Previous estradiol treatment in ovariectomized mice provides lasting enhancement of memory and brain estrogen receptor activity.

Results of a large body of literature indicate that post-menopausal estrogen therapy has the potential to enhance cognitive function in women, if administered within a critical window of estrogen sensitivity. Many early observational studies found that postmenopausal women on estrogen therapy outperformed women with no history of estrogen use on a battery of cognitive tasks (Jacobs et al., 1998; Kimura, 1995; Schmidt et al., 1996). However, the results of the Women’s Health Initiative Memory Study, conducted by the National Institutes of Health, surprisingly found that estrogen therapy modestly increased the risk for cognitive impairment in postmenopausal women who were aged 65-79 years at the time of treatment (Espeland et al., 2004; Shumaker et al., 2004). Critics of this study have cited a number of design flaws (Gleason et al., 2005; Maki, 2004; Sherwin, 2005) but the strongest criticism has been that the subjects were too old (65-79 years) and beyond a critical post-menopausal window when the treatment was initiated for it to be effective (Gibbs and Gabor, 2003). Results of the ongoing Kronos Early Estrogen Prevention Study (KEEPS) (Gleason et al., 2015) and others (Alhola et al., 2010; Henderson et al., 2016) indicate no harm, but also no benefit, of hormone therapy for cognitive function when initiated in more recently postmenopausal women. Other studies have reported beneficial effects on both activation of neural substrates of memory and cognitive outcomes when estrogen therapy is initiated in younger postmenopausal women (Dumas et al., 2010, 2008; Gleason et al., 2006; Gorenstein et al., 2011). Whether benefits will be apparent as the women continue to age remains to be determined. In spite of potential for positive effects on cognitive aging, current guidelines recommend estrogen use be limited to the smallest effective dose for the shortest time possible due to potential health risks of long-term use (Chen and Colditz, 2007; Chen et al., 2006). Initial intriguing studies in humans have reported that even short-term use of estrogen therapy within the perimenopausal window can have long-term cognitive benefits for women as they age (Bagger et al., 2005; Whitmer et al., 2011). Rodent models have been used extensively to investigate the multifaceted effects of estrogen on cognition in not only menopausal models (Daniel et al., 2015; Koebele and Bimonte-Nelson, 2016) but also long-term interaction with the cholinergic system (Gibbs, 2010), rapid effects on memory consolidation (Luine, 2016; Sheppard et al., 2017), interaction of multiple cognitive systems (Galea et al., 2017; Korol and Pisani, 2015), and how estrogen sensitivity varies across the rodent lifespan (Bean et al., 2014). Our lab has adopted a rodent model of short-term perimenopausal estrogen therapy to determine the long-term effects of short-term estrogen use on the hippocampus and hippocampus-dependent memory. We previously reported that aged rats treated for 40 days with continuous, unopposed estradiol immediately after ovariectomy significantly outperformed vehicle-treated rats on the hippocampus-dependent radial-arm maze task up to 7 months after termination of treatment (Rodgers et al., 2010). Memory enhancement coincided with lasting increased expression of estrogen receptor alpha (ERα) and the estrogen-sensitive target protein choline acetyltransferase in the hippocampus. In subsequent studies we found that antagonism of the nuclear estrogen receptors in the hippocampus following previous estradiol exposure abrogates memory enhancement (Black et al., 2016) and exogenously increasing expression of ERα in the hippocampus alone of ovariectomized rats is sufficient to induce memory enhancement (Witty et al., 2012). Together these observations indicate that estrogen receptor signaling in the hippocampus is an important mediator of the observed hippocampus-dependent memory enhancement that persists long after previous exposure to continuous, unopposed estradiol. The hippocampus of both humans and rodents has been reported to be particularly sensitive to estrogen exposure. Estrogen treatment in rodents has been shown to elicit a biased use of hippocampus-dependent memory systems at the expense of striatum-dependent memory systems (Hussain et al., 2016; Lipatova et al., 2014; Pisani et al., 2016; Quinlan et al., 2013; Zurkovsky et al., 2007). Similarly, clinical reports indicate that hippocampus-dependent verbal memory