Rapid and local neuroestrogen synthesis supports long-term potentiation of hippocampal Schaffer collaterals-cornu ammonis 1 synapse in ovariectomized mice.

In aging women, cognitive decline and increased risk of dementia have been associated with the cessation of ovarian hormones production at menopause. In the brain, presence of the key enzyme aromatase required for the synthesis of 17-ß-estradiol (E2) allows for local production of E2 in absence of functional ovaries. Understanding how aromatase activity is regulated could help alleviate the cognitive symptoms. In female rodents, genetic or pharmacological reduction of aromatase activity over extended periods of time impair memory formation, decreases spine density, and hinders long-term potentiation (LTP) in the hippocampus. Conversely, increased excitatory neurotransmission resulting in rapid N-methyl-d-aspartic acid (NMDA) receptor activation rapidly promotes neuroestrogen synthesis. This rapid modulation of aromatase activity led us to address the hypothesis that acute neuroestrogens synthesis is necessary for LTP at the Schaffer collateral-cornu ammonis 1 (CA1) synapse in absence of circulating ovarian estrogens. To test this hypothesis, we did electrophysiological recordings of field excitatory postsynaptic potential (fEPSPs) in hippocampal slices obtained from ovariectomized mice. To assess the impact of neuroestrogens synthesis on LTP, we applied the specific aromatase inhibitor, letrozole, before the induction of LTP with a theta burst stimulation protocol. We found that blocking aromatase activity prevented LTP. Interestingly, exogenous E2 application, while blocking aromatase activity, was not sufficient to recover LTP in our model. Our results indicate the critical importance of rapid, activity-dependent local neuroestrogens synthesis, independent of circulating hormones for hippocampal synaptic plasticity in female rodents.

Female mice lacking membrane estrogen receptor alpha display impairments in spatial memory.

Estrogens exert effects on cognition by acting on estrogen receptors (ER) including ERα. Activation of nuclear ERα results in classical genomic signaling leading to transcriptional changes that occur over hours to days. In contrast, activation of ERα localized to the membrane results in rapid signaling with effects occurring in seconds to minutes. The goal of the current study was to determine the role of membrane ERα in spatial memory. Female wildtype (WT) and transgenic mice that lack membrane ERα and express nuclear only ERα (NOER) were trained on an eight-arm radial-maze task. Following training, mice were tested on delay trials, in which delays ranging from 30 min to 5 h were inserted between the 4th and 5th arm choices. Performance was measured by number of proactive and retroactive errors. Proactive errors are short-term working memory errors defined by reentries into arms previously visited during the post-delay period or errors made during the pre-delay period. Retroactive errors are delay-dependent memory errors, defined as reentries into arms during the post-delay that were previously visited during the pre-delay. Consistent with a role for membrane ERα in rapid signaling, NOER mice made more proactive errors than WTs across all delays. NOER mice made more retroactive errors than WTs only after the 5-h delay. WT and NOER mice performed similarly on elevated plus maze and open field tests indicating no effects of membrane ERα on anxiety-related behavior or locomotor activity. Results reveal that membrane ERα plays important roles in both short-term and longer-term delay-dependent memory either directly or potentially indirectly through a role in the regulation of estradiol levels via the hypothalamic-pituitary-gonadal axis.

Cardiometabolic health, menopausal estrogen therapy and the brain: How effects of estrogens diverge in healthy and unhealthy preclinical models of aging.

Research in preclinical models indicates that estrogens are neuroprotective and positively impact cognitive aging. However, clinical data are equivocal as to the benefits of menopausal estrogen therapy to the brain and cognition. Pre-existing cardiometabolic disease may modulate mechanisms by which estrogens act, potentially reducing or reversing protections they provide against cognitive decline. In the current review we propose mechanisms by which cardiometabolic disease may alter estrogen effects, including both alterations in actions directly on brain memory systems and actions on cardiometabolic systems, which in turn impact brain memory systems. Consideration of mechanisms by which estrogen administration can exert differential effects dependent upon health phenotype is consistent with the move towards precision or personalized medicine, which aims to determine which treatment interventions will work for which individuals. Understanding effects of estrogens in both healthy and unhealthy models of aging is critical to optimizing the translational link between preclinical and clinical research.

History of Previous Midlife Estradiol Treatment Permanently Alters Interactions of Brain Insulin-like Growth Factor-1 Signaling and Hippocampal Estrogen Synthesis to Enhance Cognitive Aging in a Rat Model of Menopause.

