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Previous Article | Next Article
The Journal of Neuroscience, December 15, 2002, 22(24):10985-10995
Effectiveness of Estrogen Replacement in Restoration of Cognitive Function after Long-Term Estrogen Withdrawal in Aging Rats
Alicja L. Markowska and Alena V. Savonenko Neuromnemonics Laboratory, Department of Psychology, Johns Hopkins University, Baltimore, Maryland 21218
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Recent studies suggest that some aspects of learning and memory may be altered by a midlife loss of estrogen, indicating a potential causal relationship between the deficiency of ovarian hormones and cognitive aging. In this study, the effects of estrogen withdrawal and replacement were tested in middle-aged Fischer-344 rats using different memory tasks. Estrogen withdrawal accelerated the rate of cognitive aging. A deficit first occurred 4 months after ovariectomy in working memory, which was tested in a delayed-nonmatching-to-position task, and progressed from long-delay to short-delay trials. Reference memory, which was tested in a place discrimination task and a split-stem T-maze, was not affected by aging or ovariectomy. The efficacy of estrogen in ameliorating the cognitive deficit in old rats depended on the type of treatment (acute vs chronic) and whether the aging-related decline in a particular cognitive process was aggravated by estrogen withdrawal. Chronic estrogen treatment (implants) was effective in improving working memory only when primed with repeated injections of estrogen, indicating that simulating the estrogen fluctuations of the estrous cycle may be more effective than the widely used mode of chronic pharmacological treatment. A challenge with scopolamine revealed that ovariectomy-induced cognitive deterioration coincided with a compromised cholinergic system. Importantly, the estrogen treatment that had restored effectively the cognitive abilities of old ovariectomized rats did not reduce their sensitivity to scopolamine. Taking into consideration that estrogen was highly effective against the amnestic action of scopolamine when tested in young-adult rats, these data emphasize that mechanisms of the protective effect of estrogen differ in young and old rats.
Key words: ovariectomy; cholinergic system; scopolamine; working memory; attention; body weight
INTRODUCTION
Recent studies suggest that some aspects of learning and memory may be altered by the midlife loss of estrogen (Sherwin, 1988
). These changes can be ameliorated by estrogen replacement therapy (Ditkoff et al., 1991
Our previous studies have indicated that there is no cognitive deficit immediately after ovariectomy, and that the deleterious effect on cognition is delayed for a longer period of time in young females than in older females (Markowska and Grinnell, 1999
The first goal of this study was to assess the effects of estrogen withdrawal on the rate of cognitive aging in two groups of middle-aged female rats: (1) the control group that had intact ovaries and naturally decreasing estrogen levels during aging and (2) the ovariectomized rats that had undergone aging along with a surgically reduced level of estrogen. Middle age coincides with the onset of an age-related loss of ovarian hormones; therefore, middle-aged female rats may be an ideal model to test the effects of estrogen replacement on memory during aging (Markowska, 1999b
When tested in young-adult rats, the duration and modes of estrogen treatment have been shown to be critical factors in determining the physiological (Gorzalka and Moe, 1994
The third goal of this study was to evaluate the ability of estrogen to enhance cholinergic function during aging. A muscarinic antagonist, scopolamine (SCOP), was used before and after the estrogen treatment in the old females to estimate the efficacy of estrogen in counterbalancing the amnestic effects.
MATERIALS AND METHODS
Subjects
Thirteen-month-old Fischer-344 female rats (n = 30) were obtained from the colony at the National Institute of Aging. The rats (three rats per cage) were housed on a 12 hr light/dark cycle. Food and water were provided ad libitum except for the periods of behavioral testing in the appetitively motivated tasks, in which water deprivation was necessary to create appropriate motivation for the task (for details, see DNMP task). To avoid the estrogen-like effect of phytoestrogens, which are found in many laboratory animal diets (Thigpen et al., 1999
Group treatment
After a 5 d acclimation period, rats were divided randomly into two groups. One group (n = 20) received ovariectomy (OVX), whereas the other group (n = 10) received sham surgery with skin and muscles cut but ovaries spared (Sham). After the recovery period (3 d), each rat was handled for 5-10 min daily over 5 d.
