no significant
difference in LMA (F8,45 = 1.72, p = 0.12) for males and females
(F8,45 = 1.40, p = 0.22).
3.3. Experiment 3: gender effects in the spatial NOR task
Overall two-way ANOVA revealed no significant effect of
objects during the acquisition trial (F1,8 = 0.33, p = 0.57) or
object versus ITI (F1,8 = 0.20, p = 0.99) for male rats during
the acquisition phase, however, during the retention trial there
was a significant difference in object exploration (F8,45 = 105.04,
p < 0.001) and objects versus ITI (F8,45 = 101.13, p < 0.001) in
male exploration, suggesting discrimination for the moved
object. This effect was also evident in the female rats, where
there was no significant effect of objects (F1,8 = 2.34, p = 0.13)
or objects and ITI (F8,45 = 1.50, p = 0.18) during the acquisi-
tion trial, but, during the retention trial there was a significant
discrimination between objects (F1,8 = 13.39, p < 0.01) and an
effect of the ITI (F8,45 = 3.37, p = 0.01). Paired samples t-test
indicated no significant differences in the exploration of two
identical objects presented in the acquisition trial regardless of
gender or ITI experienced (data not shown) when analysed by
paired samples t-test. During the retention trial both males and
121
Fig. 4. (a). Exploration times of mature (a) female hL rats (n = 6 per group)
during the retention trial of the spatial task, where two identical objects are pre-
sented to the subjects but one of the objects has moved position with respect to
the acquisition trial. ** p < 0.01 represents significant moved object preference
compared to the stationary object and (b) exploration times of mature male hL
rats (n = 6 per group) during the retention trial of the spatial task. ** p < 0.01 rep-
resents significant discrimination between the moved object and the stationary
object. Data are shown as the mean S.E.M. and were analysed at each time
point using paired samples students t-tests (n = 6).
females exhibited significant differences in spatial exploration.
Male animals showed a significant preference for the moved
object up to a period of 3 h with respect to the stationary object
(p < 0.01; Fig. 4b), in contrast female animals only showed a
preference for the moved object at 1 h (p < 0.01; Fig. 4a) follow-
ing paired samples t-test.
A one-way ANOVA revealed a significant effect of length
of ITI on the DI value during the NOR test adapted for spatial
memory in females (F8,53 = 3.62, p < 0.05), however, post hoc
Dunnetts t-test revealed no significant differences. In males,
a significant effect of length of ITI on the DI was observed
(F8,53 = 3.62, p < 0.01) and significant differences in DI at 30 min
(p < 0.05) and 1 h (p < 0.01) ITI compared to 48 h ITI (Table 3).
One-way ANOVA showed a significant decrease in discrimina-
tion for the moved object in females at ITIs of 1 h (F1,10 = 8.69),
2 h (F1,10 = 28.7), 3 h (F1,10 = 6.4) and 4 h (F1,10 = 4.4) ITI when
compared to the DI obtained for males (p < 0.05, Table 3).
One-way ANOVA indicated no significant difference in LMA
for males and females (F8,45 = 1.38, p = 0.23) and (F8,45 = 1.41,
p = 0.22), respectively.
4. Discussion
The present study used the novelty of exploration to investi-
gate the impact of gender differences and oestrous cycle phase in
a single strain of laboratory rat, the hooded Lister, using a sim-
ple cognitive task. The hypothesis, that both gender and stage of
oestrous cycle will affect cognitive performance in the rat, was
based on neurophysiological and behavioural data suggesting
122 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125
Table 3
Discrimination Index values for mature male and female hL rats (n = 6) during the
retention trial in the spatial NOR task, where two identical objects are presented,
however, one occupies a new position in the NOR arena
Inter-trial interval (h) DI females DI males
0.05 0.10 0.11 0.35
0.5 0.19 0.10 0.38
1 0.28 0.04a 0.44
2 0.14 0.05a 0.20
3 0.11 0.05a 0.30
4 0.006 0.06a 0.20
5 0.11 0.07 0.10
24 0.07 0.07 0.10
48 0.09 0.04 0.15
Data are shown as the mean S.E.M. * p < 0.05 and ** p < 0.01 at that ITI com-
pared to the 48 h ITI for that gender.
a p < 0.05 significant difference in DI between males and females at that ITI
following one-way ANOVA.
that sex hormones will modify memory retention in rodents. All
the tests took place in an open field and were based on the nat-
ural behaviours and spontaneity of animals exploring novelty.
