Premarin improves memory, prevents scopolamine-induced amnesia and increases number of basal forebrain choline acetyltransferase positive cells in middle-aged surgically menopausal rats

Abstract

Conjugated equine estrogen (CEE) is the most commonly prescribed estrogen therapy, and is the estrogen used in the Women's Health Initiative study. While in-vitro studies suggest that CEE is neuroprotective, no study has evaluated CEE's effects on a cognitive battery and brain immunohistochemistry in an animal model. The current experiment tested whether CEE impacted: I) spatial learning, reference memory, working memory and long-term retention, as well as ability to handle mnemonic delay and interference challenges; and, II) the cholinergic system, via pharmacological challenge during memory testing and ChAT-immunoreactive cell counts in the basal forebrain. Middle-aged ovariectomized (Ovx) rats received chronic cyclic injections of either Oil (vehicle), CEE-Low (10 microg), CEE-Medium (20 microg) or CEE-High (30 microg) treatment. Relative to the Oil group, all three CEE groups showed less overnight forgetting on the spatial reference memory task, and the CEE-High group had enhanced platform localization during the probe trial. All CEE groups exhibited enhanced learning on the spatial working memory task, and CEE dose-dependently protected against scopolamine-induced amnesia with every rat receiving the highest CEE dose maintaining zero errors after scopolamine challenge. CEE also increased number of ChAT-immunoreactive neurons in the vertical diagonal band of the basal forebrain. Neither the ability to remember after a delay nor interference, nor long-term retention, was influenced by the CEE regimen used in this study. These findings are similar to those reported previously for 17 beta-estradiol, and suggest that CEE can provide cognitive benefits on spatial learning, reference and working memory, possibly through cholinergic mechanisms.

Fig 1

a) Mean ± SE distance to platform (cm) for each treatment group shown for the 5 testing days collapsed across trials, and for trial one of the retention day. b) Mean ± SE distance to the platform (cm) for each treatment group for trial 5 (days 1-4) to trial 1 of the next day (days 2-5). While the three CEE groups did not have a significant increase in distance scores from trial 5 to trial 1, the Ovx-Oil group did. Thus, all groups receiving CEE remembered the platform location overnight while the Ovx-Oil group did not. *p < 0.05.

Fig 2

Number of platform crossings±SE for each treatment group across the two 30 sec blocks during the probe trial. On Block 1 (0-30 seconds), the Ovx-CEE-High group made more platform crossings relative to the Ovx-Oil group. On Block 2 (31-60 seconds), there were no group differences.

Fig 3

a) Mean±SE total number of errors for each treatment group across the test trials (trials 2-6) for days 1-4 and baseline day 5. CEE-treated animals showed enhanced performance on trial 2 relative to Oil-treated animals for days 1-4. There were no group differences on baseline day 5. b) Mean±SE total number of errors for each treatment group after scopolamine (0.2 mg/kg, IP) challenge. The Ovx-CEE-High group made fewer errors compared to the Ovx-Oil group on the test trial after scopolamine challenge. The Ovx-Oil and Ovx-CEE-Low groups had increased errors after the scopolamine challenge relative to baseline. In contrast, the Ovx-CEE-Medium and Ovx-CEE-High groups showed no difference relative to baseline, suggesting maintained performance after cholinergic challenge. It is noteworthy that all Ovx-CEE-High animals made zero errors after scopolamine challenge.

Fig 4

Mean±SE ChAT-immunoreactive neurons in the VDB (a), and MS (b) of the BF. All CEE-treated groups were collapsed and compared to the Ovx-Oil group. CEE-treated animals had more ChAT-immunoreactive neurons relative to the Ovx-Oil group *ps < 0.05. Photomicrographs show ChAT-immunoreactive neurons in sections through the MS and VDB of the BF from animals in the Ovx-Oil (c), Ovx-CEE-Low (d), Ovx-CEE-Medium (e), Ovx-CEE-High (f).

Fig 5

Mean±SE serum estradiol (a), and estrone (b), levels for each treatment group. Log transformation was performed due to a skewed distribution. A significant linear trend for both hormones was evident (*ps = .015), showing that estradiol and estrone levels increased with treatment. In addition, the Ovx-CEE High group had higher levels of both estradiol and estrone compard to the Ovx-Oil group (ps < 0.05).