The current study expanded on our previous findings by exploring the effects of chronic exposure to physiologic doses of estradiol replacement on an operant version of the DSA task in ovariectomized young, middle-aged, and old rats. Previously, we had examined the effects of chronic exposure to a high physiologic dose of estradiol on this same task in ovariectomized young adult female Long Evans rats (Wang et al., 2008
). In that study, rats with Silastic implants containing 10% estradiol had a clear deficit in proportion correct on the DSA task relative to control rats with cholesterol vehicle implants. In the present study, a similar impairing effect of estradiol was observed in rats with either a 5 or a 10% estradiol implant. Specifically, the estradiol-treated rats performed significantly worse than did rats with cholesterol implants in all but the first block of testing, and at all but the longest (18 sec) delay, where all rats were performing close to chance. The lower proportion correct in estradiol-treated groups was the result of increases in both win-stay and lose-stay errors relative to the control group, indicating that the poorer performance of estradiol-treated rats was the result of an overall increase in the error rate, rather than an increased propensity to make a particular type of error. There were also some subtle effects of estradiol treatment in the two training stages that preceded DSA testing, suggesting that estradiol replacement may have impaired not only DSA performance, but also the acquisition of the basic alternation response.
It is important to note that the rats in this study were tested in a single day on a place vs. response dual solution T-maze task prior to DSA testing. While there is a possibility that this prior experience may have influenced performance on the subsequent DSA task, this seems unlikely given that the rats in the previous study described above did not undergo the T-maze testing, but showed very similar estradiol-related deficits on the DSA task (Wang et al., 2008
). Whether or not a single day of testing would modify estradiol effects on DSA in middle-aged and old rats is unkown.
The blood estradiol levels in both of the estradiol-treated groups tested in this study were significantly higher than levels in the vehicle control group. However, the blood estradiol levels in the two exposure groups did not differ significantly from each other. The results of the DSA task reflect this with both estradiol-treated groups showing a similar degree of impairment relative to control. Although we only assessed serum estradiol levels at one time point (2 months after implant) in the current study, in an earlier study we sampled rats with similar estradiol implants 1, 2.5 and 4 months after the capsules were implanted and did not see a significant decline in blood estradiol levels over that time period (Wang et al., 2008
It is important to note that the circulating estradiol levels measured in the vehicle implanted control rats were higher than would be expected after ovariectomy. Commercial RIA kits—which were designed for use with human samples—have been shown to produce discrepant results when used to measure estradiol in rat sera, possibly due to interactions of the immunochemical reagents with the rat serum matrix (Strom et al., 2008
). Because of the problems we encountered with the samples from this study, we recently compared the Coat-a-Count RIA kit utilized in this study (Siemens #TKE21, Los Angeles, CA) with an estradiol double antibody RIA kit available from the same company (Siemens # KE-2D1, Los Angeles, CA). We found serum estradiol levels from ovariectomized female rats with cholesterol implants similar to those used in the current study to be below the level of detection with the double antibody kit, while these same serum samples run in parallel using the Coat-a-Count RIA resulted in serum estradiol levels very similar to those reported for the cholesterol implanted rats in (Unpublished data). Unfortunately, we do not have serum left from the current study that can be reanalyzed using the double antibody kits. However, given the results from our follow-up study, we are confident that the OVX rats tested in this study had very low serum estradiol levels, and that the 5% and 10% estradiol replaced groups received physiologically relevant, albeit similar, levels of estradiol replacement.
