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European Journal of Neuroscience, Vol. 24, pp. 229–242, 2006
Progesterone reverses the spatial memoryenhancements initiated by tonic and cyclic oestrogentherapy in middle-aged ovariectomized female rats
Heather A. Bimonte-Nelson,1 Kevin R. Francis,2 Claudia D. Umphlet2 and Ann-Charlotte Granholm21Department of Psychology, Arizona State University, PO Box 871104, Tempe, AZ 85287, USA2Department of Neuroscience and the Center on Ageing, Medical University of South Carolina, Charleston, SC 29425, USA
Keywords: aging, estradiol, hippocampus, hormone replacement, reference memory
While some research has indicated that ovarian hormone therapy (HT) benefits memory and decreases risk of Alzheimer's disease inmenopausal women, several newer studies have shown null or detrimental effects. Despite the null and negative cognitive findings,the numerous studies showing positive effects beg the question of what factors determine whether HT acts as a neuroprotectant or arisk factor for brain functioning. Using middle-aged female rats, we directly compared six HTs. We evaluated the effects ofovariectomy, tonic low-dose, tonic high-dose and biweekly cyclic estradiol treatment, as well as whether progesterone altered theeffectiveness of any one of these oestrogen regimens. Animals were tested on spatial and complex cued (intramaze patterns)reference memory using variants of the Morris maze. The tonic low-dose and cyclic estradiol treatments improved spatialperformance, while the addition of progesterone reversed these beneficial cognitive effects of estradiol. Additionally, all groupslearned to locate the platform on the cued task; however, an egocentric circling strategy was used with sham ovary-intact andhormone-replacement groups showing the most efficient search strategy. Although the question of memory retention 8 weeks afterthe first cognitive assessment was addressed, a large number of animals died between the first and second test, rendering the retestuninterpretable for many group comparisons. Specifically, both doses of tonic estradiol dramatically increased the number of deathsduring the 17-week experiment, while the cyclic estradiol treatment did not. Progesterone decreased the number of deaths due totonic estradiol treatment. Our findings suggest that the dose of estradiol replacement as well as the presence of progesteroneinfluences the cognitive outcome of estradiol treatment. Further, there appears to be a dissociation between HT effects on cognitionand mortality rates.
There is abundant clinical and basic science evidence that oestrogens
investigating the relationship between ovarian hormone replacement
impact cognitive function (Dohanich, 2002). Since the first controlled
and cognition are inconsistent. Indeed, several newer clinical studies,
clinical evaluation in 1952 showing that oestrogen injections given to
including the large Women's Health Initiative Memory Study, have
75-year-old women enhanced memory task performance (Caldwell &
indicated that ovarian hormone replacement has null or detrimental
Watson, 1952), there have been numerous studies linking ovarian
effects on cognition and AD or dementia risk in postmenopausal
hormones and cognition in menopausal women. For example, women
women (Mulnard et al., 2000; Wang et al., 2000; Rapp et al., 2003;
exhibited memory decline after oopherectomy (Sherwin, 1988;
Shumaker et al., 2003, 2004). However, despite the null and negative
Phillips & Sherwin, 1992) and years since oopherectomy were
findings, the numerous studies showing positive effects of hormone
negatively correlated with memory scores (Nappi et al., 1999).
treatment beg the question of what factors determine whether hormone
Estrogen treatment benefits memory in studies of menopausal women
therapy acts as a neuroprotectant or a risk factor for brain functioning.
spanning self-report (Campbell & Whitehead, 1977) and case studies
Many variables may contribute to the discrepancy between the
(Ohkura et al., 1995) to more objective evaluations (Sherwin, 1988;
clinical studies showing positive effects of oestrogen and those
Phillips & Sherwin, 1992; Kampen & Sherwin, 1994; Jacobs et al.,
showing negative or null effects; these include age of subjects, time
1998). There is evidence that ovarian hormone replacement may
since menopause, and prior hormone replacement history. Another
decrease the risk of Alzheimer's disease (AD) or enhance memory in
factor which may impact the outcome of hormone therapy is whether
AD patients (Asthana et al., 1999; Henderson et al., 1994, 1996;
treatment includes a progestin. There is evidence that the combination
Paganini-Hill & Henderson, 1994; Tang et al., 1996; Kawas et al.,
therapy of oestrogen plus progesterone has a negative impact on
1997; Baldereschi et al., 1998). However, findings from studies
cognition, while oestrogen alone does not (Rice et al., 2000).
Preclinical data collected by us and others suggest that progesteronesupplementation is detrimental to cognition in aged ovariectomized
Correspondence: Dr Heather A. Bimonte-Nelson, as above.
(Ovx) rats (Bimonte-Nelson et al., 2004b) and that administration of
progesterone or its metabolite, allopregnanalone, impairs performance
Received 14 October 2005, revised 3 April 2006, accepted 4 April 2006
on spatial and avoidance tasks in adult rodents (Farr et al., 1995; Frye
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
H. A. Bimonte-Nelson et al.
& Sturgis, 1995; Johansson et al., 2002). Further, we have shown that
to which the subjects need to attend (for discussion, see Hyde et al.,
progesterone reverses the estradiol-induced alterations in neurotrophin
2002 and Stavnezer et al., 2002). The current study assessed the
protein levels in cognitive brain regions (Bimonte-Nelson et al.,
influence of several ovarian hormone therapies on spatial vs. complex
nonspatial (cued) learning and memory. In both tests, reference
There is also increasing evidence that whether oestrogen is experi-
memory was evaluated; the platform remained paired with the same
mentally observed to influence cognitive performance depends on the
reinforcement across all testing days. For the spatial test, the platform
type of task utilized. Interestingly, the strongest clinical evidence
remained in a constant spatial location such that rats can solve the task
supporting a role for oestrogen in cognition is within the verbal domain,
using extra-maze cues within the testing room environment. For the
with benefits seen primarily on verbal tasks and few or inconsistent
nonspatial test, the platform remained paired with an intramaze
effects on visuospatial tasks (Caldwell & Watson, 1952; Kampen &
patterned cue that was painted on the maze wall. There were four
Sherwin, 1994; Maki et al., 2001; for review, see Cholerton et al., 2002).
different patterns the rats needed to discern in order to solve the task
Paradoxically, women using hormone therapy showed the greatest
using the cue. As the intramaze cue and platform were concurrently
increase in PET cerebral blood flow over time, compared to nonhormone
rotated within the maze after every trial, the spatial and nonspatial cues
users, in the hippocampus and related brain regions; these are areas
were dissociated. This task could not be solved by using spatial cues.
which show profound alterations in AD and are intimately linked to
The current experiment was an attempt to clarify the nature of the
spatial navigation and memory (Maki & Resnick, 2000, 2001).
effects of oestrogen on cognition. We sought to decipher the cognitive
Animal researchers have exerted increased efforts over the last
effects of low- vs. high-dose tonic estradiol, of estradiol alone vs.
