Increasing hippocampal estrogen receptor alpha levels via viral vectors increases MAP kinase activation and enhances memory in aging rats in the absence of ovarian estrogens

Abstract

We previously demonstrated that aged ovariectomized rats that had received prior estradiol treatment in middle-age exhibited increased levels of estrogen receptor alpha (ERα) in the hippocampus as well as enhanced hippocampal dependent memory as compared to aged rats that had not received mid-life estradiol treatment. These effects persisted long after the estradiol treatment had been terminated. The goal of the current experiment was to determine if increased expression of ERα in the hippocampus, in the absence of exogenously administered estrogens, can impact the hippocampus and cognitive function in aging ovariectomized rats. Middle-aged rats were trained for 24 days on an eight-arm radial maze spatial memory task. All rats were then ovariectomized. Forty days later, rats received either lentiviral delivery to the hippocampus of the gene encoding ERα (lenti-ERα) or a control virus. Rats were tested on delay trials in the radial-maze in which delays of varying lengths were imposed between the fourth and fifth arm choices. Following behavior testing, hippocampi were immunostained using western blotting for ERα, the ERα-regulated protein choline acetyltransferase, and phosphorylation of the ERα-regulated kinases, ERK/MAPK and Akt. Results revealed that aging ovariectomized rats that received delivery of lenti-ERα to the hippocampus exhibited enhanced spatial memory as indicated by increased arm-choice accuracy across delays as compared to ovariectomized rats that received control virus. Western blot data revealed that lenti-ERα delivery significantly increased levels of ERα and phosphorylated ERK/MAPK and had no impact on levels of ChAT or phosphorylation of Akt. Results indicate that increasing hippocampal levels of ERα in aging females in the absence of ovarian or exogenously administered estrogens leads to increases in phosphorylation of ERK/MAPK as well as in enhanced memory.

Figure 1. Expression of lenti-viral vector.

Confocal image of granule cells of the rat dentate gyrus ∼400 µm from injection site. Top: Nuclei stained with Dapi. Bottom: ERα FLAG staining. FLAG-tagged ERα is present in nuclei and dendrites. 63× –1× digital zoom.

Figure 2. Experimental timeline.

OVX = Ovariectomy.

Figure 3. Effects of lenti-viral delivery to the hippocampus of the gene encoding ERα (Lenti-ERα) or a control virus (Lenti-Cherry) to aging ovariectomized rats on number of total errors in a spatial memory radial-maze task when various delays were imposed between the 4^th and 5^th arm choices.

(A) Mean number of total errors in the first eight arm choices (+SEM) averaged across delays. ^# P = .077 vs. Lenti-Cherry. (B) Mean number of total errors of first eight arm choices (+SEM) at each delay.

Figure 4. Effects of lenti-viral delivery to the hippocampus of the gene encoding ERα (Lenti-ERα) or a control virus (Lenti-Cherry) to aging ovariectomized rats on the number of post-delay retroactive errors in a spatial memory radial-maze task when various delays were imposed between the 4^th and 5^th arm choices.

(A) Mean number of retroactive errors (+SEM) averaged across delays. ^* P = .047 vs. Lenti-Cherry. (B) Mean number of retroactive errors (+SEM) at each delay.

Figure 5. Effects of lenti-viral delivery to the hippocampus of the gene encoding ERα (Lenti-ERα) or a control virus (Lenti-Cherry) to aging ovariectomized rats on protein expression of ERα in the hippocampus as measured by western blotting.

Mean density x area (+SEM) expressed relative to Lenti-Cherry control. * P = .03 vs. Lenti-Cherry. Representative blot images for ERα and the loading control β-actin are shown in insets above the graph.

Figure 6. Effects of lenti-viral delivery to the hippocampus of the gene encoding ERα (Lenti-ERα) or a control virus (Lenti-Cherry) to aging ovariectomized rats on protein expression of choline acetyltransferase (ChAT) in the hippocampus as measured by western blotting.

Mean density x area (+SEM) expressed relative to Lenti-Cherry control. Representative blot images for ChAT and the loading control β-actin are shown in insets above the graph.

Figure 7. Effects of lenti-viral delivery to the hippocampus of the gene encoding ERα (Lenti-ERα) or a control virus (Lenti-Cherry) to aging ovariectomized rats on protein expression of phosphorylated MAPK and Akt.

Western blot data showing the effects of Lenti-ERα or Lenti-Cherry on levels of phosphorylated p44 MAPK and phosphorylated p42 MAPK (A), and phosphorylated Akt (B) in the hippocampus. Mean density x area (+SEM) expressed relative to Lenti-Cherry control. * P = .043 vs. Lenti-Cherry. Representative blot images for phosphorylated and total p44 MAPK, p42 MAPK, and Akt are shown in insets above the graph.