Ovariectomized Rhesus Monkeys Research Articles

Mesenchymal stem cell-based bioengineered constructs enhance vaginal repair in ovariectomized rhesus monkeys

Transvaginal meshes repair for treating pelvic organ prolapse (POP) was halted by the U. S. Food and Drug Administration (FDA) because they can lead to severe complications. Therefore, investigations of new therapeutic strategies are urgently needed. Cell-based regenerative therapy holds great promise for the repair and restoration of damaged tissue. Here, we generated a bioengineered graft by seeding human umbilical cord mesenchymal stem cells (HUMSCs) on bioscaffolds to reconstruct the damaged vagina. In the in vitro study, HUMSCs proliferated well and the density was appropriate after 5 days of culture. Besides, we demonstrated that the differentiation potential of HUMSCs was maintained with external growth factor stimulation. The complete transcriptomic profile of HUMSCs revealed that HUMSCs cultured on grafts produced significantly higher levels of proangiogenic cytokines than cells cultured in tissue culture plates (TCPs). Three months after implantation of the bioengineered grafts into ovariectomized (OVX) rhesus monkeys via sacrocolpopexy, extracellular matrix reorganization, large muscle bundle formation, angiogenesis and, mechanical properties of the vagina were enhanced. To our knowledge, this is the first demonstration of the utility of stem cell-based bioengineered grafts for repairing damaged vaginal tissue in rhesus monkeys. These results elucidate a new approach for vagina repair and provide new ideas for treating POP.

Obligatory role of hypothalamic neuroestradiol during the estrogen-induced LH surge in female ovariectomized rhesus monkeys

Negative and positive feedback effects of ovarian 17β-estradiol (E2) regulating release of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) are pivotal events in female reproductive function. While ovarian feedback on hypothalamo-pituitary function is a well-established concept, the present study shows that neuroestradiol, locally synthesized in the hypothalamus, is a part of estrogen's positive feedback loop. In experiment 1, E2 benzoate-induced LH surges in ovariectomized female monkeys were severely attenuated by systemic administration of the aromatase inhibitor, letrozole. Aromatase is the enzyme responsible for synthesis of E2 from androgens. In experiment 2, using microdialysis, GnRH and kisspeptin surges induced by E2 benzoate were similarly attenuated by infusion of letrozole into the median eminence of the hypothalamus. Therefore, neuroestradiol is an integral part of the hypothalamic engagement in response to elevated circulating E2 Collectively, we will need to modify the concept of estrogen's positive feedback mechanism.

Glucocorticoid treatment facilitates development of a metabolic syndrome in ovariectomized Macaca Mulatta fed a high fat diet

Metabolic syndrome (MetS) is characterized by a cluster of key features, which include abdominal obesity, insulin resistance, hypertension, and dyslipidemia. The aim of this study was to assess the impact of elevated glucocorticoid levels on the development of MetS in middle-aged female rhesus monkeys (Macaca Mulatta) after ovariectomy. Six female ovariectomized rhesus monkeys (9–13years) were randomly assigned to either a control group (normal diet, n=3) or a group in which MetS was facilitated (n=3). The MetS group fed with HFD (15% fat) and received oral prednisone acetate treatment (50mg/day). After 24months, the GCs treatment was withdrawn with continuation of high-fat feeding for a further 12months. After 24months, the MetS group displayed a significant increase in body weight and abdominal circumference. Additionally, the MetS animals displayed abnormal serum lipids, insulin resistance and impaired glucose tolerance. Histology of liver biopsies indicated marked accumulation of lipid droplets in hepatocytes of MetS animals. Withdrawal of GCs treatment led to recovery from above-mentioned metabolic disorders. Whereas GCs treatment increased leptin expression, it lowered expression of adiponectin and other factors in adipose tissue. Expression of Hydroxy-steroid dehydrogenase-1 and glucose transporter type-4 in the livers of MetS animals were reduced. We conclude that in the context of high fat diet, high levels of exogenous GCs contribute to the development of MetS in non-human primates.