The effects of estradiol on working memory have been found to be variable and complex, likely due to the fact that working memory has many components and has been assessed using a variety of different tasks that tap different brain pathways/memory systems. Most human studies have found hormone replacement in postmenopausal women to improve verbal learning and memory (for review, see Rice et al., 1997
), as well as other prefrontally mediated aspects of working memory (Duff and Hampson, 2000
). However, recently the WHISCA has reported conflicting results, finding that women taking hormone replacement had impaired verbal working memory relative to women not on hormone replacement (Resnick et al., 2006
). Most of the women in the WHI began HRT a number of years after menopause, but recently a smaller randomized, double blind trial reported similar negative effects of HRT on verbal working memory in younger, recently postmenopausal women (Maki et al., 2007
). Studies using older female non-human primates have also reported that estrogen status of the animal has disparate cognitive effects, improving some cognitive functions but not others. Estrogens appear to improve or have no effect on performance on the prefrontally mediated delayed response working memory task (Rapp et al., 2003
; Roberts, Gilardi et al., 1997
) and also to mildly improve performance on the more hippocampal delayed non-matching to sample recognition memory test (Lacreuse et al., 2000
; Rapp et al., 2003
). These findings are in contrast to our results showing impaired performance on a prefrontally mediated working memory task in a rodent model.
Very few rodent studies have assessed the effects of estradiol treatment on prefrontal tasks in rodents. Our own research showed estradiol to impair performance on prefrontally mediated tasks in young ovariectomized females (Wang et al., 2008
), a finding that is further supported by the current results. Wide et al. (2004)
also found that chronic estradiol treatment impaired performance on a T-maze version of the DSA task. McGaughy and Sarter (1999)
found that ovariectomized rats did better than sham-operated rats on an attentional task that is sensitive to lesions of either the prefrontal cortex (Muir et al., 1996) or the medial forebrain cholinergic input to the prefrontal cortex (McGaughy et al., 2002). In contrast,, Barnes et al. (2006)
reported that attention was impaired in ovariectomized rats and that estradiol treatment ameliorated the deficit. However, unlike our studies or the McGaughy and Sarter study, the rats in Barnes study were ovariectomized after rather than before acquisition of the task. In summary, while there are some studies to the contrary (e.g. Barnes et al., 2006
), the effects of estradiol on prefrontally-mediated tasks appear to be mostly negative. This differs from the findings of numerous rodent studies where estradiol has been shown to improve performance on behaviors such as place learning, which are more hippocampally mediated (Gibbs, 2000
; Korol and Kolo, 2002
; Luine et al., 1998
; Zurkovsky et al., 2006
The rats in the current study were maintained on a soy free diet, whereas most other rodent studies have used soy-based diets containing phytoestrogens. The degree to which estrogens from the diet interact with the experimental estrogen treatments to alter behavioral outcomes is unknown and would be difficult to determine without studies specifically designed to address this question. However, it is well-established that the amount of soy phytoestrogens in rodent diets varies widely depending on the supplier and the lot number of the feed (Thigpen et al., 2004
). These differences could be one factor contributing to the variability of the results that have been observed across studies. Although, in a recent experiment, two days of systemic estradiol treatments produced robust impairments in response learning in young adult rats on phyto-free diets and these were similar to impairments seen in rats fed standard rodent chow (Pisani et al., 2009
The extent to which the use of a soy free diet may have contributed to the negative effects of estradiol reported here in comparison to the positive effects reported on cognitive tasks in many other rodent studies is difficult to determine. However, these are not the only studies to report impairing effects of estrogen treatment on cognitive functioning. For example, the results are consistent with studies showing that response learning, which is sensitive to striatal manipulations, is impaired by estradiol administration either systemically or directly to the striatum (Korol and Kolo, 2002
; Zurkovsky et al., 2007
). Collectively, the literature suggests that the effects of estrogens on cognition are complex, improving some functions and impairing others.
The pattern of effects seen in this study is suggestive of altered dopamine function in the prefrontal cortex (Kritzer et al., 2007
; Verma and Moghaddam, 1996
). Dopamine is important for accurate performance on prefrontally-mediated working memory tasks in humans and non-human primates (Luciana et al., 1998
), as well as in rodents (Bubser and Schmidt, 1990
; Luine et al., 1998
), with reductions in prefrontal dopamine producing impairments on short delay delayed response tasks, including DSA, in both rodents and nonhuman primates (Brozoski et al., 1979
; Bubser and Schmidt, 1990
; Goldman-Rakic, 1998
; Sawaguchi and Goldman-Rakic, 1991
). Prefrontal D1 receptor activation has also been implicated in accurate DSA performance in rodents (Vijayraghavan et al., 2007
; Zahrt et al., 1997
). Estradiol treatment has been shown to reduce prefrontal dopamine levels (Luine et al., 1998
). Thus, the deficits in DSA performance observed in our studies could be mediated by a loss of tonic dopaminergic neurotransmission and disrupted D1 activation in the prefrontal cortex.