decade to determine the effects that oestrogen has on cognition and
combination treatment with progesterone, and of short- vs. long- term
underlying neurobiological substrates. Like the human work (Maki &
treatment. Further, given the work of Daniel et al. (1997) showing that
Resnick, 2000, 2001), rodent studies also show oestrogenic influences
estradiol-induced spatial working memory benefits were sustained in
on the hippocampus, including dendritic spine density alterations
young Ovx rats even when treatment was terminated before
across the estrous cycle and increases in Ovx rats given oestrogen
behavioural testing ensued, estradiol may exert changes in the brain
treatment, as well as oestrogen-induced facilitation of hippocampal
resulting in cognitive enhancements that persist beyond the hormone
long-term potentiation (Gould et al., 1990; Woolley & McEwen, 1992,
administration period. Hence, the continuous oestrogen exposure of a
1994; Cordoba Montoya & Carrer, 1997; Gupta et al., 2001). In
tonic regimen may not be necessary to initiate cognitive changes;
addition, oestrogen enhances the performance of Ovx rats on tasks that
intermittent estradiol exposure may be effective. To evaluate this, we
can be solved utilizing a spatial strategy and are hippocampal-
also tested a biweekly estradiol injection regimen. The experimental
dependent, an effect seen in young (Daniel et al., 1997; Gibbs, 1999;
design of this multiple-group study was such that direct comparisons
Bimonte & Denenberg, 1999; Fader et al., 1999; Sandstrom &
could be made between the six hormone replacement groups.
Williams, 2001; Bowman et al., 2002; Heikkinen et al., 2002; Holmes
Specifically, in middle-aged female rats we evaluated (i) the effects
et al., 2002; Korol & Kolo, 2002; El-Bakri et al., 2004; Feng et al.,
of Ovx, (ii) the effects of tonic low-dose oestrogen, (iii) the effects of
2004; Daniel et al., 2005) and aged (Frick et al., 2002; Luine &
tonic high-dose oestrogen, (iv) the effects of intermittent, cyclic
Rodriguez, 1994; Gibbs, 2000; Markham et al., 2002) rodents. Still,
oestrogen and (v) whether progesterone altered the effectiveness of
several animal studies testing the cognitive effects of oestrogen have
any one of these oestrogen treatments. We questioned whether
reported null or negative results. While some of these null or negative
performance and solving strategy on spatial and complex cued
effects have been seen on spatial tasks (Singh et al., 1994; Chesler &
reference memory tasks, as assessed via variants of the Morris water
Juraska, 2000; Galea et al., 2001, 2002; Holmes et al., 2002;
maze, would be differentially influenced by these hormone regimens.
Fernandez & Frick, 2004), there is an increasing literature suggestingthat several types of nonspatial memory are negatively affected byoestrogen. There are different types of nonspatial cognition that can be
Materials and methods
tapped into during maze testing, including memory for motoric
responses as well as ability to locate a visual cue within the maze.
Only a handful of studies have compared oestrogen effects on spatial
At the start of the experiment there were 92 subjects, which were
vs. nonspatial memory. For example, compared to young Ovx rats
12-month-old (middle-aged) Fischer-344 female rats born and raised
given oil, those given estradiol injections performed better on a spatial
at the ageing colony of the National Institute on Ageing at Harlan
plus-maze task, but worse on a nonspatial response task that required
Laboratories (Indianapolis, IN, USA). Prior to surgery rats were
animals to make a turn in a consistent direction for reward (Korol &
acclimated for several weeks, and were pair-housed with an identical
Kolo, 2002). Whether estradiol influences this motor response-
treatment-assigned cage-mate in the MUSC animal facility in a barrier
dependent form of nonspatial working memory may depend on
environment. Animals had exposure to food and water ad libitum, and
estradiol dose. Indeed, high-dose estradiol injections impaired, and
were maintained on a 12-h light–dark cycle. All procedures were
low-dose estradiol injections facilitated, performance of young Ovx
approved by the local IACUC committee and adhered to NIH
rats on a delayed alternation task that utilized directional turn response
as the correct choice (Wide et al., 2004). Others have compared spatialand nonspatial maze learning via a Morris water maze with a hiddenplatform, no intramaze cues, and numerous extramaze cues (the spatial
Hormone manipulation procedures
version) or in a similar apparatus but with a platform that protrudes
Table 1 summarizes the hormone manipulation procedures for each
from the water (the nonspatial cued version). Results from these
group of subjects and Fig. 1 schematically depicts the experimental
studies range from estradiol alone enhancing both versions in Ovx
timeline. Due to the large number of subjects, animals were run in two
mice (Rissanen et al., 1999) to no effects on either version in adult
experimental waves, with each treatment group represented in each
Ovx rats (Chesler & Juraska, 2000).
wave. The descriptions below are for each wave of animals. Forty-
The visible platform version of the Morris water maze is less
seven days before behavioural testing ensued, 82 rats received Ovx
complex than the spatial version as the visible platform is the only cue
and 10 rats received sham surgery. Rats were anaesthetized with an
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 24, 229–242
Progesterone–oestrogen interactions on cognition
Table 1. Hormone manipulation procedures and attrition rate of subjects
Numbers (n) of rats
n died ⁄ n at onset
Ovx or sham surgery
(tonic treatment)
(cyclic treatment)
Ovx + vehicle inj
Week 1 Week 4
End of End of
injection injection
injection injection injection
Fig. 1. A time line summarizing sham surgery and ovariectomy, hormone replacement and behavioural testing experimental procedures.
intraperitoneal injection of a cocktail of 70 mg ⁄ kg ketamine (Fort
with each other, and the skin was stapled. The groups not receiving
Dodge Animal Health, Fort Dodge, IA, USA) and 6 mg ⁄ kg xylazine
hormone administration (Sham and Ovx groups) received a sham
(Lloyd Laboratories, Shenandoah, IA, USA). For Ovx, bilateral
pellet surgery which included identical procedures except that the
dorsolateral incisions were made in the skin and peritoneum, and the
pocket was left empty before closure. As the pellets were 60-day
ovaries and tips of uterine horns were ligatured and removed. The
release, new pellets were inserted, or sham pellet surgery was given as
muscle was then sutured and the skin stapled. Sham surgery consisted
described above, exactly 60 days after the first pellet-insertion surgery
of skin incision and staple in the same fashion.
was performed.