Effects of odanacatib on bone matrix mineralization in rhesus monkeys are similar to those of alendronate

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K which is an important enzyme for the degradation of collagen I. Aim of the present work was the head-to-head comparison between the effects of ODN and alendronate (ALN) on bone mineralization density distribution (BMDD), based on quantitative backscattered electron imaging in relation to changes in histomorphometric mineralizing surface per bone surface (MS/BS) in 12–22years old ovariectomized rhesus monkeys. Trabecular and cortical BMDD derived parameters from vertebrae and proximal tibiae were compared among vehicle (VEH, n=8), odanacatib low dose (ODN-L, n=8), odanacatib high dose (ODN-H, n=8), and alendronate (ALN, n=6) treated animals. Additionally, data from an intact, non-treated group of animals are shown (INT, n=8). In trabecular bone from the vertebra and metaphyseal tibia, the BMDD of the ODN and ALN treatment groups was shifted toward higher mineralization densities (p<0.001) consistent with the significant reduction of MS/BS (p<0.05 in ODN-H and ALN) compared to VEH. Vertebral trabecular CaMean (average degree of mineralization) was significantly higher in ODN-L (+6.5%), ODN-H (+6.1%), and ALN (+6.7%, all p<0.001). Tibial osteonal cortical bone revealed also significantly increased CaMean for ODN-L (+1.4%, p<0.05), ODN-H (+2.2%, p<0.05), and ALN (+3.4%, p<0.001) versus VEH, while primary cortical bone (devoid of secondary osteons) did not show any significant differences between the study groups. The percentage of primary bone area in the tibial cross-sections (on average 45±12%) was also not significantly different between the study groups (p=0.232). No significant differences in any BMDD parameters of all studied skeletal sites between ODN and ALN treatment were found. Correlation analysis revealed that MS/BS was highly predictive for trabecular BMDD in vertebral bone. The higher MS/BS, the lower was CaMean. Our findings are consistent with the inhibition of bone resorption of ODN and ALN in trabecular and osteonal compartments.

Diverse Synaptic Distributions of G Protein-coupled Estrogen Receptor 1 in Monkey Prefrontal Cortex with Aging and Menopause.

Age- and menopause-related impairment in working memory mediated by the dorsolateral prefrontal cortex (dlPFC) occurs in humans and nonhuman primates. Long-term cyclic 17β-estradiol treatment rescues cognitive deficits in aged ovariectomized rhesus monkeys while restoring highly plastic synapses. Here we tested whether distributions of G protein-coupled estrogen receptor 1 (GPER1) within monkey layer III dlPFC synapses are sensitive to age and estradiol, and coupled to cognitive function. Ovariectomized young and aged monkeys administered vehicle or estradiol were first tested on a delayed response test of working memory. Then, quantitative serial section immunoelectron microscopy was used to determine the distributions of synaptic GPER1. GPER1-containing nonperforated axospinous synapse density was reduced with age, and partially restored with estrogen treatment. The majority of synapses expressed GPER1, which was predominately localized to presynaptic cytoplasm and mitochondria. GPER1 was also abundant at plasmalemmas, and within cytoplasmic and postsynaptic density (PSD) domains of dendritic spines. GPER1 levels did not differ with age or treatment, and none of the variables examined were tightly associated with cognitive function. However, greater representation of GPER1 subjacent to the PSD accompanied higher synapse density. These data suggest that GPER1 is positioned to support diverse functions key to synaptic plasticity in monkey dlPFC.

Multiple clinically relevant hormone therapy regimens fail to improve cognitive function in aged ovariectomized rhesus monkeys

Preclinical studies in aged, surgically-menopausal rhesus monkeys have revealed powerful benefits of intermittent estrogen injections on prefrontal cortex–dependent working memory, together with corresponding effects on dendritic spine morphology in the prefrontal cortex. This contrasts with the inconsistent effects of hormone therapy (HT) reported in clinical studies in women. Factors contributing to this discrepancy could include differences in the formulation and sequence of HT regimens, resulting in different neurobiological outcomes. The current study evaluated, in aging surgically menopausal rhesus monkeys, the cognitive effects of 4 HT regimens modeled directly on human clinical practice, including continuous estrogen treatment opposed by progesterone. None of the regimens tested produced any cognitive effect, despite yielding physiologically relevant serum hormone levels, as intended. These findings have implications for the design of regimens that might optimize the benefits of hormone treatment for healthy aging, and suggest that common HT protocols used by women may fail to result in substantial cognitive benefit, at least via direct effects on the prefrontal cortex.