tasks are most consistently improved by postmenopausal estrogen therapy while effects on other cognitive functions are variable (Sherwin, 2003). The goal of the current work is to test the hypothesis that previous exposure to continuous, unopposed estradiol in ovariectomized female mice results in lasting enhancement of hippocampus-dependent memory and estrogen receptor-dependent gene expression in the hippocampus. We also hypothesized that continuous, unopposed estradiol exposure will have lasting effects on estrogen receptor activity in the brain that are distinct from endogenous ovarian hormone synthesis. In addition to the hippocampus, treatment with estrogens in absence of ovarian function has been reported to affect a number of brain regions. In humans, functional magnetic resonance imaging studies have reported greater activity in both the hippocampus and frontal cortex during memory tasks in postmenopausal women currently on estrogen therapy (Dumas et al., 2010; Gleason et al., 2006). Similarly, in ovariectomized mice, ongoing estradiol treatment improves novel object recognition memory through physiological effects reported in both the medial temporal lobe (Fonseca et al., 2013) and cerebral cortex (Fernandez and Frick, 2004). Estradiol treatment in ovariectomized mice has also been shown to affect brain stem (Asarian and Geary, 2007) and hypothalamus-dependent (Thammacharoen et al., 2017) feeding behavior, amygdala-dependent anxiety-like behavior (Oyola et al., 2012), and cerebellum-dependent memory (Andreescu et al., 2007). We were therefore interested to determine if previous exposure to continuous, unopposed estradiol in ovariectomized female mice results in lasting enhancement of estrogen receptor-dependent gene expression in the cortex and hypothalamus in addition to the hippocampus. The transgenic ERE-Luciferase (ERE-LUC) reporter mouse line was chosen as our model system to achieve these goals. The classic cellular function of nuclear estrogen receptors is that of a ligand-dependent nuclear transcription factor. Ligand-bound receptor enters the nucleus to interact with the RNA polymerase complex and target mRNA synthesis at specific gene promoter elements known as estrogen response elements (ERE) (Mckenna, 1999; Tsai and Malley, 1994). The ERE-LUC mouse line has been engineered to express the firefly luciferase enzyme under unique control of the consensus ERE. Quantification of luciferase enzyme protein expression in tissue from this mouse thus provides a direct measurement of the relative rate of ERE-dependent gene expression in the tissue of interest (Ciana et al., 2001). Previous work with the ERE-LUC mouse model has established that expression of the ERE-dependent luciferase transgene is readily detectable across multiple brain regions of intact female mice, varies across the estrus cycle in a brain region-specific manner, and is significantly reduced by application of the pan-estrogen receptor antagonist ICI-182,780 (Stell et al., 2008). This previous work established that ERE-Luciferase transgene expression in the ERE-LUC mouse brain responds to changes in levels of circulating estrogens, that these effects are sub-region specific, and that transgene activity is dependent on activity of nuclear estrogen receptors. Therefore, the ERE-LUC mouse line is the ideal model to study the lasting effects of our rodent model of short-term perimenopausal continuous estradiol exposure on nuclear estrogen receptor activity in the brain. The current work reports three related experiments examining the lasting effects of short-term continuous estradiol exposure in ovariectomized mice on the hippocampus and hippocampus dependent memory. In Experiment 1, we establish that this treatment paradigm results in lasting enhancement of hippocampus dependent memory in wild-type mice in agreement with previous work in rats (Rodgers et al., 2010). In Experiment 2, we applied the same rodent model of short-term perimenopausal, unopposed estradiol exposure to the transgenic ERE-Luciferase mouse line and determined the lasting effects on ERE-dependent gene expression in the hippocampus, cortex, and hypothalamus. Finally in Experiment 3, we compared the lasting effects of continuous, unopposed estradiol exposure to the lasting effects of prolonged exposure to the same duration of endogenous ovarian hormone synthesis. Results of this experiment will improve our understanding of how short-term exposure to continuous, unopposed estradiol treatment can have unique lasting effects on hippocampal function and cognition in rodents.