The rats were tested repeatedly during seven testing periods (periods 1-7), starting 1 month after ovariectomy and continuing for the next 9 months (Fig. 1). Behavioral testing included the delayed-nonmatching-to-position (DNMP) task, the spontaneous alternation task, place and visual discrimination, straight swim, and repeated acquisition in the water maze.
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Figure 1. Design of the experiment and group treatment. Sham, Control rats with sham surgery in which ovaries were spared. OVX, Ovariectomized rats. E, Treatment with 17 -estradiol: i, acute, with injections; m, chronic, with implants. Injections of SCOP were applied during periods 4 and 5.
Six months after ovariectomy, during period 5, the OVX rats were divided randomly into two subgroups. The first half of OVX rats (OVX1 group) and Sham rats were injected subcutaneously with 0.1 ml of sesame oil, whereas the second half of OVX rats (OVX2) received subcutaneous injections of estrogen (10 µg of 17
SCOP challenges
During period 4, both Sham and OVX rats received intraperitoneal injections of saline (0.9% NaCl solution; Sigma, St. Louis, MO), SCOP (0.05, 0.1, and 0.2 mg/kg in saline; Sigma), or methylscopolamine (0.2 mg/kg in saline; Sigma) 20 min before behavioral testing. Each drug and dose was applied three times in a semirandom order with intertreatment intervals of 3-4 d.
During period 5, SCOP was administered to Sham, OVX1, and OVX2 rats twice; in the OVX2 group, SCOP was administered after the second and the fourth set of estrogen injections. Because at that time all rats were already 20 months of age, only the minimal dose of SCOP (0.05 mg/kg) was used.
Ovariectomy
The rats were anesthetized with an intraperitoneal injection of Nembutal (40 mg/kg; Abbott Labs, Abbott Park, IL). Fur on both sides of the body was shaved from a hip to the lowest rib. Bilateral ovariectomies were performed using an incision 1.5 cm inferior to the palpated rib cage. Ovaries and surrounding fat tissue were removed; the incision was closed by suturing the muscles and stapling the skin. Antibiotic gel was applied on the wound, and the animal was placed on paper towels in a temperature-controlled cage until awakening.
Vaginal cytology
To monitor the stages of estrus cyclicity, cell samples were obtained by vaginal lavage, fixed, and stained in accordance with the Wright-Giemsa method (Hema 3, Fisher Scientific, Houston, TX). Next, the vaginal smears were investigated under the microscope and diagnosed for estrus stage (i.e., estrus, diestrus, or proestrus). Estrus stage was defined by the predominance of cornified cells in the vaginal lumen. The following period in which the cornified cells became less numerous and leukocytes and nucleated cells were present was defined as diestrus. Proestrus was defined as the time during which the vaginal smear was characterized by the predominance of nucleated epithelial cells (Feder, 1981
Estrogen treatments
The subcutaneous injections of estrogen contained 10 µg of 17
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Figure 2. The schedule of 17
Silastic tubing pellets (length, 5 mm; inner diameter, 1.47 mm), filled for 3 mm with 17
Sampling estradiol concentration
Once per month, the rats were anesthetized with gas anesthesia (see above), and a vein on the side of the tail was cut 5-6 cm up from the tip. Using peristaltic motion, 1 ml of blood was collected into an Eppendorf tube containing 0.1 ml of heparin. Plasma was collected by spinning the tubes for 30 min at 4°C and then stored at -70°C until ready for 125I-radioimmunoassay for 17
Behavioral testing
DNMP task
Rats were tested in a split-stem T-maze that has been described previously (Hepler et al., 1985Water maze tasks
Water maze apparatus. A large metal pool (180 cm in diameter) filled with opaque water at 24 ± 2°C and a 10 cm platform were used in place discrimination, repeated acquisition, and visual discrimination tasks. A straight plastic alley (10 × 70 cm) was placed in the large pool and used during the straight swim test to estimate swimming ability. To prevent fatigue and changes in thermoregulation, the rat was removed from the pool and dried with a towel after each trial. The tracking system (HVS Image Analysis VP-200; HVS Image, Hampton, UK) acquired images from a camera mounted 1.4 m above the surface of the water. Eight 40 W bulbs, mounted in a parallel configuration 1.2 m above the surface of the water, provided lighting. Straight swim test. This test, described in detail previously (Markowska et al., 1993Spontaneous alternation in Y-maze