Interestingly there was no differential effect of estradiol treatment in young, middle-aged and old rats. All three age groups were similarly impaired by chronic estradiol treatment. There also were not any negative effects of age, itself, on DSA performance. In fact, old rats actually performed somewhat better than young or middle-aged rats at the 6 and 9-second delays across the first three blocks of testing. The old rats also made fewer lose-stay errors than the young and middle-aged rats did. The lack of an age effect is at odds with prior studies, showing that working memory in prefrontally-mediated tasks is sensitive to aging (Gallagher and Rapp, 1997
). We had hypothesized that older rats would do more poorly on the DSA task and that estradiol treatment would result in larger DSA deficits in older rats.
There are several possible explanations for the lack of an age effect. It may be that the operant form of the DSA task used in this study is relatively insensitive to aging or that the age at which these rats were tested (18−20 months) was not old enough to see a clear age-related decline in function. Recently we tested 22−24 month-old female Long Evans rats on the same DSA task and found that these older rats did not perform as well as did young and middle-aged rats, particularly at the shortest delays and in the later blocks of testing (Neese et al., in prep). This supports the latter contention. Consistent with our results, Dunnett et al., (1988
also did not find an age-induced deficit in rodents (<21 months) on operant tests of working memory (delayed matching and nonmatching to sample) at short delays (<10 seconds: Dunnett et al., 1988
It is important to note that the young rats used in this study were nulliparous whereas the middle-aged and old rats were retired breeders. Several studies have linked reproductive experience to improved cognitive function (Pawluski et al., 2006a
). However, these effects are usually reported shortly after pup weaning (Pawluski et al., 2006a
), or in rats that have given birth to a single litter (Pawluski et al., 2006b
). Thus, it seems unlikely that differences in parity between the young and middle-aged or old rats are responsible for the lack of age-related differences in DSA performance.
Another possibility is that young rats may attend more to nonessential visual cues making memory or attentional tasks more difficult for them than for older animals. However, the rats in this study were tested in darkened testing chambers. Thus, extraneous visual cues are unlikely to have played a significant role in performance.
Finally, the rats in the current study were tested during the early part of the dark phase of the light-dark cycle, whereas many studies test animals during the light phase of the cycle (e.g. Galea et al., 2001
; Gresack and Frick, 2006
; Korol and Kolo, 2002
). Research suggests an interaction between age, time of day and delay on performance of tasks that include delays. For example, Winocur and Hasher (1999)
found that aged rats tested early in the dark phase of the cycle performed better than those tested late in the dark phase in an operant delayed alternation task (Winocur and Hasher, 1999
), although young rats still performed better than old rats, on average.
In summary, it is likely that several factors contributed to the lack of any age-related decrements in DSA performance in the current study. First, operant versions of the DSA task, particularly those employing short delays, appear to be relatively resistant to age-related decline. Second, the “aged” rats in this study were still relatively early in the aging process (18−20 months of age) at the time of testing, and third, the rats were tested during a stage of the light-dark cycle that would be likely to optimize the performance of the older animals on the task.
In conclusion, this study found that chronic estradiol replacement produced impairments in an operant version of the DSA task, independent of age. Given that the task employed short intertrial delays (less than 20 seconds), these results suggest prefrontal disruption (Maruki et al., 2001
; Sloan et al., 2006
). Dopamine neurotransmission and subsequent prefrontal D1 receptor activation have been implicated in accurate DSA performance (Vijayraghavan et al., 2007
; Zahrt et al., 1997
). Given that chronic estradiol treatment has been found to reduce basal levels of dopamine in the prefrontal cortex, the observed deficits in DSA performance could be mediated by an overall loss of tonic dopaminergic neurotransmission and disrupted D1 activation in the prefrontal cortex. Future studies should begin to address these potential underlying neurochemical mechanisms for the cognitive deficits.