Differential effects of odanacatib and alendronate on bone turnover in the femoral neck of adult ovariectomized rhesus monkeys

Differential effects of odanacatib and alendronate on bone turnover in the femoral neck of adult ovariectomized rhesus monkeys

Odanacatib: Location and timing are everything

Odanacatib: Location and timing are everything

Odanacatib reduces bone turnover and increases bone mass in the lumbar spine of skeletally mature ovariectomized rhesus monkeys

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once-weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen-deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle-treated OVX monkeys. Treatment with ODN also led to dose-dependent increases in serum 1-CTP and maintained estrogen deficiency-elevated Trap-5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L₁ to L₄) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose-dependently increased L₁ to L₄ BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX-vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX-vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN-treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long-term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates.

Head-to-head comparison of treatment effects of odanacatib to alendronate in the ovariectomized rhesus monkey: A longitudinal multi-modality imaging study

Head-to-head comparison of treatment effects of odanacatib to alendronate in the ovariectomized rhesus monkey: A longitudinal multi-modality imaging study

HR-pQCT based finite element analysis estimates of bone strength at ultra-distal radius in ovariectomized rhesus monkeys demonstrates efficacy of odanacatib and differentiation from Alendronate

HR-pQCT based finite element analysis estimates of bone strength at ultra-distal radius in ovariectomized rhesus monkeys demonstrates efficacy of odanacatib and differentiation from Alendronate

Differential effects of odanacatib compared to alendronate on bone turnover in adult ovariectomized rhesus monkeys

Differential effects of odanacatib compared to alendronate on bone turnover in adult ovariectomized rhesus monkeys

Effects of Estradiol on Cerebrospinal Fluid Levels of Agouti-Related Protein in Ovariectomized Rhesus Monkeys

Hypothalamic proopiomelanocortin (POMC)-derived MSH peptides and the melanocortin receptor antagonist, agouti-related protein (AgRP), interact to regulate energy balance. Both POMC and AgRP neurons express estrogen receptors, but little is known about estrogen regulation of the melanocortin system in the primate. We have therefore examined the effects of physiological doses of estradiol (E2) on POMC and AgRP in lumbar cerebrospinal fluid (CSF) of ovariectomized monkeys. POMC prohormone was measured by ELISA. AgRP was measured by RIA (sensitive for the more biologically active C-terminal AgRP(83-132) but also detects full-length AgRP) and by ELISA (measures primarily full length AgRP). In the first experiment, 14 animals were studied before and after 3 wk of E2. CSF POMC did not change, but AgRP(RIA) decreased from 7.9 +/- 1.2 to 4.7 +/- 1.2 fmol/ml after E2 (P = 0.03) and the POMC/AgRP(RIA) ratio increased from 4.2 +/- 0.89 to 6.8 +/- 1.04 (P = 0.04). AgRP(ELISA) did not change, but the ratio of AgRP(RIA) compared with AgRP(ELISA) was reduced after E2 (P = 0.02). In the second experiment, 11 animals were studied after 6 wk of E2, and similar changes were noted. The degree of AgRP(RIA) suppression with E2 was inversely related to body mass index (r = 0.569; P = 0.03). These results show for the first time that E2 suppresses AgRP(C-terminal) in CSF, increases the POMC to AgRP ratio, and may decrease AgRP processing, thus leading to increased melanocortin signaling. Furthermore, obesity was associated with resistance to the suppressive effects of E2 on AgRP, analogous to what is seen with obesity and leptin resistance.

