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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.
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ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 24, 229–242

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