Across species, including humans, elevated levels of brain estrogen receptor (ER) α are associated with enhanced cognitive aging, even in the absence of circulating estrogens. In rodents, short-term estrogen treatment, such as that commonly used in the menopausal transition, results in long-term increases in ERα levels in the hippocampus, leading to enhanced memory long after termination of estrogen treatment. However, mechanisms by which increased levels of brain ERα enhances cognitive aging remain unclear. Here we demonstrate in aging female rats that insulin-like growth factor-1 (IGF-1), which can activate ER via ligand-independent mechanisms, requires concomitant synthesis of brain-derived neuroestrogens to phosphorylate ERα via MAPK signaling, ultimately resulting in enhanced memory. In a rat model of menopause involving long-term ovarian hormone deprivation, hippocampal neuroestrogen activity decreases, altering IGF-1 activity and resulting in impaired memory. However, this process is reversed by short-term estradiol treatment. Forty days of estradiol exposure following ovariectomy results in maintenance of neuroestrogen levels that persist beyond the period of hormone treatment, allowing for continued interactions between IGF-1 and neuroestrogen signaling, elevated levels of hippocampal ERα, and ultimately enhanced memory. Collectively, results demonstrate that short-term estradiol use following loss of ovarian function has long-lasting effects on hippocampal function and memory by dynamically regulating cellular mechanisms that promote activity of ERα in the absence of circulating estrogens. Translational impacts of these findings suggest lasting cognitive benefits of short-term estrogen use near menopause and highlight the importance of hippocampal ERα, independent from the role of circulating estrogens, in regulating memory in aging females.SIGNIFICANCE STATEMENT Declines in ovarian hormones following menopause coincide with increased risk of cognitive decline. Because of potential health risks, current recommendations are that menopausal estrogen therapy be limited to a few years. Long-term consequences for the brain and memory of this short-term midlife estrogen therapy are unclear. Here, in a rodent model of menopause, we determined mechanisms by which short-term midlife estrogen exposure can enhance hippocampal function and memory with cognitive benefits and molecular changes enduring long after termination of estrogen exposure. Our model indicates long-lasting benefits of maintaining hippocampal estrogen receptor function in the absence of ongoing estrogen exposure and suggests potential strategies for combating age-related cognitive decline.

Previous estradiol treatment during midlife maintains transcriptional regulation of memory-related proteins by ERα in the hippocampus in a rat model of menopause.

Previous midlife estradiol treatment, like continuous treatment, improves memory and results in lasting increases in hippocampal levels of estrogen receptor (ER) α and ER-dependent transcription in ovariectomized rodents. We hypothesized that previous and continuous midlife estradiol act to specifically increase levels of nuclear ERα, resulting in transcriptional regulation of proteins that mediate estrogen effects on memory. Ovariectomized middle-aged rats received estradiol or vehicle capsule implants. After 40 days, rats initially receiving vehicle received another vehicle capsule (ovariectomized controls). Rats initially receiving estradiol received either another estradiol (continuous estradiol) or a vehicle (previous estradiol) capsule. One month later, hippocampi were dissected and processed. Continuous and previous estradiol increased levels of nuclear, but not membrane or cytosolic ERα and had no effect on Esr1. Continuous and previous estradiol impacted gene expression and/or protein levels of mediators of estrogenic action on memory including ChAT, BDNF, and PSD-95. Findings demonstrate a long-lasting role for hippocampal ERα as a transcriptional regulator of memory following termination of previous estradiol treatment in a rat model of menopause.

Medroxyprogesterone opposes estradiol-induced renal damage in midlife ovariectomized Long Evans rats.

