Scavenging of bacteria or bacterial products by magnetic particles functionalized with a broad-spectrum pathogen recognition receptor motif offers diagnostic and therapeutic applications.

Sepsis is a dysregulated host response of severe bloodstream infections, and given its frequency of occurrence and high mortality rate, therapeutic improvements are imperative. A reliable biomimetic strategy for the targeting and separation of bacterial pathogens in bloodstream infections involves the use of the broad-spectrum binding motif of human GP-340, a pattern-recognition receptor of the scavenger receptor cysteine rich (SRCR) superfamily that is expressed on epithelial surfaces but not found in blood. Here we show that these peptides, when conjugated to superparamagnetic iron oxide nanoparticles (SPIONs), can separate various bacterial endotoxins and intact microbes (E. coli, S. aureus, P. aeruginosa and S. marcescens) with high efficiency, especially at low and thus clinically relevant concentrations. This is accompanied by a subsequent strong depletion in cytokine release (TNF, IL-6, IL-1ß, Il-10 and IFN-γ), which could have a direct therapeutic impact since escalating immune responses complicates severe bloodstream infections and sepsis courses. SPIONs are coated with aminoalkylsilane and capture peptides are orthogonally ligated to this surface. The particles behave fully cyto- and hemocompatible and do not interfere with host structures. Thus, this approach additionally aims to dramatically reduce diagnostic times for patients with suspected bloodstream infections and accelerate targeted antibiotic therapy. STATEMENT OF SIGNIFICANCE: Sepsis is often associated with excessive release of cytokines. This aspect and slow diagnostic procedures are the major therapeutic obstacles. The use of magnetic particles conjugated with small peptides derived from the binding motif of a broad-spectrum mucosal pathogen recognition protein GP-340 provides a highly efficient scavenging platform. These peptides are not found in blood and therefore are not subject to inhibitory mechanisms like in other concepts (mannose binding lectine, aptamers, antibodies). In this work, data are shown on the broad bacterial binding spectrum, highly efficient toxin depletion, which directly reduces the release of cytokines. Host cells are not affected and antibiotics not adsorbed. The particle bound microbes can be recultured without restriction and thus be used directly for diagnostics.

Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles Enable a Stable Non-Spilling Loading of T Cells and Their Magnetic Accumulation.

T cell infiltration into a tumor is associated with a good clinical prognosis of the patient and adoptive T cell therapy can increase anti-tumor immune responses. However, immune cells are often excluded from tumor infiltration and can lack activation due to the immune-suppressive tumor microenvironment. To make T cells controllable by external forces, we loaded primary human CD3+ T cells with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs). Since the efficacy of magnetic targeting depends on the amount of SPION loading, we investigated how experimental conditions influence nanoparticle uptake and viability of cells. We found that loading in the presence of serum improved both the colloidal stability of SPIONs and viability of T cells, whereas stimulation with CD3/CD28/CD2 and IL-2 did not influence nanoparticle uptake. Furthermore, SPION loading did not impair cytokine secretion after polyclonal stimulation. We finally achieved 1.4 pg iron loading per cell, which was both located intracellularly in vesicles and bound to the plasma membrane. Importantly, nanoparticles did not spill over to non-loaded cells. Since SPION-loading enabled efficient magnetic accumulation of T cells in vitro under dynamic conditions, we conclude that this might be a good starting point for the investigation of in vivo delivery of immune cells.

Sex-specific features of spine densities in the hippocampus.

Previously, we found that in dissociated hippocampal cultures the proportion of large spines (head diameter ≥ 0.6 µm) was larger in cultures from female than from male animals. In order to rule out that this result is an in vitro phenomenon, we analyzed the density of large spines in fixed hippocampal vibratome sections of Thy1-GFP mice, in which GFP is expressed only in subpopulations of neurons. We compared spine numbers of the four estrus cycle stages in females with those of male mice. Remarkably, total spine numbers did not vary during the estrus cycle, while estrus cyclicity was evident regarding the number of large spines and was highest during diestrus, when estradiol levels start to rise. The average total spine number in females was identical with the spine number in male animals. The density of large spines, however, was significantly lower in male than in female animals in each stage of the estrus cycle. Interestingly, the number of spine apparatuses, a typical feature of large spines, did not differ between the sexes. Accordingly, NMDA-R1 and NMDA-R2A/B expression were lower in the hippocampus and in postsynaptic density fractions of adult male animals than in those of female animals. This difference could already be observed at birth for NMDA-R1, but not for NMDA-R2A/B expression. In dissociated embryonic hippocampal cultures, no difference was seen after 21 days in culture, while the difference was evident in postnatal cultures. Our data indicate that hippocampal neurons are differentiated in a sex-dependent manner, this differentiation being likely to develop during the perinatal period.