These results demonstrate that performance in the NOR and
the spatial NOR vary significantly between the sexes in hooded
Lister rats of the same age (i.e. young mature animals). There
were several novel findings in this study: females show improved
object recognition memory compared to males as assessed by
the NOR task over longer retention periods (up to 3 h compared
with 30 min in males). Males showed better spatial recognition
than females in the spatial NOR task (up to 3 h compared with
1 h in females). Performance of females in the NOR task was
unaffected by stage of oestrous cycle when tested after a 1 h
inter-trial interval; spatial memory over the oestrous cycle was
shown to be significantly better during vaginal oestrus than dur-
ing other stages of the cycle, when tested after a 1 h retention
period.
Experiment one was designed to evaluate the sensitivity of
the NOR and spatial NOR to detect subtle changes in hormone
levels across the oestrous cycle in the female rat. During the
standard NOR female rats were able to significantly identify the
novel object assessed at each stage of the oestrous cycle follow-
ing a period of 1 h after first exposure in the acquisition trial
(Fig. 1). When the spatial NOR paradigm was used females in
di-oestrus (late and early) and pro-oestrus, there was no signif-
icant discrimination for the moved object suggesting that the
elevation of both oestrogen and progesterone [52] is detrimen-
tal to object location memory and it has been found that task
performance is slower during pro-oestrous [44]. A significant
discrimination for the moved object was only observed during
the oestrous phase by which time oestradiol levels have declined
and progesterone levels are also decreasing rapidly (Fig. 2), sug-
gesting that oestrogen and/or progesterone may play a role in
object location memory. The finding from experiment one is both
in agreement and contrast with previously published findings.
Walf et al. [50] have recently demonstrated a significant effect of
the oestrous cycle during the NOR in female Long-Evans rats.
Female animals were tested in the NOR during different stages
of the oestrous cycle and it was found that during behavioural
oestrus (pro-oestrus) there was significant object discrimination
compared to other stages of the cycle when tested 4 h post-
training, suggesting beneficial effects of the observed oestrogen
peak at pro-oestrus. Results from our study do not confirm their
findings. This may be due to a different inter-trial interval (4 h
0.07 compared with 1 h) essentially making the task harder in their
0.06* study. Experiment 2 in the present study confirms that female
0.04** rats can successfully identify a novel object following an inter-
0.04
0.08
trial interval of 3 h but not 4 h. It may be that increased oestrogen
0.06 levels during pro-oestrus enables object recognition beyond 3 h.
0.06 However, no attempt was made to identify stage of the oestrous
0.08 cycle during this particular experiment, although no oestrous
0.06 cycle effects were found following a 1 h inter-trial interval in
Experiment 1; subsequent studies are required to clarify this
issue. In the Walf [50] study, the rats were only 55 days old at
the start of the study, the weight at time of experimentation was
not stated, although biochemical parameters were measured. It
is important to only use animals with a regular oestrous cycle
to avoid endocrinological anomalies. Our laboratory has shown
(unpublished observations) that animals under 200220 g have
irregular oestrous cycles. Walf [50] present their data as a per-
centage and grouped late and early di-oestrus as one measure
only which makes comparisons to the present study more diffi-
cult. In addition, the exploration time in seconds is low compared
to those in this study. Further studies would clarify these discrep-
ancies. Another study, Frye [17], found that sex differences in
water maze tasks were limited to differences between females in
oestrous and males, but not when the females were in di-oestrus
(i.e. when oestrogen levels are at their lowest and progesterone
is elevated) suggesting that oestrogen has an inhibitory effect on
spatial memory in such tasks, thus supporting the spatial differ-
ences found in this study.
Laboratory studies resemble human studies in women, where
some researchers have found differences in cognitive function
over the stage of the menstrual cycle [25,29] but others have
found no significant differences [24]. Oestrous cycle differ-
ences may be difficult to measure because the sensitivity of the
behavioural measure may be insufficient to detect the subtle
differences in hormones and so brain-behaviour relationships
pose a difficult problem. It must also be remembered that, if
behavioural responses are subject to hormonal influence via clas-
sical genomically mediated steroid receptor pathways then there
will be a significant lag time between serum level changes and
target response.