Each arm of the Y maze was 55 × 15 cm. The rat was placed in one arm and allowed to move freely through the maze for a 5 min test session. The sequence of arm entries was recorded. An alternation was defined as the number of triads containing entries into all three arms divided by the maximum possible alternations (Stone et al., 1992Data analysis
The effect of ovariectomy during periods 1-4 was analyzed as a between-subject main effect, with ANOVA conducted for each period separately. The effects of ovariectomy and estrogen treatments during periods 5-7 were analyzed by ANOVA with a main effect of groups (Sham, OVX1, and OVX2) and were followed by post hoc tests. The variables of performance in different sessions or delays, or after different doses of SCOP treatment, were used as repeated measures in a mixed-design ANOVA. The performance in consecutive testing periods was analyzed in a repeated-measures omnibus ANOVA. Post hoc Newman-Keuls tests were applied to significant effects and interactions to estimate the differences in the focused set of means. Some differences in the levels of degrees of freedom reported in ANOVAs reflected the data withdrawal of rats excluded from the study because of temporal or permanent health problems (fur appearance, food consumption, cataracts, etc.). When ANOVA yielded a significant effect for a set of variables (for example, all variables of platform trials), F and p values are presented as a range from minimum to maximum (F(df) = minimum
maximum, minimum < p < maximum). Body weight was used as a covariance to adjust the significance of between-group differences in speed of swimming. Because the effect of covariance may be overestimated if the group means for the measure used as a covariance are significantly different, additional analysis was performed using Pearson's correlation coefficient. The correlation between body weight and speed of swimming was assessed separately in Sham and OVX rats to avoid the potential confounding effect of between-group differences in body weight. All statistical analyses were conducted using Statistica 8.0 (StatSoft, Tulsa, OK).
RESULTS
Changes in body weight in the course of estrogen withdrawal and replacement
Beginning at 10 weeks after surgery, OVX rats were significantly heavier than age-matched control rats (F(1,27) = 4.12; p < 0.05) (Fig. 3). This difference was observed during both food deprivation periods and food ad libitum periods. The estrogen injections (group OVX2, period 5) quickly abolished the effect of ovariectomy on body weight. Following treatment with implants (period 6) further decreased body weight below the level of the Sham group (F(1,11) = 12.32; p < 0.01). Estrogen implants also decreased body weight when applied in the OVX1 group (period 7); therefore, OVX1 rats were no longer different from Sham rats (p > 0.8). Therefore, the gain in body weight in OVX rats was reversed easily by both modes of estrogen treatment, injections and implants, indicating that the mechanisms of regulating of body weight by estrogen remained spared with aging.
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Figure 3. The dynamics of body weight in control (sham surgery, open squares) and experimental [ovariectomized OVX1 (gray circles) and OVX2 (open triangles) ] groups of Fischer-344 rats during periods 1-7 of behavioral testing. A decrease in body weight during every period was attributable to food deprivation during testing in the DNMP task. Note that OVX rats gained weight compared with Sham rats, and this effect of ovariectomy was abolished quickly by both types of estrogen treatment (injections and implants).
Changes in estrous cycle and in plasma estrogen levels in the course of estrogen withdrawal and replacement
At the beginning of the experiment, 13-month-old female rats showed irregular cyclicity with a high variability of the cycle duration (range, 3-10 d). However, a majority of cycles (79%) contained a proestrus stage. In Sham rats, the number of cycles with proestrus substantially decreased at the age of 15 months (36%) and was not detectable at the age of 18-19 months. The estrus cycles of 20- to 22-month-old Sham rats contained the long periods of diestrus (8-12 d), infrequently followed by the short estrous stages.
Ovariectomy in 13-month-old females resulted in cessation of estrus cycle, and the vaginal smears of OVX rats were characterized by a predominance of leukocytes (88% of slides). Estrogen treatment (injections and/or implants) administered 7-8 months after ovariectomy resulted in vaginal cytology with a prevalence of cornified cells (90% of slides).