Autoantibodies against thrombospondin type 1 domain-containing 7A induce membranous nephropathy.

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, and one-third of patients develop end-stage renal disease (ESRD). Circulating autoantibodies against the podocyte surface antigens phospholipase A2 receptor 1 (PLA2R1) and the recently identified thrombospondin type 1 domain-containing 7A (THSD7A) are assumed to cause the disease in the majority of patients. The pathogenicity of these antibodies, however, has not been directly proven. Here, we have reported the analysis and characterization of a male patient with THSD7A-associated MN who progressed to ESRD and subsequently underwent renal transplantation. MN rapidly recurred after transplantation. Enhanced staining for THSD7A was observed in the kidney allograft, and detectable anti-THSD7A antibodies were present in the serum before and after transplantation, suggesting that these antibodies induced a recurrence of MN in the renal transplant. In contrast to PLA2R1, THSD7A was expressed on both human and murine podocytes, enabling the evaluation of whether anti-THSD7A antibodies cause MN in mice. We demonstrated that human anti-THSD7A antibodies specifically bind to murine THSD7A on podocyte foot processes, induce proteinuria, and initiate a histopathological pattern that is typical of MN. Furthermore, anti-THSD7A antibodies induced marked cytoskeletal rearrangement in primary murine glomerular epithelial cells as well as in human embryonic kidney 293 cells. Our findings support a causative role of anti-THSD7A antibodies in the development of MN.

Control of aromatase in hippocampal neurons.

Our knowledge on estradiol-induced modulation of synaptic function in the hippocampus is widely based on results following the application of the steroid hormone to either cell cultures, or after the treatment of gonadectomized animals, thus ignoring local neuronal estrogen synthesis. We and others, however, have shown that hippocampus-derived estradiol also controls synaptic plasticity in the hippocampus. Estradiol synthesis in the hippocampus is regulated by several mechanisms, which are reviewed in this report. The regulation of the activity of aromatase, the final enzyme of estrogen biosynthesis, by Ca(2+) transients, is of particular interest. Aromatase becomes inactivated as soon as it is phosphorylated by Ca(2+)-dependent kinases upon calcium release from internal stores. Accordingly, thapsigargin dephosphorylates aromatase and stimulates estradiol synthesis by depletion of internal Ca(2+) stores. Vice versa, letrozole, an aromatase inhibitor, phosphorylates aromatase and reduces estradiol synthesis. Treatment of the cultures with 17ß-estradiol results in phosphorylation of the enzyme and increased aromatase protein expression, which suggests that estradiol synthesis in hippocampal neurons is regulated in an autocrine manner.

Sexual neurosteroids and synaptic plasticity in the hippocampus.

Sexual neurosteroids (SN), namely 17ß-estradiol (E2) and 5α-dehydrotestosterone (DHT), are synthesized in the hippocampus, where they induce circuit modifications by changing the number of excitatory spine synapses in a paracrine and sex-specific manner. The mechanisms of this sex-specific synapse turnover, which are likely to affect cognitive functions, are poorly understood. We found that hippocampal neurons synthesize estradiol, which maintains LTP and synapses in females but not in males. In females, inhibition of estradiol synthesis results in impairment of LTP and synapse loss. These effects were not seen in males. The essential role of local estrogen on the stability and maintenance of connectivity in the hippocampus is consistent with age-related cognitive decline in women after menopause. In male animals the regulation of synaptic stability and plasticity by locally synthesized sexual steroids remains to be clarified. This article is part of a Special Issue entitled SI: Brain and Memory.

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism.

INTRODUCTION: Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17ß-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue. METHODS: We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes. RESULTS: Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻7M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-ß. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole. CONCLUSIONS: We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-ß. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.

Estradiol responsiveness of synaptopodin in hippocampal neurons is mediated by estrogen receptor ß.