OBJECTIVE: Our laboratory previously published that long-term administration of estradiol (E2) was detrimental to the kidneys of midlife ovariectomized Long Evans rats, contrasting clinical studies in showing that menopausal hormone therapy is associated with decreased albuminuria. However, it is unknown whether this renal benefit was due to estrogen and/or the combination with progestogen. Therefore, the objective of the current study was to determine the impact of medroxyprogesterone (MPA) on E2-mediated renal damage using a rodent model. METHODS: Female Long Evans retired breeders underwent ovariectomy at 11 months of age and were treated for 40 days with subcutaneous E2, E2+MPA or vehicle at doses mimicking that of menopausal hormone therapy (N = 5-7 per group). Systolic blood pressure was measured along with indices of renal damage and function to investigate the impact of MPA on E2-mediated renal outcomes. Renal estrogen receptor alpha and G protein-coupled estrogen receptor transcript copy numbers were measured in all treatment groups through droplet digital PCR. RESULTS: Middle-aged female Long Evans rats displayed spontaneous hypertension with similar systolic blood pressures and heart weights between groups. Even though blood pressure was comparable, E2 reduced glomerular filtration rate and increased proteinuria indicating pressure-independent renal damage. Coadministration with MPA prevented E2-induced glomerular filtration rate impairment and proteinuria by promoting renal hypertrophy and preventing renal interstitial fibrosis. Both E2 and E2+MPA reduced renal estrogen receptor alpha (ERα) and increased renal G protein-coupled estrogen receptor mRNA, but neither ERα nor ERß protein was different between groups. CONCLUSION: MPA was protective against E2-induced renal damage and dysfunction in middle-aged female Long Evans rats. Assessing the impact of hormone therapy on renal outcomes may be an important clinical factor when considering treatment options for postmenopausal women.

Neuroestrogen-Dependent Transcriptional Activity in the Brains of ERE-Luciferase Reporter Mice following Short- and Long-Term Ovariectomy.

Previous work has demonstrated that estrogen receptors are transcriptionally active in the absence of ovarian estrogens. The current work aims to determine whether brain-derived estrogens influence estrogen receptor-dependent transcription after short- or long-term loss of ovarian function. Experiments were conducted using estrogen response element (ERE)-Luciferase reporter mice, which express the gene for luciferase driven by consensus ERE, allowing for the quantification of ERE-dependent transcription. Brain regions examined were hippocampus, cortex, and hypothalamus. In Experiment 1, short-term (10 d) ovariectomy had no impact on ERE-dependent transcription across brain regions compared with sham surgery. In Experiment 2, chronic intracerebroventricular administration of the aromatase inhibitor letrozole significantly decreased transcriptional activity in 10-d-old ovariectomized mice across brain regions, indicating that the sustained transcription in short-term ovariectomized mice is mediated at least in part via actions of neuroestrogens. Additionally, intracerebroventricular administration of estrogen receptor antagonist ICI-182,780 blocked transcription in 10-d-old ovariectomized mice across brain regions, providing evidence that sustained transcription in ovariectomized mice is estrogen receptor dependent. In Experiment 3, long-term (70 d) ovariectomy significantly decreased ERE-dependent transcription across brain regions, though some residual activity remained. In Experiment 4, chronic intracerebroventricular letrozole administration had no impact on transcription in 70 d ovariectomized mice across brain regions, indicating that the residual ERE-dependent transcription in long-term ovariectomized mice is not mediated by neuroestrogens. Overall, the results indicate that ERE-dependent transcription in the brain continues after ovariectomy and that the actions of neuroestrogens contribute to the maintenance of ERE-dependent transcription in the brain following short-term, but not long-term, loss of ovarian function.

Nuclear estrogen receptor activation by insulin-like growth factor-1 in Neuro-2A neuroblastoma cells requires endogenous estrogen synthesis and is mediated by mutually repressive MAPK and PI3K cascades.

Non-canonical mechanisms of estrogen receptor activation may continue to support women's cognitive health long after cessation of ovarian function. These mechanisms of estrogen receptor activation may include ligand-dependent actions via locally synthesized neuroestrogens and ligand-independent actions via growth factor-dependent activation of intracellular kinase cascades. We tested the hypothesis that ligand-dependent and ligand-independent mechanisms interact to activate nuclear estrogen receptors in the Neuro-2A neuroblastoma cell line in the absence of exogenous estrogens. Transcriptional output of estrogen receptors was measured following treatment with insulin-like growth factor-1 (IGF-1) in the presence of specific inhibitors for mitogen-activated protein kinase (MAPK), phosphoinositde-3 kinase (PI3K), and neuroestrogen synthesis. Results indicate that IGF-1-dependent activation of nuclear estrogen receptors is mediated by MAPK, is opposed PI3K, and requires concomitant endogenous neuroestrogen synthesis. We conclude that both cellular signaling context and endogenous ligand availability are important modulators of ligand-independent nuclear estrogen receptor activation.