Because 13-month-old females at the beginning of the experiment already showed irregular cyclicity, and because the probability of proestrus stage decreased further with more advanced age, the plasma was collected only during diestrus. The estrogen plasma level in the Sham group was ~10 ± 1 pg/ml during diestrus and did not change significantly across all ages tested (Fig. 4). Ovariectomized rats showed a very low level of estrogen, close to the minimum detectable by the assay used. Both types of estrogen treatments, injections and implants (Fig. 4), created estrogen concentration in plasma similar to physiological levels during proestrus (Feder, 1981
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Figure 4. Plasma level of estrogen in Sham (diestrus) and OVX rats during behavioral testing (periods 1-7). The gray bar indicates the level of estrogen 1 hr after injection of 10 µg of 17
DNMP task
Ovariectomy increases the rate of aging-associated decline in working memory
Ovariectomy did not affect the rate of acquisition in the DNMP task. The number of sessions it took to reach the criteria of 85% of correct arm choices in 2 consecutive days was 11.5 ± 1.6 and 11.7 ± 1.1 for the Sham and OVX groups, respectively. The percentage of correct alternations of the arms increased significantly during the first 6 d of training (F(5,140) = 8.12; p < 0.0001) and was not different between groups (F(1,28) = 1.57; p < 0.22). The acquisition of correct choices in the stem of the T-maze (reference memory) was also similar in Sham and OVX rats (F(1,28) = 0.60; p < 0.44). The accuracy of correct choices in the stem of the T-maze as a measure of reference memory remained very high in the course of aging (range, 90-100%; effect of testing periods; p > 0.6) and was not different among Sham, OVX, and estrogen-treated rats (p values > 0.7). There were no group differences in retention of correct stem choices between consecutive periods of testing (p values > 0.3). The number of correct arm choices as a measure of working memory decreased with aging. The omnibus ANOVA of choice accuracy in short-delay trials (intertrial interval, 30 sec) revealed a significant effect of period of testing (F(6,102) = 4.35; p < 0.001) and group (F(2,17) = 6.10; p < 0.01). However, there were no significant differences between OVX and Sham rats until 7 months after ovariectomy (group × period interaction: F(12,102) = 1.98; p < 0.01). The choice accuracy of Sham rats gradually declined after 19 months of age followed by a significant decrease at 22 months (post hoc test; p < 0.02) (Fig. 5). The OVX rats showed a significant age-related deterioration in working memory at 20 months of age (post hoc test; p < 0.01), i.e., 2 months earlier than Sham rats (Fig. 5).
When the demands on working memory were increased by the introduction of longer intertrial delays (1-30 min), ovariectomized rats showed a deficit in performance 4 months after ovariectomy, much earlier than that in regular, short-delay trials (Fig. 6). The introduction of 1 and 5 min delays 1 month after surgery (period 1) decreased the accuracy of arm choices (effect of delay: F(2,56) = 19.48; p < 0.0001) in both Sham and OVX groups (Fig. 6A). Importantly, the accuracy of choice after the 5 min delay was close to a chance level, yielding no baseline for using longer delays. Repeated testing decreased the sensitivity of performance to delays (periods 1 and 2) (effect of retesting: F(1,28) = 5.75; p < 0.03), which allowed the inclusion of 15 and 30 min delays during period 3. The most challenging 30 min delay resulted in significant differences between OVX and Sham groups (simple main effect: F(1,27) = 4.83; p < 0.05) (Fig. 6C). Even more robust between-groups differentiation was revealed in the variable of maximum consecutive correct choices (Fig. 6F). OVX rats showed significantly lower values of maximum consecutive correct choices in 5, 15, and 30 min delay trials (simple main effect; p values < 0.05). It is important to note that the between-group differences in this variable were revealed not only in the longest-delay trials (30 min), where the total number of correct choices was significantly lower in OVX rats, but also in trials where no deficit occurred in the number of correct choices.
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Figure 5. Aging-related changes in working memory tested in the DNMP task. Each point represents the average percentage of correct arm choices per group. Only sessions with regular, short-delay trials and without SCOP treatment are shown. The dashed line for the OVX2 group (open triangles) indicates the age-related decline before the estrogen treatment. The caret indicates the onset of the age-related decline in the appropriate group as a result of a post hoc test (p < 0.05). The asterisk indicates a significant difference from the OVX1 group (open circles) during the period (simple main-effect ANOVA; p < 0.05). Open squares, Sham.