In the hippocampus, synaptic proteins, as studied so far, have been shown to be upregulated by 17ß-estradiol, while inhibition of local estradiol synthesis consistently downregulates them. As an exception to this rule, we have previously shown that synaptopodin, an actin-associated postsynaptic protein, is downregulated in response to estradiol in dissociated cultured hippocampal neurons. In this study, we show, unexpectedly, that synaptopodin is downregulated in the hippocampus of aromatase knock-out mice and that inhibition of neuronal estradiol synthesis using the aromatase inhibitor letrozole also downregulates synaptopodin in these cultures. Moreover, the effects of estradiol and letrozole are additive, suggesting a subtle balance between available ligand and receptor. Using selective estrogen receptor agonists and antagonists, we consequently studied the effects of estrogen receptor subtypes on synaptopodin expression in our hippocampal cultures. We found that estradiol-induced downregulation of synaptopodin is mediated by estrogen receptor ß. Estrogen receptor ß in turn, is upregulated in response to intracellular estradiol ablation following inhibition of estradiol synthesis by letrozole in dissociated hippocampal cultures, as well as in the hippocampus of the aromatase knock-out mouse. Thus, it appears that both the application of estradiol, via binding to estrogen receptor ß, and letrozole, via upregulation of estrogen receptor ß, eventually result in a downregulation of synaptopodin. Our data show that the synaptic plasticity caused by estradiol is subject to a subtle balance of the levels of estrogen receptor subtypes regulated by the available ligands. In addition, both seem to be part of a homeostatic feedback mechanism.

Endocrine regulation of estrogen synthesis in the hippocampus?

Estradiol synthesis in the ovaries is regulated via feedback mechanisms mediated by gonadotrophin-releasing hormone (GnRH) and gonadotrophins, secreted by the hypothalamus and the pituitary, respectively. Estradiol synthesis also takes place in the hippocampus. In hippocampal slice cultures of female animals, GnRH regulates estradiol synthesis dose-dependently. Hence, both hippocampal and ovarian estradiol synthesis are synchronized by GnRH. Hippocampus-derived estradiol is essential to synapse stability and maintenance because it stabilizes the spine cytoskeleton of hippocampal neurons. Inhibition of hippocampal estradiol synthesis in mice, however, results in loss of spines and spine synapses in females, but not in males. Stereotaxic application of GnRH to the hippocampus of female rats confirms the regulatory role of GnRH on estradiol synthesis and synapse density in the female hippocampus in vivo. This regulatory role of GnRH necessarily results in estrus cyclicity of spine density in the hippocampus of females.

Aromatase inhibition abolishes LTP generation in female but not in male mice.

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

Estrogen-regulated synaptogenesis in the hippocampus: sexual dimorphism in vivo but not in vitro.

Hippocampal neurons are capable of synthesizing estradiol de novo. Estradiol synthesis can be suppressed by aromatase inhibitors and by knock-down of steroid acute regulatory protein (StAR), whereas elevated levels of substrates of steroidogenesis enhance estradiol synthesis. In rat hippocampal cultures, the expression of estrogen receptors (ERs) and synaptic proteins, as well as synapse density, correlated positively with aromatase activity, regardless of whether the cultures originated from males or females. All effects induced by the inhibition of aromatase activity were rescued by application of estradiol to the cultures. In vivo, however, systemic application of letrozole, an aromatase inhibitor, induced synapse loss in female rats, but not in males. Furthermore, in the female hippocampus, density of spines and spine synapses varied with the estrus cycle. In addressing this in vivo-in vitro discrepancy, we found that gonadotropin-releasing hormone (GnRH) regulated estradiol synthesis via an aromatase-mediated mechanism and consistently regulated spine synapse density and the expression of synaptic proteins. Along these lines, GnRH receptor density was higher in the hippocampus than in the cortex and hypothalamus, and estrus cyclicity of spinogenesis was found in the hippocampus, but not in the cortex. Since GnRH receptor expression also varies with the estrus cycle, the sexual dimorphism in estrogen-regulated spine synapse density in the hippocampus very likely results from differences in the GnRH responsiveness of the male and the female hippocampus. This article is part of a Special Issue entitled 'Neurosteroids'.

Estrogen synthesis in the hippocampus.

Estradiol plays essential roles in the modulation of synaptic plasticity and neuroprotection in males as well as in females, as has been shown particularly in the hippocampus. Although it has long been known that aromatase, the final enzyme in estrogen synthesis, is expressed in the hippocampus, a new paradigm emerged when it was shown that estradiol is actually synthesized de novo in this part of the brain. Increasing evidence indicates that hippocampus-derived estradiol plays a role in synaptic plasticity and neuroptrotection, rather than estradiol originating from the gonads. In recent years, a number of in vivo and in vitro studies have shown that hippocampus-derived estradiol substantially contributes to hippocampal function, in particular to structural synaptic plasticity.

Roles of 17ß-estradiol involve regulation of reelin expression and synaptogenesis in the dentate gyrus.