According to the manufacturer (Innovative Research of America,
Sarasota, FL, USA), each low-dose estradiol, high-dose estradiol and
Tonic ovarian hormone treatment
progesterone pellet releases a continuous dose of hormone resulting in
In addition to the Sham and Ovx groups, which received no hormone
levels approximating 20 pg ⁄ mL, 40 pg ⁄ mL and 12 ng ⁄ mL, respect-
administration, there were four tonic hormone replacement groups:
ively, of circulating hormone. Thus, the estimated circulating estradiol
Ovx given low-dose tonic estradiol (Ovx-LoE), Ovx given low-dose
level in rats receiving low-dose treatment was 20 pg ⁄ mL, or high-
tonic estradiol plus progesterone (Ovx-LoE + P), Ovx given high-
dose treatment was 40 pg ⁄ mL, and progesterone level in rats
dose tonic estradiol (Ovx-HiE), and Ovx given high-dose tonic
receiving two progesterone pellets was 24 ng ⁄ mL. These ovarian
estradiol plus progesterone (Ovx-HiE + P). Twenty-two days after
hormone levels are within the physiological range seen across the
Ovx or sham surgery (25 days before test), pellet insertion surgeries
estrous cycle in young rats (Butcher et al., 1974). Furthermore, this
were performed. After ketamine plus xylazine anaesthesia, a small
approximate progesterone dose has been shown to counteract
incision was made in the scruff of the neck and a subcutaneous pocket
oestrogen's effects on growth factors in cognitive brain regions, as
was created with blunt dissection scissors. For animals receiving tonic
well as impair spatial memory in aged rats (Bimonte-Nelson et al.,
estradiol, one estradiol pellet of the appropriate dose (0.25 mg, 60-day
2004a, 2004b).
release, i.e. 0.25 mg released over a period of 60 days, for the lowdose or 0.50 mg, 60-day release for the high dose) was inserted intothe pocket and the skin was stapled. For animals receiving tonic
Intermittent (cyclic) estradiol treatment
estradiol plus progesterone, one estradiol pellet of the appropriate dose
To evaluate the cognitive effectiveness of cyclic estradiol treatment
and two progesterone pellets (each progesterone pellet was 200 mg,
with or without progesterone, three additional groups were included in
60-day release) were inserted such that the pellets were not in contact
this study: Ovx given cyclic estradiol (Ovx-CycE), Ovx given cyclic
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 24, 229–242
H. A. Bimonte-Nelson et al.
estadiol plus progesterone (Ovx-CycE + P), and Ovx given vehicle
Spatial Morris maze
injection as the injection control (Ovx-Veh). The Ovx-CycE and Ovx-
The Morris maze (Morris et al., 1982) consisted of a round tub (188 cm
CycE + P groups received one 10-lg injection of 17b-estradiol
in diameter) that was filled with room-temperature water made opaque
(Sigma) in 0.1 mL sesame oil (Sigma) into the scruff of the neck every
with black nontoxic paint. A black platform (10 cm wide) was
other Friday afternoon. The 10-lg injection dose was based on
submerged just below the water surface. There were numerous
findings that two consecutive daily injections resulted in dendritic
extramaze cues throughout the room including posters, heat lamps,
spine increases 48 h after the second injection (Woolley & McEwen,
tables and a sink, and there were no obvious intramaze cues. The rat was
1994) as well as potentiated hippocampal ACh release during spatial
placed in the tub of water from any of four locations (North, South, East,
task training as assessed 24 h after the second injection (Marriott &
or West) and had 60 s to locate the hidden platform, which remained in a
Korol, 2003). The Ovx-Veh rats received an injection of 0.1 mL
fixed location (in the Northeast quadrant) throughout testing. If the rat
sesame oil at the same time as the Ovx-CycE and Ovx-CycE + P rats.
did not find the platform, it was gently guided to it using a black plastic
The Ovx-CycE + P rats received two progesterone pellets in the scruff
rod. Once the rat found the platform the trial was terminated. After 15 s
of the neck at the same time as the tonic hormone-treated animals, as
on the platform, the rat was gently removed from the maze and placed
described above. The Ovx-Veh and Ovx-CycE groups were given
into its heated cage until the next trial. The rats were given three trials per
sham pellet surgery at the same time as well. In addition, as described
day for 5 days; the drop-off location for each trial varied semirandomly
above for the tonic treatment groups, new pellets were inserted, or
so that no two identical drop-off locations were given on the same day.
sham surgeries were given, exactly 60 days after the first pellet
Animals were tested in squads (seven or eight rats in each squad) so that
surgery. The surgeries performed on the injected groups were executed
trial 1 was completed for each rat in the group, then trial 2, then trial 3,
at the same time as the tonic treatment groups.
as in prior studies (Schrott et al., 1992; Hyde et al., 2002; Stavnezer
For each wave, all nine treatment groups were given surgery within
et al., 2002). The approximate intertrial interval was 5–8 min. After
the same 2-day span at the exact same interval before testing. The
the last trial on day 5, each rat was given a 60-s probe trial in which the
resulting nine treatment groups were: Sham, Ovx, Ovx-LoE, Ovx-
platform was removed from the maze. A video camera suspended on the
LoE + P, Ovx-HiE, Ovx-HiE + P, Ovx-Veh, Ovx-CycE and Ovx-
ceiling above the maze tracked the rat's path during each trial and a
CycE + P. The number of subjects for each treatment group at the first
tracking system (Smart System; San Diego Instruments) was used to
group of testing sessions and at the second group of testing sessions,
analyse each rat's tracing.
8 weeks later, is shown in Table 1. In addition, the Ovx and Ovx-Vehgroups did not differ on any measure, suggesting that vehicle injectionevery other week did not influence maze performance. Hence, these
two groups were combined and will be referred to as Ovx hereafter.
This nonspatial cued version of the Morris maze was based on our priorresearch using a similar apparatus in mice (Hyde et al., 2002). Figure 2a
Apparatus and testing procedures
is a photograph of the cued Morris maze. Cued Morris maze testing was
Variations of the Morris water maze were used to evaluate spatial and
performed in a different room than spatial Morris maze testing. The
cued reference memory. Eight weeks after initial spatial and cued
testing apparatus and procedure for the cued Morris maze were as similar
Morris maze testing, animals were retested on the spatial Morris maze
as possible to the spatial Morris maze; rats were tested in squads for three
to evaluate spatial retention and relearning. The retest procedure was
trials per day for 5 days. The tub was identical to the spatial version
exactly the same as the initial testing procedure.
except that there was a painted movable insert made of stainless steel that
Fig. 2. (a) A picture of the cued Morris water maze. The maze insert, which had patterns painted on the walls, was rotated after every trial. The platform remainedpaired with a rat's positive pattern throughout testing, thereby making this a reference memory task. (b) A schematic diagram showing how each maze was dividedinto quadrants and annuli for the probe trial quantification (although the inner sector is circular rather than annular, it is described as annular for simplicity).
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 24, 229–242
Progesterone–oestrogen interactions on cognition
hugged the inside wall of the maze. Each quadrant of the insert was
variables which may influence motor or sensory function resulting in
painted with a different black-and-white pattern to serve as intramaze
effects on reaction times (e.g. Gallagher et al., 1993; Foster et al., 2003).
cues. The patterns, each separated by a region of solid black, were
Given the differences in sensory and motor responses across the estrous
horizontal stripes, dark solid grey, vertical stripes and solid white. The
cycle and after oestrogen treatment (e.g. Becker, 1999; Diaz-Veliz et al.,
walls of the testing room were bare and overhead lights were turned off
1999; Estrada-Camarena et al., 2003), for both spatial and cued versions
during testing to minimize extramaze cues. The maze was illuminated by
of the Morris maze performance was assessed by swim path distance (in
indirect lighting via lights placed on the floor around the maze directed
inches; 1 inch 25.4 mm) to the platform. We also report swim speed
toward the ceiling (required for the tracking system).