Much research has focused on manipulating gonadal
hormone levels in female rodents either by testing at different
stages throughout the oestrous cycle or through ovariectomy
and subsequent exogenous hormone replacement at both
physiological and supraphysiological levels [35,36]. Although
a gonadal hormone treatment in gonadectomised animals may
dramatically modify a particular response it cannot be assumed
that this itself is a robust gender difference (i.e. there is no real
evidence that this effect could happen in an intact animal due
to the often complex interactions between genomic and non-
genomic actions of hormones, in addition to the homeostatic
influence of feedback control) [27]. One major advantage of
the NOR task is that it is based purely on working memory
 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125
and the task depends on natural behaviours as opposed to any
reinforcement or deprivation (i.e. no reward or punishment). In
fact, the majority of previous studies have used food deprivation,
often to 90% of free feeding body weight (to ensure performance
in various mazes) which may complicate the interpretation of
results, since such practices can affect the oestrous cycle, food
consumption and dietary preference [55]. Additionally, the
Morris water maze may be regarded as stressful, so that, true sex
differences cannot be attributed solely to gonadal hormones as
corticosteroid levels will increase which may play a significant
role in learning and memory. It is well established that stress and
arousal affect performance on learning tasks [46]. The stress
response as measured by corticosterone and ACTH following
restraint stress varies across the oestrous cycle. Thus, at pro-
oestrus, when oestrogen levels are at their maximum, there is
an elevated stress response compared to during di-oestrus. This
may affect performance in cognitive tasks (no major changes at
pro-oestrus were seen in this study). Such stress responses were
avoided in the current study by regular handling and adequate
habituation. In addition, it was noted that no animals defecated
during any of the trials, again strengthening the hypothesis that
the rats were not under any undue stress.
Experiment 2 was aimed at investigating sex differences over
increasing inter-trial intervals in the NOR paradigm. We were
able to show a clear sexual dimorphism, where females were
able to significantly discriminate the novel object up to a period
of 3 h post-training (Fig. 3a), in contrast, male rats were unable
to identify the novel object from the familiar object after an
ITI of 30 min (Fig. 3b). The novel object recognition task is a
relatively new paradigm to assess cognitive variables, and thus
there is a lack of information available supporting sexual dimor-
phism in this task, although this paradigm is currently subject
to much research. One study which attempts to define gender
differences in a similar object recognition task used C57BL/6J
mice [16]; however, results disagree with those presented in this
study where male mice indicated a preference for the novel
object compared to the females after a 24 h inter-trial interval
but not at a 7-day inter-trial delay. Shorter inter-trial intervals
between the sexes were not studied and therefore it is possi-
ble that some differences due to a shorter delay in trials were
missed when comparing the effect of gender in mice. Addi-
tionally, the female mice would be in a different stage of their
oestrous cycle during the retention period compared to during
the initial phase, which is true of the longer ITIs in this study. In
another study [19], using a different object recognition task to
evaluate male and female differences in Wistar rats, inter-trial
intervals of 30, 60, 90 and 120 min were used. The results from
our study are partly in agreement with those shown in that study;
males could not identify the novel object after 1 h where females
could remember up to 90 min. During our object recognition task
the females were shown to remember the familiar object up to
3 h (180 min) following the initial acquisition trial and although
the results obtained by Ghi et al. [19] cannot be directly com-
pared to the NOR task, since the radial maze task was used which
may be influenced by the progressive learning of the task, this
study is still supportive of our results. Consistent with our pre-
clinical observations, human studies have shown that females
123
are superior to males in object array tasks [34,37], suggesting
that men encode less information about specific visual features.
The majority of the literature, however, focuses on differences
between the sexes for object location memory/spatial memory,
which have also been tested in this study.
Experiment 3 was designed to evaluate gender differences
in the spatial NOR paradigm. This task, like the standard NOR
paradigm, is not dependent on reinforcement and is purely based
on natural exploratory behaviours of rats. Instead of a novel
object presented in the retention trial, copies of both objects
were presented in the retention trial; the only difference being
that one object is occupying a different position with respect
to the previous trial. Female rats were only able to identify the
moved object after an inter-trial period of 1 h (Fig. 4a) in con-
trast male rats identified the moved object up to an ITI of
3 h (Fig. 4b). These findings support previous laboratory work
where a clear sexual dimorphism in spatial working memory
is seen in both humans and other laboratory animals [9,10,17].
In both human and rodent females poor spatial ability has been
linked to high levels of circulating oestrogen during pro-oestrous
or prior to ovulation or when pharmacologically manipulated
[3,5,6,14,17,18,22]. Sex differences in Long-Evans hooded rats
have previously been reported on the Morris water maze, where
males perform better than females [51] which is in agreement
with the current study, although there is some conflicting data
where no sex differences have been observed [54]. In the Long-
Evans rat at 7 days of age, males tend to perform better on
spatial mazes than females [17] which has also been observed
in the present behavioural paradigm where male hooded Lis-
ter rats of the same age show an enhanced exploration of the
moved object over longer inter-trial intervals (Fig. 4b) this
sex difference may be attributed to the organizational effects
of sex hormones during growth and development of the animal
[54] and later in adulthood, where circulating androgens, oestro-
gens and progestagens will exert activational effects in steroid
responsive neural circuits (e.g. behaviours related to oestrus).