The data showed that the onset of ovariectomy-induced impairment in working memory was first revealed in the trials with long delays and high demands on working memory (4 months after ovariectomy, or period 3) and was followed by an additional decline in regular, short-delay trials with lower demand on working memory (7 months after ovariectomy, or period 5).
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Figure 6. Delay sensitivity of working memory in Sham (open bars) and OVX (shaded bars) rats. A-C represent the average percentage of correct arm choices in trials with different delays. D-F represent the maximum of consecutive correct choices. Asterisks indicate significant effects of ovariectomy as a result of a simple main-effect ANOVA (p < 0.05).
Ovariectomy increases cognitive sensitivity to SCOP in aging rats
To estimate the effect of compromised cholinergic functioning on T-maze alternation, both ovariectomized and control rats were injected with SCOP. Different doses of SCOP (0.05, 0.1, and 0.2 mg/kg) were administered systemically during period 4 of behavioral testing, when the performance of OVX rats was not different from Sham rats in regular, short-delay trials. Ovariectomized 19-month-old rats were more sensitive to the effect of SCOP than age-matched control rats. In contrast to Sham rats, OVX rats already had shown a significant decline in choice accuracy after the lowest dose of SCOP (effect of dose: F(3,42) = 9.39; p < 0.0001) (Fig. 7). Sham rats decreased their choice accuracy after an increasing dose of SCOP, but this decline did not reach significance (p > 0.07). Moreover, the decline in choice accuracy induced by SCOP treatment remained in OVX rats for at least 3 d, resulting in significant differences between OVX and Sham rats in sessions without SCOP injections (F(1,18) = 5.29; p < 0.05).
However, when treatment with estrogen improved the choice accuracy in OVX rats (period 5), the lowest dose of SCOP abolished the effect of estrogen (Fig. 7). SCOP was administered after the second and fourth sets of estrogen injections. However, in both cases the estrogen-injected OVX2 rats significantly decreased the accuracy of choices (F(1,7) = 6.92; p < 0.05) and were not different from OVX1 rats, with a performance close to a chance level.
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Figure 7. Effect of systemic SCOP (Sc) administration on the accuracy of correct arm choices in OVX (gray circles) and Sham (open squares) groups (19-month-old rats, 6 months after ovariectomy). The inset shows the effect of the lowest dose of SCOP tested 1 month later. Note that OVX rats that received estrogen treatment (group OVX2) were still more sensitive to SCOP than Sham rats. Single and double asterisks indicate a significant SCOP-induced decline as a result of a post hoc test applied to the significant effect of dose in the OVX group (p < 0.01 or 0.001, respectively). A decline in the performance of Sham rats did not reach significance (p > 0.07). The pound sign indicates a significant effect of SCOP as a result of a one-way, repeated-measures ANOVA (p < 0.05).
Efficacy of different modes of estrogen treatment in ameliorating the cognitive deficit
After OVX rats showed a significant age-related decline in working memory in regular, short-delay probe trials (Fig. 5, period 5), one-half of ovariectomized rats (OVX2) received subcutaneous injections of 17
During period 6, the OVX2 rats, which previously received injections of estrogen, were implanted with pellets containing 17
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Figure 8. Effect of 17
Spontaneous alternation in the Y-maze task
No differences were observed between Sham and OVX groups, indicating that the ovariectomized rats showed deficits in working memory compared with control rats only when higher demands on working memory were imposed, such as in the DNMP task.
Place discrimination in the water maze
No differences in spatial reference memory were observed between control and OVX rats up to 9 months after surgery (Fig. 9A,B). The only ovariectomy-induced difference in place discrimination performance was observed for the variable of speed of swimming (Fig. 9C). Starting 1 month after surgery, OVX rats swam significantly slower than Sham rats (F(1,28) = 4.69-33.32 for periods 1-7; 0.001 < p < 0.05). Estrogen replacement did not reduce existing differences in swim speed, although it brought back the body weight to the level of Sham rats.
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Figure 9. Performance of Sham and ovariectomized (groups OVX1 and OVX2) rats in platform (A) and probe (B) trials of the place discrimination task. The dotted line in B indicates chance level. The average speed of swimming during platform trials is shown in C. For treatments of OVX1 (gray circles) and OVX2 (gray triangles) groups, see Figure 1. Pound signs indicate a significant improvement in place discrimination attributable to retesting compared with period 1 (post hoc test applied to a significant effect of period; p < 0.05). Asterisks indicate a significant difference between control and ovariectomized rats (OVX1 and OVX2) as a result of a post hoc test applied to a significant effect of group (p < 0.05). Open squares, Sham.