(distance ⁄ time). Distance and speed scores were analysed separately for
Prior to testing, each rat was randomly assigned one of the four
each version of the maze using one between-subjects factor (Treatment)
patterns as their positive stimulus. The platform remained with that
and two within-subjects factors (Days and Trials) in repeated-measures
pattern throughout testing for a given subject. The rats were always
anova. Probe-trial data were analysed using repeated-measures anova
released from South, and had 60 s to locate the platform. Once the
with Treatment as the between-subjects variable and Quadrant as the
platform was found, the rat remained on it for 15 s. After each trial,
repeated measures. The maze was divided into specific quadrant and
the platform and tub insert were semirandomly rotated such that the
annulus designations as shown in Fig. 2b.
platform remained paired with its positive pattern, but the positivepattern was in a different place in space. This was done to dissociateextramaze from intramaze cues. After the last trial on day 5, the
platform was removed from the maze for the probe trial. A video
As there were no main effects of Testing Wave, nor were there Testing
camera suspended on the ceiling above the maze tracked the rat's path
Wave · Treatment interactions for any measure, the two waves were
during each trial and a tracking system (Ethovision system; Noldus
combined for all analyses.
Information Technology) was used to analyse the tracing of each rat.
Spatial Morris maze testing
Dependent variables and statistical analyses
Latency to reach the platform has been argued to be a poor measure of
Figure 3 shows the distance-to-platform scores for each treatment
learning, especially when there may be treatment group differences in
group for each testing day, and Fig. 4 shows the main effects for
Fig. 3. Mean ± SEM distance-to-platform scores (inches) across days of testing for the spatial version of the task for (a) Ovx vs. Sham groups, (b) Ovx, Ovx-LoE+P groups, (c) Ovx, Ovx-HiE, and Ovx-HiE+P groups, and (d) Ovx, Ovx-CycE, and Ovx-CycE+P groups.
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 24, 229–242
H. A. Bimonte-Nelson et al.
groups did not differ from each other, the intermittent cyclic estradiol
treatment was as effective as the low-dose tonic estradiol treatment on
this measure. In addition, it is noteworthy that there was aTreatment · Trial interaction for the Ovx vs. Ovx-LoE repeated-
measures anova (F2,46 ¼ 4.55, P < 0.05). Further evaluationsshowed that the Ovx-LoE group showed better performance than the
Ovx group on trial 1 collapsed across days 2–5 (main effect of
Treatment: F1,23 ¼ 9.49, P < 0.01) thereby indicating that the LoE
treatment aided in remembering the platform location overnight. This
effect was marginal for the Ovx-CycE vs. Ovx comparison
(F1,26 ¼ 3.24, P ¼ 0.08).
Concomitant treatment with progesterone reversed the beneficial
effects of estradiol. Indeed, Ovx female rats given tonic low-dose
estradiol plus progesterone swam a longer distance to the platformthan Ovx rats given low-dose estradiol alone (F1,18 ¼ 5.82; P < 0.05).
Similarly, Ovx female rats given cyclic oestrogen plus progesteroneswam a longer distance to the platform than Ovx rats given cyclic
oestrogen alone (F1,19 ¼ 5.56; P < 0.05). Progesterone completely
reversed these beneficial effects so that the animals receiving thecombination treatments did not differ from animals receiving Ovx
Fig. 4. Mean ± SEM distance-to-platform scores (inches) for each treatment
alone, as represented by the lack of significant statistical differences
group collapsed across all testing days for the spatial version of the task. Low-dose tonic estradiol and cyclic estradiol treatments enhanced spatial maze
between the Ovx and Ovx-LoE + P groups, as well as between the
performance in middle-aged Ovx females, while the addition of progesterone
Ovx and Ovx-CycE + P groups. Interestingly, while both low-dose
reversed these beneficial cognitive effects; *P < 0.05.
tonic and cyclic estradiol treatment enhanced spatial referencememory performance, neither high-dose tonic oestrogen nor high-dose tonic oestrogen plus progesterone had effects on distance-to-
distance-to-platform scores collapsed across days of testing. The
platform scores as compared to Ovx alone. There were clear
distance scores of Sham and Ovx females did not differ, nor did Trial
differences in the effectiveness of the high vs. low tonic estradiol
or Days interact with Treatment, indicating that removal of the ovaries
treatments as the Ovx-HiE group swam a longer distance to the
did not influence performance in 14-month-old female rats. There
platform than the Ovx-LoE group (F1,16 ¼ 6.40, P < 0.05).
were marked positive effects of oestrogen replacement on distance
Upon further inspection of the spatial maze data it was clear that
scores, which were negatively influenced by the additional presence of
some groups showed lower distance scores on day 1. As animals had
progesterone. Ovx females receiving low-dose tonic or cyclic estradiol
no prior exposure to the maze before day 1, an assessment of
treatment swam shorter distances to the platform than did females that
individual trials within this first testing day may reveal group
received Ovx with no hormone supplementation (Ovx vs. low-dose
differences in response to novelty of the maze situation. This analysis
tonic estradiol treatment, main effect: F1,23 ¼ 5.36, P < 0.05; Ovx vs.
revealed that there were no group differences upon the rats' first
cyclic estradiol treatment, main effect: F1,26 ¼ 5.65, P < 0.05),
exposure to the maze (day 1, trial 1). However, as shown in Fig. 5,
suggesting that both low-dose tonic and cyclic estradiol treatment
there were group differences on trial 2 for this first day of testing.
enhanced spatial reference memory. As the Ovx-LoE and Ovx-CycE
Specifically, t-tests showed that, compared to Ovx rats not given
Fig. 5. Mean ± SEM distance-to-platform scores (inches) for each treatment group across trials for day 1 of testing for the spatial version of the task. While therewere no significant group differences for trials 1 and 3 on day 1, all but the Ovx-CycE + P group swam a shorter distance to the platform than did the Ovx group fortrial 2; *P < 0.05.
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 24, 229–242
Progesterone–oestrogen interactions on cognition
hormone, sham rats (t24 ¼ 2.38, P < 0.05) and Ovx rats given tonic
low-dose estradiol (t23 ¼ 3.19, P < 0.005), tonic low-dose estradiol
To evaluate whether rats localized the platform to the spatial location,
plus progesterone (t25 ¼ 2.28, P < 0.05), tonic high-dose estradiol
the platform was removed after all testing trials were completed, i.e.
(t23 ¼ 2.41, P < 0.05), tonic high-dose estradiol plus progesterone
after trial 3 on day 5. The percentage of the total distance moved in
(t23 ¼ 2.47, P < 0.05) or cyclic estradiol (t26 ¼ 3.16, P < 0.005)
the platform quadrant was compared to that of the quadrant diagonally
swam less distance to reach the platform on trial 2. There were no
opposite the platform. Rats that learned the location of the platform
group differences on trial 3 for day 1.
were expected to spend the greatest percentage of their total distancein the quadrant that had contained the platform during the prior trials.