During growth and development of any animal, steroids have
been shown to be responsible for alterations in regional cell
growth, proliferation and even death which will then influence
cell number, size and packing density [13], so, it is likely that
later in life pre-defined neuronal circuitry should have a signifi-
cant impact on subsequent cognitive abilities due to fundamental
gender differences or hormonal fluctuations during the oestrous
cycle.
A possible variable which may affect performance in the
NOR task is that gender differences may be viewed due to
specific sexually dimorphic behaviours other than memory abil-
ity. Female rats have increased locomotor activity (LMA),
enhanced limb co-ordination and improved sensory percep-
tion and enhanced attentional mechanistics on the night of
behavioural oestrous and it is such natural hormone fluctuations
that may influence observations in most behavioural paradigms
[12]. Non-cognitive factors such as locomotor activity (LMA)
differences were ruled out in this study by the lack of group
differences, assessed by number of lines crossed, for both sexes
over both trials. One study has shown long-term (6+ months)
environmental enrichment compared to no enrichment attenu-
124 J.S. Sutcliffe et al. / Behavioural Brain Research 177 (2007) 117125
ates the effects of oestrogen on novel object recognition [21].
Since the animals in this study received bedding and cardboard
tubes for enrichment as standard practice, a greater effect of gen-
der and inter-trial interval may be seen had these animals been
housed on wood chips only (Beta-bed-grade 6, Datesand Ltd).
There have been several proposed mechanisms for gender dif-
ferences seen in this study and others of its type. The hippocam-
pus is a neural structure often associated with the mediation
of spatial learning. Interestingly, the structure of the hippocam-
pus has been shown to undergo significant structural changes
throughout the oestrous cycle showing significantly more den-
dritic spines at pro-oestrus when there is elevated oestrogen
than during the oestrous phase where hormone levels are at
their nadir, consistent with the view that HPA fluctuations
across the oestrous cycle may be involved in spatial perfor-
mance [1,2,47,48,55,57,58]. This suggests that the structural
features of the brain may rely on the gonadal hormone levels
which may in turn mediate performance in different behavioural
tasks. However, sex hormone receptors (ER, ER and proges-
terone receptors) in the hippocampus are low [53] and sparse
in the striatum [42]; so these differences could be due to the
fluctuations in corticosteroids which are also altered across the
oestrous cycle [3,4,17]. Oestrogen and progesterone have also
been shown to influence the concentration of enzymes, receptors
and transporter mechanisms associated with neurotransmission,
which are vital for the adequate storage and processing of mem-
ories. Such neurotransmitters include the primary excitatory
neurotransmitter, glutamate, the primary inhibitory neurotrans-
mitter, gamma amino butyric acid (GABA) and acetylcholine
[54]. Further research into the impact of ovarian steroids on
cognition is vital to ensure the many potential interactions
between hormones and neurotransmitter systems are eluci-
dated, especially regarding the biological basis of learning and
memory.
To our knowledge, this is the first study to document a
direct comparison between the sexes in object recognition mem-
ory and spatial recognition memory as assessed by the NOR
task; to assess optimal inter-trial intervals over which both
male and female hooded Lister rats can hold and retrieve the
information for later trials and one of the first to investigate
the influence of the oestrous cycle in both types of task for
females. The proposal that sex differences in behavioural perfor-
mance may be mediated by physiological correlates of various
endocrine states has been clearly demonstrated in this study.
Recent work has shown clear sexual dimorphisms in a multi-
tude of neurotransmitter systems, including serotonin, GABA
(-aminobutyric acid), acetylcholine, vasopressin, opioids and
monoamines [3,4,8], which will evidently affect performance
in numerous behavioural paradigms. Regretfully, much work
has focused on just one of the sexes and mounting evidence
suggests that, in pharmacological and behavioural studies espe-
cially, both genders must be considered as separate entities to
benefit research interests. Future studies are aimed at further
evaluating the influence of hormones on cognition as assessed
by the NOR following a variety of pharmacological treatments
with concurrent elucidation of biochemical mechanisms. Pre-
vious work from our laboratory has shown that the NOR is a
suitable, well-validated behavioural paradigm to assess changes
in cognitive function following a variety of pharmacological
insults [20,45]. These studies may help to clarify the relation-
ship between object recognition memory and spatial ability due
to different sex hormones of which, to date, there is limited
comparative data in the NOR. Despite significant progress in
the influence of hormonal factors in cognitive processes and
neuronal protection, the value of hormones as an adjunct ther-
apy for the treatment of cognitive impairments associated with
age, disease and physical injury remains an under-explored and
exciting field.
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