Lasting impact on memory of midlife exposure to exogenous and endogenous estrogens.

We previously demonstrated that 40 days of prior midlife estradiol treatment results in enhanced spatial memory in aging ovariectomized rats long after termination of the estradiol treatment. Our current goal was to determine whether this benefit is due to lasting impacts on memory specifically of previous exogenous estradiol treatment or simply due to delaying cognitive deficits that occur following loss of ovarian hormones. Middle-aged rats were ovariectomized or underwent sham surgery. Ovariectomized rats received estradiol (Previous Estradiol) or vehicle (Previous Vehicle) implants. Rats undergoing sham surgery (Previous Intact) received vehicle implants. Forty days later, Previous Intact rats were ovariectomized, the other 2 groups underwent sham surgeries, and all implants were removed. Thus, no ovarian or exogenously administered hormones were present during behavior testing. Rats underwent 24 days of acquisition training on an 8-arm radial maze. Following acquisition and again 2 months later, rats were tested on delay trials, during which animals had to remember the location of food rewards across time delays inserted between fourth and fifth arm choices. During acquisition, rats that had previous extended exposure to exogenous estradiol (Previous Estradiol) and endogenous ovarian hormones (Previous Intact) significantly outperformed rats that did not experience extended hormone exposure (Previous Vehicle). However, during delays trials the Previous Estradiol group significantly outperformed both the Previous Vehicle and Previous Intact groups. Results demonstrate that whereas extended exposure to endogenous ovarian hormones may provide short-term cognitive benefits, midlife estradiol treatment following ovariectomy provides additional benefits that persist for months following termination of treatment. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Long- but not short-term estradiol treatment induces renal damage in midlife ovariectomized Long-Evans rats.

Clinical recommendations limit menopausal hormone therapy to a few years, yet the impact of a shorter treatment duration on cardiovascular health is unknown. We hypothesized that both short- and long-term estradiol (E2) treatment exerts positive and lasting effects on blood pressure, vascular reactivity, and renal health. This study was designed to mimic midlife menopause, followed by E2 treatment, that either followed or exceeded the current clinical recommendations. Female Long-Evans retired breeders were ovariectomized (OVX) at 11 mo of age and randomized into three groups: 80-day (80d) vehicle (Veh>Veh), 40-day (40d) E2 + 40d vehicle (E2>Veh), and 80d E2 (E2>E2). In comparison to Veh>Veh, both the E2>Veh and E2>E2 groups had lower systolic blood pressure and enhanced mesenteric relaxation in response to estrogen receptor-α stimulation. Despite the reduced blood pressure, E2>E2 induced renal and cardiac hypertrophy, reduced glomerular filtration, and increased proteinuria. Interestingly, kidneys from E2>Veh rats had significantly fewer tubular casts than both of the other groups. In conclusion, long-term E2 lowered blood pressure but exerted detrimental effects on kidney health in midlife OVX Long-Evans rats, whereas short-term E2 lowered blood pressure and reduced renal damage. These findings highlight that the duration of hormone therapy may be an important factor for renal health in aging postmenopausal women.

Circulating Estradiol Regulates Brain-Derived Estradiol via Actions at GnRH Receptors to Impact Memory in Ovariectomized Rats.

Systemic estradiol treatment enhances hippocampus-dependent memory in ovariectomized rats. Although these enhancements are traditionally thought to be due to circulating estradiol, recent data suggest these changes are brought on by hippocampus-derived estradiol, the synthesis of which depends on gonadotropin-releasing hormone (GnRH) activity. The goal of the current work is to test the hypothesis that peripheral estradiol affects hippocampus-dependent memory through brain-derived estradiol regulated via hippocampal GnRH receptor activity. In the first experiment, intracerebroventricular infusion of letrozole, which prevents the synthesis of estradiol, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task. In the second experiment, hippocampal infusion of antide, a long-lasting GnRH receptor antagonist, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory. In the third experiment, hippocampal infusion of GnRH enhanced hippocampus-dependent memory, the effects of which were blocked by letrozole infusion. Results indicate that peripheral estradiol-induced enhancement of cognition is mediated by brain-derived estradiol via hippocampal GnRH receptor activity.

Evidence for Ligand-Independent Activation of Hippocampal Estrogen Receptor-α by IGF-1 in Hippocampus of Ovariectomized Rats.