A repeated-measures anova (with Quadrant as the repeated measure
Analysis of swim speed showed clear dissociations between swim
and Treatment group as the between-subjects variable) showed that a
speed and performance (as measured by distance to the platform).
higher percentage of the distance was spent in the platform quadrant
Specifically, while only cyclic and low-dose tonic estradiol treatments
improved maze performance, all estradiol treatments decreased swim
F1,77 ¼ 238.50, P < 0.0001). As represented in Fig. 6, this pattern
speed. Low-dose tonic (F1,23 ¼ 21.29, P < 0.0001) and high-dose
was seen for all treatment groups, thereby indicating that all groups,
tonic (F1,23 ¼ 30.28, P < 0.0001) estradiol significantly, and cyclic
regardless of hormone status, localized the platform location by the
estradiol marginally (F1,26 ¼ 3.87, P ¼ 0.06), decreased swim speed
end of testing (Quadrant · Treatment group interaction, P > 0.89).
relative to Ovx rats without hormone treatment (data not shown).
Progesterone reversed this effect on swim speed for rats treated withhigh-dose tonic or cyclic estradiol, as the Ovx group did not differ
Cued Morris maze testing
Ovx-HiE + P or Ovx-CycE + P groups
Ps > 0.36). However, progesterone did not reverse the effect on swim
Distance to platform
speed in rats treated with low-dose tonic estradiol, as the Ovx-
For the cued version of the maze, Fig. 7 shows the distance-to-
LoE + P group had a slower swim speed than the Ovx group
platform scores for each treatment group for each testing day and
(F1,25 ¼ 10.68, P < 0.005). Further, Ovx and Sham groups did not
Fig. 8 shows the main effects for distance-to-platform scores collapsed
differ, thereby suggesting that Ovx did not influence swim speed in
across days of testing. Low-dose tonic estradiol treatment improved
middle-aged rats.
nonspatial performance, as the Ovx-LoE group had lower distance
To determine whether any group exhibited a change in speed across
scores than the Ovx group (F1,22 ¼ 4.80; P < 0.05). On the other
trials for the spatial task, thereby providing an evaluation of
hand, the combination of low-dose tonic estradiol plus progesterone
exhaustion, we ran a separate repeated-measures anova for each
did not enhance performance. Indeed, the Ovx-LoE + P group
group with Trial as the within-factor variable. The Trial variable was
exhibited distance scores that were intermediate between the Ovx
not significant for any group (all Ps > 0.18), indicating that none of
and Ovx-LoE groups. As a result, the Ovx-LoE + P group did not
the groups showed a change in swim speed across trials.
differ significantly from the Ovx or the Ovx-LoE groups (P > 0.12).
Platform quadrant
Opposite quadrant
Fig. 6. Mean ± SEM percentage distance spent in the quadrant containing the platform vs. the diagonally opposite quadrant for each treatment group for the probetrial on the spatial Morris water maze. All groups spent more time in the target (platform) quadrant during the probe trial, in which the platform was removed fromthe maze; ***P < 0.0001.
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 24, 229–242
H. A. Bimonte-Nelson et al.
Fig. 7. Mean ± SEM distance-to-platform scores (inches) across days of testing for the nonspatial, cued version of the task for (a) Ovx vs. Sham groups, (b) Ovx,Ovx-LoE and Ovx-LoE+P groups, (c) Ovx, Ovx-HiE, and Ovx-HiE+P groups, and (d) Ovx, Ovx-CycE, and Ovx-CycE+P groups.
enhanced cued performance to the same degree, as the Ovx-HiE and
Ovx-LoE distance scores did not differ. Neither Ovx, cyclic estradiol,
nor cyclic estradiol plus progesterone influenced distance scores on the
The swim speed rankings of the groups were remarkably similar
between the spatial and cued tasks. Moreover, as seen in the spatial
version of the task, there was a dissociation between performance and
swim speed for this task as well. Low-dose tonic (F1,22 ¼ 13.69;
P < 0.005) and high-dose tonic (F1,22 ¼ 44.77; P < 0.0001) estradiolsignificantly decreased swim speed relative to Ovx rats withouthormone treatment (data not shown). As seen on the spatial version of
the task, progesterone reversed the swim speed effect for high-dose,but not low-dose, tonic estradiol treatment. Indeed, the Ovx-HiE + Pgroup did not differ from the Ovx group (P > 0.15), while the Ovx-
LoE + P and Ovx groups did differ (F1,25 ¼ 19.62, P < 0.0005).
Neither cyclic estradiol treatment, cyclic estradiol plus progesteronetreatment nor Ovx influenced swim speed.
Fig. 8. Mean ± SEM distance-to-platform scores (inches) for each treatment
To evaluate whether any group exhibited a change in speed across
group collapsed across all testing days for each treatment group for thenonspatial version of the task; *P < 0.05.
trials for the nonspatial task, we ran a repeated-measures anova on eachgroup with Trials as the within-subject variable. The Trials variable wasnot significant for any group (all Ps < 0.07), indicating that none of the
High-dose tonic estradiol treatment also improved nonspatial per-
groups showed a significant change in swim speed across trials.
formance, as the Ovx-HiE group had lower distance scores than theOvx group (F1,22 ¼ 4.57, P < 0.05). While the high-dose tonic
estradiol plus progesterone treatment group exhibited somewhat lowerdistance scores than Ovx alone, this comparison did not reach
To determine whether subjects localized the platform to their assigned
statistical significance (P ¼ 0.11). However, this concomitant prog-
patterned cue, a probe trial was performed. As in the spatial version,
esterone treatment clearly did not completely reverse the beneficial
the platform was removed after all testing trials were completed. In
effects of high-dose tonic estradiol as the Ovx-HiE and Ovx-HiE + P
contrast to the probe trial results for the spatial version of the task, on
groups did not differ. Both high- and low-dose tonic estradiol
the cued task rats did not spend a higher percentage of their total
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 24, 229–242
Progesterone–oestrogen interactions on cognition
distance traveled in the positive stimulus location than in the opposite
quadrant (main effect of Quadrant collapsed across treatment group,
There was a high degree of animal attrition during this study, the
P > 0.65) thereby indicating that rats did not learn to associate the
majority of which occurred between the first and second testing
platform with the nonspatial patterned cue provided within the maze.
phase. As the study progressed it became increasingly clear that the
This lack of platform association was consistent across treatment
mortality rate was higher in particular groups (see Table 1). By the
groups given the lack of significant Treatment · Quadrant interaction
completion of the study, after the second behavioural testing phase
(P > 0.92), as represented in Fig. 9a. Further evaluations showed that
was completed, the percentages of rats that died were as follows:
rats did not spend a higher proportion of their distance swum in the
Sham, 0%; Ovx (not injected with vehicle), 11%; Ovx + veh, 0%;
positive stimulus quadrant than in the other three quadrants
Ovx-LoE, 89%; Ovx-LoE + P, 55%; Ovx-HiE, 78%; Ovx-HiE + P,
(P > 0.59), while most treatment groups did spend a significantly
50%; Ovx-CycE, 0%; Ovx-CycE + P, 22%. Statistical tests (v2)
higher percentage of their distance swum in the platform annulus (see
showed that tonic oestrogen treatment increased the number of
Fig. 9b). Specifically, repeated-measures anovas run separately for
deaths (Ovx-LoE + Ovx-HiE vs. the Ovx group with no vehicle
each group comparing the inner to the platform annulus showed that
injection: v2 ¼ 28.18, P < 0.005), while the addition of progester-
all groups spent more of their percentage distance in the platform than
one was protective (Ovx-LoE + P + Ovx-HiE + P vs. Ovx-LoE +
in the inner annulus (all Ps < 0.05). The same analyses comparing the
Ovx-HiE: v2 ¼ 4.18, P < 0.05). In addition, it is especially
platform annulus to the outer annulus showed that all but the Ovx
noteworthy that by the completion of the study while eight of
group spent more of their percentage distance in the platform than in
the nine rats treated with low-dose-tonic oestrogen alone died, and
the outer annulus (each P-value for the groups with hormone < 0.05).