In the range of ages tested (13-22 months), no effect of aging was revealed. Moreover, repeated testing improved the performance of old rats in both platform (Fig. 9A) and probe (Fig. 9B) trials compared with middle-aged naive rats (effect of period for different variables: F(4,68) = 12.34-45.98; p values < 0.01).
Although the place discrimination task is one of the most sensitive tasks to detect the effect of aging on reference memory (Gage et al., 1984
Repeated acquisition in the water maze
The interaction of the effects of aging and repeated testing in the repeated acquisition task was more complex than in the place discrimination task. At 17 months of age, the rats were tested in this task for the first time (Fig. 10, period 3). Sham and ovariectomized rats learned the new positions of the platform and did not differ with regard to the time spent in the correct quadrant (Fig. 10A,B).
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Figure 10. The preference for the quadrants with and without the platform tested after the first (A) and second (B) session in the repeated acquisition task. Each bar indicates the time spent in the quadrant with the most recent platform location (mean ± SEM). The time spent in the quadrant with a previous platform location is indicated as a marker (mean). Thick lines indicate a range of preferences for two other nonplatform quadrants. The previous correct quadrant for session 1 was a quadrant with a platform location used in the place discrimination task. See Figure 1 for treatments for the OVX1 and OVX2 groups. Significant between-groups differences in the preference for the correct quadrant were revealed only in the first session, in which a change in the platform location coincided with partial changes in spatial cues. Asterisks indicate a significant difference from Sham rats as a result of a post hoc test applied to a significant effect of group (p < 0.05).
When tested at 21 months of age, preference to the correct quadrant significantly declined in both Sham and OVX1 rats (session 1) (effect of period: F(1,16) = 9.32; p < 0.01) (Fig. 10A). Importantly, the memory of old rats for the new platform location was not accurate, because preference was distributed between two quadrants (Fig. 10A, period 6). Importantly, the second quadrant with a high preference was one of the adjacent quadrants but not the one with the old platform location. This indicates that poor memory for the platform location after the first session of the repeated acquisition task was not a result of interference with memories for the old platform location but, instead, of difficulties in learning the new position of the platform.
In the first session the change in the platform location was accompanied by the alteration in spatial cues. Notably, because only some of the spatial cues were changed, whereas others such as the geometry of the room and the location of light sources were the same, the rat's navigation could have been affected by the interference between the new and the old set of spatial cues. This additional complexity of the first session, when learning of the new platform location required the complex differentiation between interfering sets of spatial cues, was the most challenging stage of learning for 21-month-old rats.
Estrogen replacement in the OVX2 rats that received both types of treatment (injection and implants, period 6) increased the preference of the correct quadrant at the end of the first session (effect of group: F(2,32) = 77.52; p < 0.0001) (Fig. 10A). However, 21-month-old OVX2+Ei+Em rats, which had been equally successful in the preference of the correct quadrant as 17-month-old rats, showed as high a preference of one of the adjacent quadrants as was observed in 21-month-old Sham and OVX1 rats. This indicates that the estrogen treatment ameliorated the aging-related decline but did not change markedly the strategy used by old rats to cope with a poor memory of the new platform location.
During the second session of period 6 (Fig. 10B), when a set of spatial cues remained the same and only the platform location was changed, Sham and OVX1 rats learned the new platform location more successfully than during the first session and were no longer different from estrogen-treated OVX2+Ei+Em rats (session × group interaction: F(4,32) = 3.84; p < 0.02). Compared with period 3, sham and ovariectomized rats improved their performance (session 2) (effect of period: F(1,16) = 12.42; p < 0.01) because of the effect of retesting and/or because of a different strategy than that used by 17-month-old naive rats (see different ranges of time spent in nonplatform quadrants in Fig. 10B, periods 3-6).