Social Subordination and Polymorphisms in the Gene Encoding the Serotonin Transporter Enhance Estradiol Inhibition of Luteinizing Hormone Secretion in Female Rhesus Monkeys1

Psychosocial factors, particularly social stress, may compromise reproduction. However, some individuals may be more susceptible to socially induced infertility. The present study used group-housed, adult, ovariectomized rhesus monkeys to test the hypothesis that exposure to psychosocial stress, imposed by social subordination, would enhance estradiol (E2)-negative feedback inhibition of LH. Because polymorphisms in the gene encoding the serotonin transporter (SLC6A4) may contribute to individual differences in response to adverse environments, we determined whether subordinate females with the short-promoter-length allele (s-variant) would show greater suppression of LH. Subordinate females, particularly those with the s-variant SLC6A4 genotype, received significantly higher rates of noncontact aggression from more dominant cage mates and had consistently lower body weights. Serum LH was not influenced by social status in the absence of E2. In contrast, subordinate females were hypersensitive to E2-negative feedback inhibition of LH. Furthermore, serum LH in subordinate females with s-variant SLC6A4 genotype was maximally suppressed by Day 4 of treatment, whereas nadir concentrations were not reached until later in treatment in other females. Finally, pharmacological elevation of serum cortisol potentiated E2-negative feedback inhibition in all females. The current data suggest that infertility induced by psychosocial stressors may be mediated by hypersensitivity to E2-negative feedback and that polymorphisms in the SLC6A4 gene may contribute to differences in reproductive compromise in response to chronic stress.

Intratrabecular tunneling increases trabecular number throughout the skeleton of ovariectomized rhesus monkeys treated with parathyroid hormone 1–84

Daily treatment of ovariectomized (OVX) adult rhesus monkeys with human parathyroid hormone (PTH) 1–84 for 16 months increases trabecular bone volume (BV/TV), number (Tb.N) and connectivity at lumbar vertebra-3 (L3) and thoracic vertebra-10. We proposed that the increased Tb.N and connectivity was achieved by stimulation of intratrabecular tunneling. Using histomorphometry to determine frequency of events, we have now quantified intratrabecular tunneling at L3 and extended it to investigate the effects of PTH(1–84) treatment on trabecular bone at the proximal femur, distal radius and iliac crest of these animals. At L3, tunneling frequency was low in control sham and OVX animals (∼ 0.05/mm 2) but increased significantly in PTH(1–84)-treated animals (0.27, 0.49 and 0.95/mm 2 with the 5, 10 and 25 μg/kg doses, respectively). Very similar tunneling frequencies were observed at all skeletal sites in all groups. Iliac crest biopsies were also collected at baseline and after 6 months of treatment and showed significant time- and dose-related increases in tunnels. Although the pattern and magnitude of response varied slightly from site to site, PTH(1–84) treatment significantly increased Tb.N, as well as BV/TV and bone formation rate at all skeletal sites. A modest but statistically significant increase in trabecular thickness occurred only at the iliac crest. In summary, intratrabecular tunneling is rare in control monkeys, but increased substantially with PTH(1–84) treatment. This phenomenon provides a plausible explanation for the PTH(1–84)-induced increase in Tb.N observed in OVX monkeys. Moreover, these analyses allowed a comparison of the effects PTH(1–84) treatment on trabecular bone at multiple locations.

Effects of Treatment with Parathyroid Hormone 1–84 on Quantity and Biomechanical Properties of Thoracic Vertebral Trabecular Bone in Ovariectomized Rhesus Monkeys