seven of the nine treated with high-dose-tonic oestrogen alone died,
Hence, the Ovx rats appeared to be less specific in their search strategy
none of the 12 rats receiving cyclic oestrogen treatment died.
by swimming mainly in the middle and outer annuli, while all groupswith hormone (Sham, Ovx-LoE, Ovx-LoE + P, Ovx-HiE, Ovx-HiE + P, Ovx-CycE and Ovx-CycE + P) localized their search
strategy to the platform annulus. Qualitative inspection of the swimpatterns confirmed an egocentric circling strategy to locate the
The current report suggests that both tonic and cyclic estradiol
platform in this nonspatial patterned Morris maze, an effect noted in
replacement enhance spatial reference memory. Our data indicate that
all treatment groups within this study and previously reported in mice
several factors influence this positive outcome, including dose of
(Stavnezer et al., 2002). This pattern was not seen in the spatial task.
estradiol for the tonic treatment and presence of progesterone for boththe tonic and cyclic hormone regimens. Specifically, tonic low-doseand cyclic estradiol treatments improved spatial reference memory in
Retention effects: spatial Morris maze testing
middle-aged Ovx rats. The addition of progesterone to these estradiolregimens significantly reversed these benefits. The effects of tonic
As shown in Table 1, a large number of subjects died between the
estradiol were dose-specific, as high-dose estradiol with or without
initial test and the retention test. Due to the especially high attrition
progesterone had no effect on spatial reference memory performance.
rate in the low- and high-dose tonic estradiol groups, the final subject
Also, low-dose estradiol facilitated overnight retention of the platform
number in these two tonic estradiol groups (for Ovx-LoE, n ¼ 3 and
location on the spatial task, an effect shown previously for acute
for Ovx-HiE, n ¼ 2) was too small to provide a meaningful
(2 days) and chronic (28 days) estradiol replacement (Markham et al.,
interpretation of potential effects. Hence, the data for these two
groups are not reported for the retention test.
Our finding that oestrogen enhances spatial reference memory in
middle-aged female rats is consistent with studies in young adult (El-
Distance to platform
Bakri et al., 2004; Feng et al., 2004) and middle-aged or aged (Frick
To evaluate group differences in retention of spatial information, for
et al., 2002; Markham et al., 2002) rodents showing positive effects on
each group comparison we analysed the data using a repeated-measures
spatial reference memory. The current experiment also reports the
anova using day 5, trial 3 of original testing and day 1, trial 1 of
novel finding that biweekly cyclic estradiol enhanced spatial reference
retention testing (Test trial) as the within-subject variable and Treatment
memory performance to that of the tonic low-dose treatment. Given
as the between-subjects variable. We also analysed group comparisons
that spatial tasks rely heavily on the hippocampus and related
using the first trial of the first day of the retest alone using t-tests. There
structures, our data suggest that the cyclic treatment influences the
were no group differences in distance scores for any analysis, thereby
hippocampal system. Others have shown that the oestrogen injection
suggesting that neither Ovx, tonic low- or high-dose estradiol plus
dose used in the current study potentiated ACh release during spatial
progesterone, cyclic estradiol, nor cyclic estradiol plus progesterone
task training (Marriott & Korol, 2003) as well as increased
influenced memory retention of the platform location across the 8-week
hippocampal dendritic spine density in young rats (Woolley &
period. Overall, all groups showed excellent retention of the platform
McEwen, 1994) when given 48 and 24 h before assessment. While
location, with mean group values on testing day 5, trial 3 similar to
there is limited research evaluating changes in cognition due to
values on retention day 1, trial 1. Mean distance values ± SEM for each
repeated cyclic estradiol treatment, all but one study has found
group for day 1, trial 1 of the retention test were: Sham, 109.24 ± 46.44;
positive effects. Indeed, weekly injections of estradiol plus progester-
Ovx, 126.01 ± 25.76; Ovx-LoE + P, 198.51 ± 96.74; Ovx-HiE + P,
one enhanced performance of Ovx rats on a delayed match-to-position
72.58 ± 20.95; Ovx-CycE, 73.90 ± 35.90; Ovx-CycE + P, 115.66 ±
T-maze task (Gibbs, 2000) and cyclic estradiol injections given every
45.83. There were also no group differences collapsed across all five
3 weeks enhanced spatial working memory performance in aged Ovx
testing days for the retest. Mean distance values ± SEM for each group
rhesus monkeys (Rapp et al., 2003). There is also evidence that
collapsed across all five days of the retention test were: Sham,
priming with oestrogen injections enhances cognitive responsiveness
149.42 ± 12.97; Ovx, 141.94 ± 10.02; Ovx-LoE + P, 172.95 ± 19.93;
to tonic oestrogen treatment (Markowska & Savonenko, 2002). In the
Ovx-HiE + P, 154.27 ± 18.38; Ovx-CycE, 170.63 ± 14.03; Ovx-
one study that did not find cyclic estradiol-induced spatial memory
CycE + P, 176.81 ± 18.50.
enhancements, young and middle-aged rats were trained on the land
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 24, 229–242
H. A. Bimonte-Nelson et al.
Platform quadrant
Opposite quadrant
Fig. 9. (a) Mean ± SEM percentage distance spent in the quadrant containing the platform vs. the diagonally opposite quadrant for each treatment group for theprobe trial on the nonspatial Morris water maze. As rats did not spend a greater percentage distance in their positive stimulus pattern quadrant, subjects did not appearto associate their positive stimulus pattern with the location of the platform. (b) Mean ± SEM percentage distance swum in each annulus for each treatment groupfor the probe trial on the nonspatial Morris water maze. All of the groups with hormone spent the majority of their percentage distance swimming in the platformannulus for the probe trial, while the Ovx group swam equal percentages of their total distance in the outer and the platform annulus.
radial-arm maze before and after Ovx surgery, followed by an
may have been due to extended training before oestrogen treatment. In
examination of cyclic estradiol effects on the same maze with 60-s or
fact, Daniel et al. (1997) showed that estradiol enhanced acquisition of
6-h delays (Ziegler & Gallagher, 2005). Lack of effects in this study
the land radial-arm maze, an effect that would have been missed in the
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 24, 229–242
Progesterone–oestrogen interactions on cognition
Ziegler & Gallagher (2005) study because oestrogen effects were
The current study attempted to evaluate how various ovarian
evaluated after rats had been trained.
hormone replacement regimens influence spatial and complex non-
spatial memory using variants of the Morris maze. Our data clearly
specifically spatial vs. nonspatial cued memory, an alternative
show that unopposed tonic low-dose as well as cyclic estradiol
explanation of the specificity of these effects might be related to the
treatment enhanced performance on the spatial reference memory task.
temporal proximity of the injections to initiation of testing.