Studies on the role of 17ß-estradiol (E2) in the hippocampus have mainly focused on CA1 and CA3 regions, whereas in dentate gyrus (DG), its role is largely unknown. Here, we examined potential functions of E2 in DG, particularly during development. Immunohistochemistry and in situ hybridization revealed abundance of estrogen receptor (ER)α, but not ERß, expression in DG. Similar to CA1, analysis of synapse densities revealed a reduction in spine synapse number in DG molecular layer of immature rats and adult mice after inhibition of estradiol synthesis using letrozole. Interestingly, strong expression of ERα was found in Cajal-Retzius (CR) cells, which regulate neuronal migration and synaptogenesis via the extracellular matrix protein reelin. Immunoreactivity of aromatase, the final enzyme of estradiol synthesis, was strongest in mature granule cells. In hippocampal slice cultures, exogenous application of E2 caused an increase in reelin expression in CR cells, which was abolished after blockade of ERs using ICI182,780. Vice versa, inhibition of aromatase activity by letrozole resulted in reduced reelin expression, suggesting that E2 deriving from hippocampal sources contributes to the regulation of reelin as well as to the maintenance of spine synapses in DG. E2 further regulated Notch1, a signaling protein involved in neuronal differentiation.

Aromatase inhibitors induce spine synapse loss in the hippocampus of ovariectomized mice.

Recently, inhibition of estrogen synthesis by aromatase inhibitors has become a favored therapy for breast cancer in postmenopausal women. Estrogen is, however, important for synapse formation in the hippocampus. Inhibition of aromatase induces spine synapse loss in organotypic hippocampal slice cultures. We therefore studied the effect of systemic treatment with the potent aromatase inhibitor letrozole on spine synapse formation and synaptic proteins in the hippocampi of female mice for periods of 7 d and 4 wk. In cyclic, letrozole-treated females and in ovariectomized, letrozole-treated females, the number of spine synapses was significantly reduced in the hippocampus but not in the prefrontal or cerebellar cortex. Consequently, the expression of the N-methyl-D-aspartate receptor NR1 was significantly down-regulated after treatment with letrozole. In cyclic animals the expression of the synaptic proteins synaptophysin and spinophilin was down-regulated in response to letrozole. In ovariectomized animals, however, protein expression was down-regulated after 7 d of treatment, whereas the expression was up-regulated after 4 wk of treatment. Our results indicate that systemic inhibition of aromatase in mice affects structural synaptic plasticity in the hippocampus. This may contribute to cognitive deficits in postmenopausal women treated with aromatase inhibitors.

The opposing roles of estradiol on synaptic protein expression in hippocampal cultures.

Estrogen-induced synaptic plasticity was frequently shown by an increase of spines at apical dendrites of CA1 pyramidal neurons after systemic application of estradiol to ovariectomized rats. Surprisingly, exogenous application of estradiol to hippocampal cultures had no effect on spines and on spine synapses, although quantitative immunohistochemistry revealed an upregulation of spinophilin and of synaptophysin, in these cultures. The role of synaptophysin as a presynaptic marker and of spinophilin as a postsynaptic marker, appears questionable from these discrepancies. In contrast, synaptopodin, a marker protein of "mature" mushroom-shaped spines, was downregulated after treatment of hippocampal cultures with estradiol. Synaptopodin is strongly associated to the spine apparatus, a spine-specific cell organelle, which is present in 80% of all mushroom-shaped spines. Consistently, we found a reduction in the number of spines, containing a spine apparatus in response to estradiol, suggesting that the presence of a spine apparatus in many but not all spines is very likely a result of their dynamic character. In summary, synaptic proteins appear to be regulated by estradiol, independent of its function on spine and spine synapse formation.

Estrus cyclicity of spinogenesis: underlying mechanisms.

