We previously demonstrated that aged ovariectomized rats that had received prior estradiol treatment in middle age exhibited enhanced spatial memory and increased levels of estrogen receptor (ER)-α in the hippocampus long after estradiol treatment was terminated. The implication for cognition of increased levels of ERα resulting from prior estradiol exposure is unknown. In the absence of estrogens, growth factors, including IGF-I, can induce ERα-mediated transcription through ligand-independent mechanisms. Our current goal was to determine whether IGF-I mediates the ability of short-term exposure to estradiol to exert long-term effects on cognition and the hippocampus of aging females. Ovariectomized middle-aged rats were implanted with estradiol or cholesterol vehicle capsules. After 40 days, all capsules were removed and drug treatments were initiated. Half of each hormone treatment group received chronic intracerebroventricular delivery of the IGF-I receptor antagonist JB1, and the other half received artificial cerebrospinal fluid vehicle. Rats were tested on a spatial memory radial-arm maze task and hippocampi were immunostained for proteins of interest by Western blotting. As expected, previous treatment with estradiol enhanced spatial memory and increased levels of ERα in the hippocampus. JB1 reversed these effects. Previous treatment with estradiol resulted in lasting increases in levels of IGF-I receptors and phosphorylation of ERK/MAPK, a downstream signaling molecule of both ERα and IGF-I receptors, and increased levels of the ERα-regulated protein, choline acetyltransferase. JB1 blocked effects on ERK/MAPK and choline acetyltransferase. Results indicate that activation of IGF-I receptors is necessary for prior estradiol exposure to exert lasting impact on the hippocampus and memory.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

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.

Many of the biochemical, structural, and functional changes that occur as the female brain ages are influenced by changes in levels of estrogens. Administration of estrogens begun during a critical window near menopause is hypothesized to prevent or delay age-associated cognitive decline. However, due to potential health risks women often limit use of estrogen therapy to a few years to treat menopausal symptoms. The long-term consequences for the brain of short-term use of estrogens are unknown. Interestingly, there are preliminary data to suggest that short-term use of estrogens during the menopausal transition may afford long-term cognitive benefits to women as they age. Thus, there is the intriguing possibility that short-term estrogen therapy may provide lasting benefits to the brain and cognition. The focus of the current review is an examination of the long-term impact for cognition of midlife use of estrogens. We review data from our lab and others indicating that the ability of midlife estrogens to impact estrogen receptors in the hippocampus may contribute to its ability to exert lasting impacts on cognition in aging females.

Ovariectomized rats that received previous administration of oestradiol in midlife display enhanced cognition and increased hippocampal levels of oestrogen receptor alpha (ERα) months after oestradiol treatment ended as compared to ovariectomized controls. Objectives of the current work were to investigate mechanisms by which ERα levels are maintained following midlife oestradiol exposure and the role of ERα in memory in aging females in the absence of circuiting oestrogens. Unliganded ERα has increased interaction with the ubiquitin ligase, C terminus of Hsc-70 interacting protein (CHIP) leading to increased degradation of the receptor. In our first experiment, we tested the hypothesis that midlife oestradiol exposure in ovariectomized rats results in decreased interaction between CHIP and hippocampal ERα, leading to increased levels of ERα. Middle-aged rats were ovariectomized and received oestradiol or vehicle implants. After 40 days, implants were removed. One month later, rats were killed and hippocampi processed for whole protein western blotting and co-immunoprecipitation, in which ERα was immunoprecipitated from lysate. As expected, ERα protein expression was increased in rats previously treated with oestradiol compared to vehicle-treated rats. In rats treated with oestradiol, there was a decrease in CHIP-ERα interaction, suggesting that previous oestradiol treatment reduces interaction, slowing degradation of ERα. In a second experiment, we determined the impact on memory of antagonism of ER in the absence of circulating oestrogens. Rats were ovariectomized and implanted with oestradiol capsules. Capsules were removed after 40 days. Rats received chronic i.c.v. infusion of ER antagonist, ICI 182,780 or aCSF vehicle and were tested on a spatial memory radial maze task. Rats treated with ICI 182,780 had significantly worse performance (more errors). These experiments provide evidence that previous midlife oestradiol treatment maintains hippocampal ERα by decreasing its interaction with CHIP and that activation of these receptors provides cognitive benefits in the absence of circulating oestrogens.