However, the effects of ovarian hormones on cued memory are more
Estradiol injections were administered every other week on Fridays,
difficult to interpret given that probe trial data suggest that rats did not
and testing began on Mondays. Hence, injections occurred 2 days
attend to intramaze patterned cues to find the platform. Rather, they
before spatial testing and 10 days before cued testing. While there
used an egocentric strategy of circling through the platform annulus.
is one study indicating that estradiol-induced spatial memory
We found that mice exhibit a similar strategy to solve this cued maze
benefits are sustained even when treatment is terminated before
(Stavnezer et al., 2002) and others have reported that female rats
behavioural testing begins (Daniel et al., 1997), it is nonetheless
exhibit strong thigmotaxic swimming patterns in the visible platform
plausible that there is a limited time window after estradiol
Morris maze (Beiko et al., 2004). Hence, in the current experiment it
injection during which positive cognitive effects are observed and
appears that all groups learned to locate the platform in both the spatial
that this window had closed by the time our cued maze testing
and cued test versions; in the spatial version they used extramaze cues
began. Future studies will evaluate whether the dissociation in
while for the cued version they used a circling strategy presumably not
spatial and cued performance in the present experiment is due to
dependent on specific intramaze or extramaze visual cue(s). Moreover,
timing of estradiol injections.
while data indicate that no treatment group utilized the patterned cue
The current experiment also demonstrated that progesterone
painted on the maze wall to find the platform, there appear to be group
reversed the beneficial effects of estradiol on spatial reference
differences in efficiency of search strategy. Notably, the Ovx group
memory. In fact, progesterone counteracted the effects of estradiol in
swam mainly in the outer and platform annuli to locate the platform,
every case where estradiol improved performance, regardless of the
and all groups with hormone utilized a more efficient strategy by
cyclic or tonic estradiol regimen. There is clinical evidence that high
localizing swimming efforts to the platform annulus.
levels of progesterone negatively influence cognition (Freeman et al.,
Despite the many studies demonstrating oestrogen-induced positive
1992, 1993; Brett & Baxendale, 2001). Further, we have shown that
effects on maze tasks, some research shows impairment or null effects
aged Ovx rats given progesterone exhibited poorer spatial working
due to ovarian hormone replacement on maze tasks in young (Singh
and reference memory performance than aged Ovx rats not given
et al., 1994; Galea et al., 2001, 2002; Holmes et al., 2002) and middle-
progesterone (Bimonte-Nelson et al., 2004a). Other rodent studies
aged (Chesler & Juraska, 2000; Fernandez & Frick, 2004) rodents.
have also demonstrated cognitive impairments due to administration
While many factors may be postulated to account for the complexity
of progesterone or its metabolites (Farr et al., 1995; Johansson et al.,
of oestrogen's effects on cognition, there are few that comprehensively
2002). Investigations across the estrous cycle and in estropausal intact
explain the disparate findings within the literature. It is compelling to
rodents further support the idea that high progesterone levels are
question whether the task motivator accounts for this variability. In the
associated with poor spatial maze performance (Warren & Juraska,
current study ovarian hormone status did not influence scores upon the
1997; 2000).
first exposure to the maze situation (day 1, trial 1), while on
While most experiments evaluating the behavioural outcome of
the second exposure (day 1, trial 2) all but one group with hormone
oestrogen–progesterone interactions have been within the realm of
replacement had a different pattern of performance than nonhormone-
sexual behaviours, there is an increasing literature dedicated to
treated Ovx rats. These findings may speak to other results that intact
evaluating these interactions on cognitive function as well as
females may be especially sensitive to water-escape tasks resulting in a
associated neurobiological effects. Studies to date indicate that
more profound acute stress response than males (Perrot-Sinal et al.,
progesterone can influence oestrogen's effects on cognition, although
1996; Beiko et al., 2004). However, given that oestrogen-induced
the extent and direction of the phenomenon is unclear. For example,
enhancements have been shown on similar tasks that use different
while the current study found that progesterone counteracted estradi-
motivators, for example the water-escape (Bimonte & Denenberg,
ol's beneficial effects on spatial reference memory in aged Ovx rats,
1999) and appetitively motivated (Luine & Rodriguez, 1994; Daniel
another study showed that weekly injections of estradiol plus
et al., 1997; Fader et al., 1999) radial-arm maze, it is unlikely that the
progesterone enhanced spatial T-maze performance of aged Ovx rats
motivator per se fully accounts for the disparate effects seen in the
relative to nonhormone-treated aged Ovx rats (Gibbs, 2000). However,
literature. Whether a task taps into working or reference memory also
because in the latter study no group received weekly injections of
does not organize the results on oestrogen and cognition, as positive
estradiol alone, it cannot be determined how the estradiol–progester-
effects have been seen on a variety of working memory (Bimonte &
one treatment group performed relative to a group receiving solely
Denenberg, 1999; Daniel et al., 1997, 2005; Fader et al., 1999;
estradiol. There is also evidence that oestrogen–progesterone interac-
Sandstrom & Williams, 2001; Bowman et al., 2002; Heikkinen et al.,
tions depend on age. Indeed, in middle-aged Ovx rats estradiol plus
2002; Holmes et al., 2002) as well as reference memory (El-Bakri
progesterone enhanced spatial reference memory (Markham et al.,
et al., 2004; Frick et al., 2002; Markham et al., 2002; Feng et al.,
2002) while, in contrast, in young Ovx rats estradiol plus progesterone
2004) tasks.
impaired spatial reference memory (Chesler & Juraska, 2000). As in
Some of the most compelling data in helping to clarify the
the latter study neither estradiol nor progesterone alone had effects,
oestrogen and cognition literature is that ovarian hormone milieu
their interaction was probably responsible for the observed deficits.
influences the strategy used to solve maze tasks. Daniel & Lee (2004)
Other rat studies have shown that progesterone injection several hours
recently showed that Ovx rats without estradiol treatment exhibited
before conditioned avoidance testing enhanced performance at the
compromised performance after removal of an intramaze static cue
estrous, but not the diestrous, estrous cycle phase, thereby suggesting
that was paired with the escape platform throughout testing, while Ovx
that circulating hormone levels at the time of, or immediately prior to,
rats given tonic estradiol were not disrupted. These data indicate that
progesterone exposure modulates progesterone's effects (Diaz-Veliz
estradiol may bias females against using intramaze information to
et al., 1994).
solve a maze task that could be solved spatially. Other data concur.