When tested 1 month later (period 7), all three groups improved performance during the first session (effect of period: F(1, 16) = 21.39; p < 0.001) (Fig. 10A). With additional training, 22-month-old Sham rats were able to learn the new position of the platform at a level similar to 17-month-old rats. These data are consistent with our previous results, which showed a protective effect of training against an aging-related decline in the repeated acquisition task in Fischer male rats (Markowska and Savonenko, 2002
An alternative explanation of the performance differences between periods 6 and 7 during the second session is that the strategy applied by the rats in period 7 was different and presumably less effective when the platform was changed repeatedly. During period 6, the high preference for the correct quadrant (group OVX2+Ei+Em in session 1 and all groups in session 2) coincided with a low visiting (below the chance level) of the two other quadrants (the previous correct quadrant was one of those quadrants). During period 7, only one of the nonplatform quadrants was rejected, and two others (including the previous correct quadrant) were visited at the chance level. This switch in the rejection of the previous correct quadrant resulted in a strategy that would be less and less effective with consecutive changes in the platform location. As was shown in male rats, an ability to reject the previous platform location significantly declines between 18 and 24 months of age (Markowska and Savonenko, 2002
Importantly, 22-month-old OVX2 rats that previously received both types of estrogen treatment were able to reject the previous platform location during the first session. However, these rats switched to the strategy inherent to age-matched controls when the platform location was changed again (session 2). These data suggest that estrogen treatment may partially ameliorate the aging-related changes in strategy rather than cause the differences in strategies between old Sham and estrogen-treated OVX rats (Fig. 10A, period 7).
There were no between-group differences in latency and distance in the platform trials in any of the testing periods. All groups shortened distance and latency to find the platform during every period of testing (effect of trials; p values < 0.01). The pattern of differences in swim speed was similar to that in the place discrimination task.
Compared with the DNMP task, the aging-related decline in the repeated acquisition task was less robust and was ameliorated by additional training. A relatively lower demand on working memory in the repeated acquisition task, in which the information changes between sets of trials but not in each trial as in the DNMP task, could contribute to this difference. Importantly, unlike the DNMP task, the age-related deficit in the repeated acquisition task was not aggravated by ovariectomy. In this case, the chronic estrogen treatment (implants) was sufficient to ameliorate the aging-related changes.
Straight swim and visual discrimination in the water maze
A decrease in swim speed in the straight alley was detected in OVX rats 4 months after surgery (period 3) (F(1,27) = 8.56; p < 0.01). It is noteworthy that an ovariectomy-induced alteration in swim speed was detected earlier in the place discrimination task, in which an uncertainty of the situation is higher and consequently more stressful for rats than in the straight alley. This suggests that ovariectomy in middle-aged rats affected an emotional rather than a motivational component of escaping from the water.
The other factor that could affect the speed of swimming is body weight (Markowska, 1999a
The estrogen treatment (injections and implants; OVX2 group; period 6) did not significantly affect the swim speed compared with the nontreated ovariectomized rats (p > 0.2). Furthermore, at 22 months of age (period 7), when both groups of ovariectomized rats (OVX1 and OVX2) received estrogen replacement for >1 month, the body weight of the OVX1+Em and OVX2+Em rats was similar to or lower than the body weight of Sham rats (Fig. 3). However, the speed of swimming of estrogen-treated rats was still significantly lower than that of Sham rats (effect of group; p < 0.01).
There was no significant effect of ovariectomy or estrogen treatment on performance in the visual discrimination task (distance to visible platform; effect of group; p > 0.3).
DISCUSSION
This study demonstrated that the rate of age-related decline in various cognitive functions was differentially affected by estrogen withdrawal. Accelerated aging in working memory function, but not in reference memory, was the most prominent consequence of estrogen withdrawal in middle-aged females. The cognitive impairment after ovariectomy was gradual (taking several months to be detected), initially occurred in tasks that placed more demands on working memory, and then was detected progressively in the easier tasks. The efficacy of estrogen in reversing the deficit in working memory as tested in the DNMP task was increased by priming the chronic estrogen administration (implants) with repeated injections, which more closely mimicked the natural estrogen cyclicity. The age-related deficit in working memory, as tested in the repeated acquisition task, was not accelerated further by estrogen withdrawal and was more responsive to both modes of estrogen replacement (injections and implants). Finally, a challenge with the muscarinic antagonist SCOP during estrogen withdrawal revealed that the ovariectomy-induced cognitive deterioration coincided with a compromised cholinergic system. Nonetheless, the estrogen treatment that had effectively restored working memory in old ovariectomized rats failed to block the amnestic action of SCOP.