Osteoporosis is characterized by impaired bone quality leading to increased susceptibility to fracture, particularly of the thoracic spine. However, the lumbar spine is studied most commonly. We investigated the effects of 16 months of treatment with full-length parathyroid hormone (PTH) 1-84 (5, 10, or 25 microg/kg) on bone mineral density (BMD) and on architecture and biomechanical properties of trabecular bone at the thoracic spine of ovariectomized (OVX) adult rhesus monkeys and compared the results with those from the lumbar spine. At baseline, 9 months after surgery, dual-energy X-ray absorptiometric BMD at T9-T12 was 7% lower in OVX than in sham animals. All PTH(1-84) doses increased BMD to sham levels within 7 months. Micro-computed tomography of T10 vertebrae showed that trabecular bone volume and connectivity were higher in PTH(1-84)-treated animals than in sham controls, primarily through a significantly greater trabecular number. Peripheral quantitative computed tomography of trabecular bone cores from T11 and T12 confirmed that PTH(1-84) increased BMD. Compression testing of the cores showed that PTH(1-84) treatment increased stiffness, modulus, yield load, and yield stress to levels significantly greater than in sham animals, with the largest effect in the 10 microg/kg group (35-54% greater than in OVX controls). Thus, PTH(1-84) treatment increased BMD and the biomechanical properties of trabecular bone at the thoracic spine of OVX rhesus monkeys. The 10 microg/kg dose produced the greatest effect on trabecular strength, possibly because the highest dose stimulated bone remodeling excessively. Importantly, the changes observed were similar to those in lumbar vertebrae, thereby validating extrapolation of results from the lumbar to the thoracic spine.

Astressin B, a Nonselective Corticotropin-Releasing Hormone Receptor Antagonist, Prevents the Inhibitory Effect of Ghrelin on Luteinizing Hormone Pulse Frequency in the Ovariectomized Rhesus Monkey

Administration of ghrelin, a key peptide in the regulation of energy homeostasis, has been shown to decrease LH pulse frequency while concomitantly elevating cortisol levels. Because increased endogenous CRH release in stress is associated with an inhibition of reproductive function, we have tested here whether the pulsatile LH decrease after ghrelin may reflect an activated hypothalamic-pituitary-adrenal axis and be prevented by a CRH antagonist. After a 3-h baseline LH pulse frequency monitoring, five adult ovariectomized rhesus monkeys received a 5-h saline (protocol 1) or ghrelin (100-microg bolus followed by 100 microg/h, protocol 2) infusion. In protocols 3 and 4, animals were given astressin B, a nonspecific CRH receptor antagonist (0.45 mg/kg im) 90 min before ghrelin or saline infusion. Blood samples were taken every 15 min for LH measurements, whereas cortisol and GH were measured every 45 min. Mean LH pulse frequency during the 5-h ghrelin infusion was significantly lower than in all other treatments (P < 0.05) and when compared with the baseline period (P < 0.05). Pretreatment with astressin B prevented the decrease. Ghrelin stimulated cortisol and GH secretion, whereas astressin B pretreatment prevented the cortisol, but not the GH, release. Our data indicate that CRH release mediates the inhibitory effect of ghrelin on LH pulse frequency and suggest that the inhibitory impact of an insufficient energy balance on reproductive function may in part be mediated by the hypothalamic-pituitary-adrenal axis.

Effects of daily treatment with parathyroid hormone 1–84 for 16 months on density, architecture and biomechanical properties of cortical bone in adult ovariectomized rhesus monkeys

Treatment with parathyroid hormone 1–84 (PTH) or teriparatide increases osteonal remodeling and decreases bone mineral density (BMD) at cortical (Ct) bone sites but may also increase bone size. Decreases in BMD and increases in size exert opposing effects on bone strength. In adult ovariectomized (OVX) rhesus monkeys, we assessed the effects of daily PTH treatment (5, 10 or 25 μg/kg) for 16 months on BMD at the radial, tibial and femoral diaphyses, and on biomechanical properties (3-point bending) of radial cortical bone and the femoral diaphysis. PTH treatment did not affect areal BMD measured by dual-energy X-ray absorptiometry at the tibial diaphysis but caused a rapid, dose-related decrease at the distal radial diaphysis. Peripheral quantitative computed tomography at the radial and femoral diaphyses confirmed a significant PTH dose-related decrease in volumetric Ct.BMD caused primarily by increased cortical area. Significant increases in cortical thickness were the result of nonsignificant increases in periosteal length and decreases in endocortical length. Histomorphometry revealed increased endocortical bone formation at the tibial diaphysis and rib, higher Haversian remodeling at the rib and increased cortical porosity at the rib and tibia. Biomechanical testing at the femoral diaphysis showed that PTH treatment had no effect on peak load, but significantly decreased stiffness and increased work-to-failure (the energy required to break the bone). Similar changes occurred in radial cortical beams but only stiffness was changed significantly. Thus, PTH treatment of OVX rhesus monkeys decreased BMD and stiffness of cortical bone but did not affect peak load, likely because of increased bone size. However, PTH treatment increased the energy required to break the femur making it more resistant to fracture.