In the absence of ovarian estrogens, increased levels of estrogen receptor (ER)α in the hippocampus are associated with improvements in cognition. In vitro evidence indicates that under conditions of low estrogen, growth factors, including Insulin-Like Growth Factor 1 (IGF-1), can activate ERα and regulate ERα-mediated transcription through mechanisms that likely involve modification of phosphorylation sites on the receptor. The goal of the current work was to investigate a role for IGF-1 in ligand-independent activation of ERα in the hippocampus of female rats. Ovariectomized rats received a single intracerebroventricular infusion of IGF-1 and hippocampi were collected 1 or 24 hours later. After 1 h, IGF-1 increased hippocampal levels of phosphorylated ERα at serine 118 (S118) as revealed by Western blotting. Coimmunoprecipitation revealed that at 1 hour after infusion, IGF-1 increased association between ERα and steroid receptor coactivator 1, a histone acetyltransferase that increases transcriptional activity of phosphorylated ERα. IGF-1 infusion increased levels of the ERα-regulated proteins ERα, choline acetyltransferase, and brain-derived neurotrophic factor in the hippocampus 24 hours after infusion. Results indicate that IGF-1 activates ERα in ligand-independent manner in the hippocampus via phosphorylation at S118 resulting in increased association of ERα with steroid receptor coactivator 1 and elevation of ER-regulated proteins. To our knowledge, these data are the first in vivo evidence of ligand-independent actions of ERα and provide a mechanism by which ERα can impact memory in the absence of ovarian estrogens.

Long-term consequences of estrogens administered in midlife on female cognitive aging.

This article is part of a Special Issue "Estradiol and cognition". 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.

Antagonism of brain insulin-like growth factor-1 receptors blocks estradiol effects on memory and levels of hippocampal synaptic proteins in ovariectomized rats.

RATIONALE: Treatment with estradiol, the primary estrogen produced by the ovaries, enhances hippocampus-dependent spatial memory and increases levels of hippocampal synaptic proteins in ovariectomized rats. Increasing evidence indicates that the ability of estradiol to impact the brain and behavior is dependent upon its interaction with insulin-like growth factor-1 (IGF-1). OBJECTIVE: The goal of the current experiment was to test the hypothesis that the ability of estradiol to impact hippocampus-dependent memory and levels of hippocampal synaptic proteins is dependent on its interaction with IGF-1. METHODS: Adult rats were ovariectomized and implanted with estradiol or control capsules and trained on a radial-maze spatial memory task. After training, rats were implanted with intracerebroventricular cannulae attached to osmotic minipumps (flow rate 0.15 µl/h). Half of each hormone treatment group received continuous delivery of JB1 (300 µg/ml), an IGF-1 receptor antagonist, and half received delivery of aCSF vehicle. Rats were tested on trials in the radial-arm maze during which delays were imposed between the fourth and fifth arm choices. Hippocampal levels of synaptic proteins were measured by western blotting. RESULTS: Estradiol treatment resulted in significantly enhanced memory. JB1 blocked that enhancement. Estradiol treatment resulted in significantly increased hippocampal levels of postsynaptic density protein 95 (PSD-95), spinophilin, and synaptophysin. JB1 blocked the estradiol-induced increase of PSD-95 and spinophilin and attenuated the increase of synaptophysin. CONCLUSIONS: Results support a role for IGF-1 receptor activity in estradiol-induced enhancement of spatial memory that may be dependent on changes in synapse structure in the hippocampus brought upon by estradiol/IGF-1 interactions.

Short-term estradiol administration in aging ovariectomized rats provides lasting benefits for memory and the hippocampus: a role for insulin-like growth factor-I.

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.

Estrogens, estrogen receptors, and female cognitive aging: the impact of timing.

Estrogens have been shown to be protective agents against neurodegeneration and associated cognitive decline in aging females. However, clinical data have been equivocal as to the benefits to the brain and cognition of estrogen therapy in postmenopausal women. One factor that is proposed to be critical in determining the efficacy of hormone therapy is the timing of its initiation. The critical period or window of opportunity hypothesis proposes that following long-term ovarian hormone deprivation, the brain and cognition become insensitive to exogenously administered estrogens. In contrast, if estrogens are administered during a critical period near the time of cessation of ovarian function, they will exert beneficial effects. The focus of the current review is the examination of evidence from rodent models investigating the critical period hypothesis. A growing body of experimental data indicates that beneficial effects of 17ß-estradiol (estradiol) on cognition and on cholinergic function and hippocampal plasticity, both of which have been linked to the ability of estradiol to exert beneficial effects on cognition, are attenuated if estradiol is administered following a period of long-term ovarian hormone deprivation. Further, emerging data implicate loss of estrogen receptor alpha (ERα) in the brain resulting from long-term hormone deprivation as a basis for the existence of the critical period. A unifying model is proposed by which the presence or absence of estrogens during a critical period following the cessation of ovarian function permanently alters the system resulting in decreased or increased risk, respectively, of neurodegeneration and cognitive decline.