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 24, 229–242
H. A. Bimonte-Nelson et al.
Indeed, estradiol may bias females against using a response-based
This tenet is supported by the facts that the first portion of the
strategy and toward using a spatial strategy (Korol & Kolo, 2002;
experiment evaluating short-term treatment effects was not affected by
Davis et al., 2005). The probe trial results in our study indicate that all
mortality as the increase in death rates occurred after this portion of
female groups, with or without ovarian hormones, utilized a spatial
the study was completed, and, perhaps more importantly, we found a
strategy on the spatial task, and that none of the groups utilized the
dissociation between maze performance and mortality. Of the low- and
intramaze patterned cues on the cued task. However, while in the
high-dose tonic estradiol treatments, only low-dose estradiol enhanced
Daniel & Lee (2004) study rats could utilize either extramaze or
spatial reference memory. However, both tonic doses of estradiol
intramaze cue(s) to solve the Morris maze task, extramaze cues could
increased rate of death compared to Ovx rats not given estradiol.
not be successfully utilized to solve the cued task in the current study
Moreover, while progesterone counteracted estradiol's positive effects
as the intramaze and extramaze cues were dissociated by rotating the
on spatial performance, it also counteracted estradol's negative effects
intramaze cue patterns after every trial. Given the probe trial results for
on mortality. In fact, the total number of deaths was significantly lower
the cued task in the current study, it appears that when forced to use
in rats given estradiol and progesterone treatment than in those given
intramaze cues to locate a hidden platform, the presence of ovarian
estradiol alone. We find it especially remarkable that while middle-
hormones facilitates use of a more efficient motoric search strategy.
aged Ovx rats treated with tonic low- and high-dose oestrogen had
Our findings that estradiol enhances performance on a spatial memory
death rates of 89 and 78%, respectively, the death rate of rats given
task, but has limited effects on nonspatial cued learning in a task with
cyclic estradiol was 0%. None of the 12 cyclic estradiol rats died
similar procedural demands, add to the accumulating data to suggest
during the 17-week study. As cause of death was not determined, it is
that estradiol facilitates spatial but not cued or response maze
unclear why tonic low- and high-dose estradiol increased the death
performance in the rodent model. The group differences in scores may
rate while cyclic treatment did not, as well as why progesterone
in fact be due to the differences in strategy use between Ovx females
decreased death rate in estradiol-treated subjects. Other studies
with and without oestrogen replacement which, in turn, would result in
evaluating long-term effects of hormones have reported a high
what we interpret as better or worse scores based on our operational
mortality rate as well (e.g. Gibbs, 2000). Further, while it may be
definition of performance.
tempting to speculate that the cause of death resulting from the tonic
Data suggest several possible neurobiological mechanisms whereby
estradiol treatment in the current and other rat experiments may relate
ovarian hormones might affect learning and memory. The accumula-
to issues regarding mortality rates and disease occurrence in meno-
ting literature points to a role for the cholinergic and neurotrophic
pausal women taking hormone replacement, the lack of autopsy in our
systems as a target of ovarian hormone actions. We have recently
deceased subjects limits extrapolation of the reported rat mortality
shown that tonic estradiol treatment increases the neurotrophins NGF,
findings to those of more clinical contexts. Clearly, studies should
BDNF and NT3 in the entorhinal cortex of aged Ovx rats, and that
further evaluate these effects.
progesterone reversed these effects (Bimonte-Nelson et al., 2004a).
Finally, while many clinical studies have shown positive effects of
Neurotrophins have been linked to memory function, and the
ovarian hormone treatment in menopausal women with or without
entorhinal cortex is functionally related to processing in the hippo-
Alzheimer's disease, several recent larger controlled clinical studies
campus, a brain region intimately involved in spatial learning and
report null or negative effects (Cholerton et al., 2002 for review; see
memory. Hence, given the recurrent findings that oestrogen enhances
also Rapp et al., 2003 and Shumaker et al., 2003, 2004). Thus far, few
spatial cognition, ovarian hormone effects on neurotrophic factors in
studies have directly compared hormone replacement therapies with
the entorhinal cortex may be one mechanism underlying the observed
and without progesterone. In fact, one study that did separate
benefits by estradiol as well as our new finding of progesterone's
oestrogen- from combination oestrogen- and progesterone-treated
counteractive effects. Furthermore, the oestrogen-induced spatial
subjects found that the combined treatment negatively influenced
memory enhancement seen in the current and other reports coincides
cognition while oestrogen alone did not (Rice et al., 2000). Results
with evidence that oestrogen has effects on physiology, connectivity
from the current and previous basic science reports add to the
and function of the hippocampus, including increases in dendritic
accumulating literature identifying progesterone as a potentially
spine density and reductions in GABA neurotransmission (Gould
crucial factor that could affect the neural and cognitive outcome of
et al., 1990; Woolley & McEwen, 1992; Murphy et al., 1998). It is
hormone replacement therapy (Woolley & McEwen, 1992; Johansson
intriguing that some of these estradiol-induced neural effects are
et al., 2002; Nilsen & Brinton, 2002a,b; Bimonte-Nelson et al.,
counteracted by concurrent treatment with progesterone, as seen, for
2004a,b). Future research testing specific cognitive and neural effects
example, in studies showing that estradiol plus progesterone treatment
of progesterone, alone and with oestrogen, will be crucial to
did not yield an increase in hippocampal dendritic spine density as did
understanding the complex effects of ovarian hormone loss and
estradiol alone (Gould et al., 1990; Woolley & McEwen, 1992). Other
replacement. Identifying the effects of the various components of
work has shown that, while oestrogen protected against glutamate
hormone therapies, individually and in combination, would be the
toxicity in hippocampal and cortical neuron cultures, some progestin
optimal approach in attempting to converge the findings that appear
preparations enhanced this neuroprotection and another counteracted
contradictory. In fact, as new data emerge it may become clear that the
its effects (Singer et al., 1996; Nilsen & Brinton, 2002a, 2002b).
cognitive effects of hormone therapy are not contradictory at all.
Collectively these findings suggest that progesterone might have
Rather, they may be dependent on numerous variables not yet taken
different neural effects than estradiol, and that progesterone can
into account in many studies, including age at treatment, baseline
decrease estradiol's effects in some circumstances.
hormone status, time since onset of reproductive senescence, dose and
In the current report we sought to compare short- vs. long-term
type of hormone therapy and the specific memory task examined.
cognitive effects of hormone replacement. However, because therewas a dramatic loss of rats within specific groups between the first andsecond testing sessions many group comparisons from the second test
are rendered uninterpretable. We believe that the high mortality rate
This research was funded by grants awarded to H.A.B.-N. [RO3 grant from the
seen in specific groups in the current experiment does not undermine
National Institute on Ageing (AG023925-01) and a Specialized Center of
the observed cognitive effects for the short-term treatment assessment.
Research Pilot Grant cofunded by the Office of Research on Women's Health at
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 24, 229–242
Progesterone–oestrogen interactions on cognition
MUSC and NIDA (P50 DA16511)]. The authors wish to thank Dr Victor
Diaz-Veliz, G., Benavides, M.S., Butron, S., Dussaubat, N. & Mora, S. (1999)
Denenberg for valuable input on the mortality data, Dr Craig Enders for advice
Behavioral effects of dopamine agonists and antagonists: influence of estrous
regarding potential ipsative data issues and Dr Karyn Frick for valuable
cycle, ovariectomy, and estrogen replacement in rats. Pharmacol. Biochem.
information regarding the oestrogen injection protocol.
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given cyclic estadiol plus progesterone; Ovx-CycE, Ovx given cyclic estradiol;
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