Treatment of Skeletally Mature Ovariectomized Rhesus Monkeys With PTH(1-84) for 16 Months Increases Bone Formation and Density and Improves Trabecular Architecture and Biomechanical Properties at the Lumbar Spine

Histomorphometric studies of treatments for osteoporosis in humans are restricted to iliac crest biopsies. We studied the effects of PTH(1-84) treatment at the lumbar spine of skeletally mature ovariectomized rhesus monkeys. PTH increased bone turnover, rapidly normalized BMD, and increased vertebral compressive strength. PTH increased trabecular bone volume primarily by increasing trabecular number by markedly increasing intratrabecular tunneling. Histomorphometric studies of the anabolic properties of PTH(1-84) (PTH) and related peptides in human bone are restricted to iliac crest biopsies. The ovariectomized (OVX) monkey is an accepted model of human postmenopausal bone loss and was used to study the effects of PTH treatment at clinically relevant skeletal sites. Skeletally mature rhesus monkeys were OVX or sham-operated and, after a bone depletion period of 9 months, treated daily for 16 months with PTH (5, 10, or 25 microg/kg). Markers of bone formation (serum osteocalcin) and resorption (urine N-telopeptide [NTX]) and lumbar spine BMD were measured throughout the study. Trabecular architecture and vertebral biomechanical properties were quantified at 16 months. PTH treatment induced dose-dependent increases in bone turnover but did not increase serum calcium. Osteocalcin was significantly increased above OVX controls by 1 month. NTX was significantly elevated at 1 month with the highest dose, but not until 12 months with the 5 and 10 microg/kg doses. Lumbar spine BMD was 5% lower in OVX than in sham animals when treatment was started. All PTH doses increased BMD rapidly, with sham levels restored by 3-7 months with 10 and 25 microg/kg and by 16 months with 5 microg/kg. PTH treatment increased trabecular bone volume (BV/TV), primarily by increasing trabecular number, and dose-dependently increased bone formation rate (BFR) solely by increasing mineralizing surface. The largest effects on BV/TV and yield load occurred with the 10 microg/kg dose. The highest dose reduced trabecular thickness by markedly increasing intratrabecular tunneling. PTH treatment of OVX rhesus monkeys increased bone turnover and increased BV/TV, BMD, and strength at the lumbar spine. All PTH doses were safe, but the 10 microg/kg dose was generally optimal, possibly because the highest dose resulted in too marked a stimulation of bone remodeling.

Estrogen Alters Spine Number and Morphology in Prefrontal Cortex of Aged Female Rhesus Monkeys

Long-term cyclic treatment with 17beta-estradiol reverses age-related impairment in ovariectomized rhesus monkeys on a test of cognitive function mediated by the prefrontal cortex (PFC). Here, we examined potential neurobiological substrates of this effect using intracellular loading and morphometric analyses to test the possibility that the cognitive benefits of hormone treatment are associated with structural plasticity in layer III pyramidal cells in PFC area 46. 17beta-Estradiol did not affect several parameters such as total dendritic length and branching. In contrast, 17beta-estradiol administration increased apical and basal dendritic spine density, and induced a shift toward smaller spines, a response linked to increased spine motility, NMDA receptor-mediated activity, and learning. These results document that, although the aged primate PFC is vulnerable in the absence of factors such as circulating estrogens, it remains responsive to long-term cyclic 17beta-estradiol treatment, and that increased dendritic spine density and altered spine morphology may contribute to the cognitive benefits of such treatment.