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.

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.

A role for hippocampal actin rearrangement in object placement memory in female rats.

Actin rearrangement, the polymerization of globular actin (G-actin) to filamentous actin, causes morphological changes in dendritic spines and is hypothesized to be a substrate of learning and memory. The ovarian hormone estradiol promotes hippocampal actin rearrangement and enhances performance on hippocampus-dependent tasks, including object placement memory. The goals of the current study were to determine a role for actin rearrangement and its regulatory pathway in object placement memory in female rats and to determine if estradiol impacts actin rearrangement in ovariectomized rats during the performance of the task. In an initial experiment, young adult Long-Evans rats were ovariectomized and implanted with capsules containing either cholesterol vehicle or estradiol. Bilateral intrahippocampal infusions of aCSF vehicle or the actin rearrangement inhibitor, latrunculin A, were administered 15 min prior to initiation of the object placement task. Latrunculin A dose-dependently impaired object placement memory. Estradiol had no impact on the ability of latrunculin A to affect performance. In a second experiment, rats were ovariectomized and received implants containing cholesterol or estradiol. Half of each hormone treatment group was exposed to the object placement memory task and half underwent control procedures. Immediately following completion of behavior, rats were euthanized and hippocampi removed. Western blotting was used to measure hippocampal levels of phosphorylated and total levels of a regulator of actin polymerization, the actin depolymerization factor cofilin. Exposure to the object placement memory task resulted in significant increases in phosphorylated levels of cofilin. Estradiol treatment had no impact on protein levels. These data support a role for hippocampal actin rearrangement and its regulatory proteins in object placement memory in female rats and suggest that chronic estradiol treatment does not impact hippocampal actin arrangement.

Long-term behavioral and pharmacodynamic effects of delta-9-tetrahydrocannabinol in female rats depend on ovarian hormone status.

Abuse of Δ9-THC by females during adolescence may produce long-term deficits in complex behavioral processes such as learning, and these deficits may be affected by the presence of ovarian hormones. To assess this possibility, 40 injections of saline or 5.6 mg/kg of Δ9-THC were administered i.p. daily during adolescence to gonadally intact or ovariectomized (OVX) female rats, yielding four treatment groups (intact/saline, intact/THC, OVX/saline, and OVX/ THC). Δ9-THC (0.56-10 mg/kg) was then re-administered to each of the four groups during adulthood to examine their sensitivity to its disruptive effects. The behavioral task required adult subjects to both learn (acquisition component) different response sequences and repeat a known response sequence (performance component) daily. During baseline (no injection) and control (saline injection) sessions, OVX subjects had significantly higher response rates and lower percentages of error in both behavioral components than the intact groups irrespective of saline or Δ9-THC administration during adolescence; the intact group that received Δ9-THC had the lowest response rates in each component. Upon re-administration of Δ9-THC, the groups that received adolescent ovariectomy alone, adolescent Δ9-THC administration alone, or both treatments were found to be less sensitive to the rate-decreasing effects, and more sensitive to the error-increasing effects of Δ9-THC than the control group (i.e. intact subjects that received saline during adolescence). Neurochemical analyses of the brains from each adolescent-treated group indicated that there were also persistent effects on cannabinoid type-1 (CB-1) receptor levels in the hippocampus and striatum that depended on the brain region and the presence of ovarian hormones. In addition, autoradiographic analyses of the brains from adolescent-treated, but behaviorally naïve, subjects indicated that ovariectomy and Δ9-THC administration produced effects on receptor coupling in some of the same brain regions. In summary, chronic administration of Δ9-THC during adolescence in female rats produced long-term effects on operant learning and performance tasks and on the cannabinoid system that were mediated by the presence of ovarian hormones, and that altered their sensitivity to Δ9-THC as adults.