Hippocampal spine density varies with the estrus cycle. The cyclic change in estradiol levels in serum was hypothesized to underlie this phenomenon, since treatment of ovariectomized animals with estradiol induced an increase in spine density in hippocampal dendrites of rats, as compared to ovariectomized controls. In contrast, application of estradiol to hippocampal slice cultures did not promote spinogenesis. In addressing this discrepancy, we found that hippocampal neurons themselves are capable of synthesizing estradiol de novo. Estradiol synthesis can be suppressed by aromatase inhibitors and by knock-down of Steroid Acute Regulatory Protein (StAR) and enhanced by substrates of steroidogenesis. Expression of estrogen receptors (ERs) and synaptic proteins, synaptogenesis, and long-term potentiation (LTP) correlated positively with aromatase activity in hippocampal cultures without any difference between genders. All effects due to inhibition of aromatase activity were rescued by application of estradiol to the cultures. Most importantly, gonadotropin-releasing hormone (GnRH) increased estradiol synthesis dose-dependently via an aromatase-mediated mechanism and consistently increased spine synapse density and spinophilin expression. As a consequence, our data suggest that cyclic fluctuations in spine synapse density result from pulsative release of GnRH from the hypothalamus and its effect on hippocampal estradiol synthesis, rather than from varying levels of serum estradiol. This hypothesis is further supported by higher GnRH receptor (GnRH-R) density in the hippocampus than in the cortex and hypothalamus and the specificity of estrus cyclicity of spinogenesis in the hippocampus, as compared to the cortex.

Cholesterol-promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus.

Cholesterol of glial origin promotes synaptogenesis (Mauch et al., (2001) Science 294:1354-1357). Because in the hippocampus local estradiol synthesis is essential for synaptogenesis, we addressed the question of whether cholesterol-promoted synapse formation results from the function of cholesterol as a precursor of estradiol synthesis in this brain area. To this end, we treated hippocampal cultures with cholesterol, estradiol, or with letrozole, a potent aromatase inhibitor. Cholesterol increased neuronal estradiol release into the medium, the number of spine synapses in hippocampal slice cultures, and immunoreactivity of synaptic proteins in dispersed cultures. Simultaneous application of cholesterol and letrozole or blockade of estrogen receptors by ICI 182 780 abolished cholesterol-induced synapse formation. As a further approach, we inhibited the access of cholesterol to the first enzyme of steroidogenesis by knock-down of steroidogenic acute regulatory protein, the rate-limiting step in steroidogenesis. A rescue of reduced synaptic protein expression in transfected cells was achieved by estradiol but not by cholesterol. Our data indicate that in the hippocampus cholesterol-promoted synapse formation requires the conversion of cholesterol to estradiol.

Inhibition of hippocampal estrogen synthesis causes region-specific downregulation of synaptic protein expression in hippocampal neurons.

Previous studies have shown that synapses and expression of synaptic proteins in hippocampal neurons are regulated by hippocampus-derived estradiol. Here, we compared the effects of this paracrine regulation in different hippocampal regions. In tissue sections, immunohistochemistry followed by semiquantitative image analysis revealed a three-fold higher expression of steroidogenic acute regulatory protein (StAR) and aromatase in neurons of the CA3 than that of the CA1 region and in granule cells. Next, we treated hippocampal cell cultures with letrozole, an aromatase inhibitor, which resulted in a dose-dependent decrease in the release of 17beta-estradiol into the medium and in a dose-dependent downregulation of spinophilin and synaptophysin expression in dissociated hippocampal neurons. The downregulation of synaptic protein expression was restored by simultaneous application of letrozole together with estradiol. In response to a defined dose of letrozole, the downregulation of spinophilin expression was significantly stronger in CA1 neurons and in granule cells, than in cells of the CA3 region in slice cultures. With synaptophysin, downregulation was stronger in stratum lucidum of CA3 than in stratum radiatum of CA1. Both region-specific expression of steroidogenic enzymes and region-specific downregulation of synaptic proteins in response to a defined dose of letrozole may suggest different levels of estrogen concentrations within the hippocampus. Varying concentrations of estradiol in the hippocampus in turn may contribute to region-specific differentiation of hippocampal neurons.

Hippocampal synapses depend on hippocampal estrogen synthesis.

Estrogens have been described to induce synaptogenesis in principal neurons of the hippocampus and have been shown to be synthesized and released by exactly these neurons. Here, we have focused on the significance of local estrogen synthesis on spine synapse formation and the synthesis of synaptic proteins. To this end, we reduced hippocampal estrogen synthesis in vitro with letrozole, a reversible nonsteroidal aromatase inhibitor. In hippocampal slice cultures, letrozole treatment resulted in a dose-dependent decrease of 17beta-estradiol as quantified by RIA. This was accompanied by a significant decrease in the density of spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a downregulation of spinophilin, a marker of dendritic spines, and synaptophysin, a protein of presynaptic vesicles, in response to letrozole. Surprisingly, no increase in the density of spines, boutons, and synapses and in spinophilin expression was seen after application of estradiol to the medium of cultures that had not been treated with letrozole. However, synaptophysin expression was upregulated under these conditions. Our results point to an essential role of endogenous hippocampal estrogen synthesis in the maintenance of hippocampal spine synapses.