These results support the hypothesis that the sensitivity of a particular cognitive process to estrogen withdrawal parallels its sensitivity to aging. In a longitudinal design of this experiment, where the same rats are tested repeatedly, the DNMP task was the first, from a battery of cognitive tasks, to show a detrimental effect of ovariectomy. This task was also the most sensitive to the effect of aging. The cognitive sensitivity to both aging and estrogen withdrawal may depend on a working memory component in the task, with the highest demand in the DNMP task and the lowest demand in the place discrimination task. The effects of estrogen withdrawal in aging rats seemed to mirror the effects of estrogen replacement in young animals, in which estrogen facilitates performance as working memory loads increase (Bimonte and Denenberg, 1999
Importantly, the measure of maximum consecutive correct choices in the DNMP task was the most sensitive to ovariectomy-induced changes in cognition. In contrast to regular measures of working memory (the percentage of correct choices), this measure was significantly affected in OVX rats in a wide range of delays (5-30 min). This indicated that the ovariectomy-induced alteration in the maximum of consecutive correct choices was not attributable to the lower level of correct choices in OVX rats. The maximum number of consecutive correct choices reflects the extent to which correct performance can be maintained over a prolonged period of time. Although the DNMP task was not designed specifically to test attention span, this variable can be interpreted as a measure of sustained attention. The assessment of attention may be conducted not only by using a particular behavioral task but also by measuring the commonality engaged in different types of procedures (Bushnell, 1998
An alternative explanation of ovariectomy-induced differences in the consecutive correct choices is a possible effect of estrogen withdrawal on strategies during performance in the DNMP task. One may suggest that ovariectomy could affect the balance between place/nonplace strategies that is shown to be sensitive to estrogen manipulations (Korol and Kolo, 2002
At the age at which memory impairment was detected only in OVX rats but not in Sham rats (17-20 months), the functions of ovaries in Sham rats already had been compromised substantially by aging, as indicated by the termination of the proestrus stage. Thus, the differences in working memory between old OVX and Sham rats were not caused by the actual drop in estrogen at the time of testing but rather by consequences of estrogen withdrawal at a younger age, when ovaries were more functional. The persistence of the effect of estrogen well beyond the period of exposure has also been documented in the working memory of young rats (Daniel et al., 1997
The central cholinergic system is a good example of a system in which the interaction of the effects of the aging processes (Biegon et al., 1986
The question of whether the deficit in working memory induced by long-term estrogen withdrawal can be ameliorated or reversed by estrogen treatment if applied at advanced ages has very important clinical implications. The enhancing effect of estrogen on working memory was documented repeatedly in young rats (O'Neal et al., 1996
However, when treatment with implants was primed with estrogen injections, the working memory of old ovariectomized rats was improved significantly, indicating that cognitive sensitivity to estrogen treatment at advanced age could be restored. This also indicated that a low effectiveness of estrogen implants in old rats was not caused by an insufficient level of estrogen but, rather, by a lack of readiness of the organism to respond to such treatment. The mechanisms of priming with estrogen injections might be related to the cyclical nature of the replacement as well as to the high levels (although brief) of estrogen in the first minutes after injections. Although additional studies are needed to differentiate between these possibilities, the present data clearly indicate that the efficacy of estrogen treatment on the cognition of old rats is highly successful with a phasic but not tonic replacement paradigm. In view of the negative results of the recently halted clinical trial in which estrogen (together with progesterone) was administered daily (Fletcher and Colditz, 2002
FOOTNOTES Received May 20, 2002; revised Oct. 3, 2002; accepted Oct. 3, 2002.
This study was supported by National Institute on Aging Grant AG15947 to A.L.M. We thank Y. Aguirre, J. L. Morton, K. Phelan, and D. Waters for their assistance with behavioral testing and J. L. Morton and L. Sims for editorial assistance.
Correspondence should be addressed to Alicja L. Markowska, National Institute on Aging, Gateway Building, Suite 2C-212, Bethesda, MD 20892. E-mail: markowsa{at}nia.nih.gov.
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