Estrogen Receptor ß Contributes to Both Hypertension and Hypothalamic Plasticity in a Mouse Model of Peri-Menopause.

Hypertension susceptibility in women increases at the transition to menopause, termed perimenopause, a state characterized by erratic estrogen fluctuation and extended hormone cycles. Elucidating the role of estrogen signaling in the emergence of hypertension during perimenopause has been hindered by animal models that are confounded by abrupt estrogen cessation or effects of aging. In the present study, accelerated ovarian failure (AOF) in estrogen receptor ß (ERß) reporter mice was induced by 4-vinylcyclohexene diepoxide in young mice to model early-stage ovarian failure (peri-AOF) characteristic of peri-menopause. It was found that administering ERß agonists suppressed elevated blood pressure in a model of neurogenic hypertension induced by angiotensin II (AngII) in peri-AOF, but not in age-matched male mice. It was also found that ERß agonist administration in peri-AOF females, but not males, suppressed the heightened NMDAR signaling and reactive oxygen production in ERß neurons in the hypothalamic paraventricular nucleus (PVN), a critical neural regulator of blood pressure. It was further shown that deleting ERß in the PVN of gonadally intact females produced a phenotype marked by a sensitivity to AngII hypertension. These results suggest that ERß signaling in the PVN plays an important role in blood pressure regulation in female mice and contributes to hypertension susceptibility in females at an early stage of ovarian failure comparable to human perimenopause.

Estrogen Effects on Cognitive and Synaptic Health Over the Lifecourse.

Estrogen facilitates higher cognitive functions by exerting effects on brain regions such as the prefrontal cortex and hippocampus. Estrogen induces spinogenesis and synaptogenesis in these two brain regions and also initiates a complex set of signal transduction pathways via estrogen receptors (ERs). Along with the classical genomic effects mediated by activation of ER α and ER ß, there are membrane-bound ER α, ER ß, and G protein-coupled estrogen receptor 1 (GPER1) that can mediate rapid nongenomic effects. All key ERs present throughout the body are also present in synapses of the hippocampus and prefrontal cortex. This review summarizes estrogen actions in the brain from the standpoint of their effects on synapse structure and function, noting also the synergistic role of progesterone. We first begin with a review of ER subtypes in the brain and how their abundance and distributions are altered with aging and estrogen loss (e.g., ovariectomy or menopause) in the rodent, monkey, and human brain. As there is much evidence that estrogen loss induced by menopause can exacerbate the effects of aging on cognitive functions, we then review the clinical trials of hormone replacement therapies and their effectiveness on cognitive symptoms experienced by women. Finally, we summarize studies carried out in nonhuman primate models of age- and menopause-related cognitive decline that are highly relevant for developing effective interventions for menopausal women. Together, we highlight a new understanding of how estrogen affects higher cognitive functions and synaptic health that go well beyond its effects on reproduction.

Redistribution of NMDA Receptors in Estrogen-Receptor-ß-Containing Paraventricular Hypothalamic Neurons following Slow-Pressor Angiotensin II Hypertension in Female Mice with Accelerated Ovarian Failure.

Hypertension in male and aging female rodents is associated with glutamate-dependent plasticity in the hypothalamus, but existing models have failed to capture distinct transitional menopausal phases that could have a significant impact on the synaptic plasticity and emergent hypertension. In rodents, accelerated ovarian failure (AOF) induced by systemic injection of 4-vinylcyclohexane diepoxide mimics the estrogen fluctuations seen in human menopause including the perimenopause transition (peri-AOF) and postmenopause (post-AOF). Thus, we used the mouse AOF model to determine the impact of slow-pressor angiotensin II (AngII) administration on blood pressure and on the subcellular distribution of obligatory N-methyl-D-aspartate (NMDA) receptor GluN1 subunits in the paraventricular hypothalamic nucleus (PVN), a key estrogen-responsive cardiovascular regulatory area. Estrogen-sensitive neuronal profiles were identified in mice expressing enhanced green fluorescent protein under the promoter for estrogen receptor (ER) ß, a major ER in the PVN. Slow-pressor AngII increased arterial blood pressure in mice at peri- and post-AOF time points. In control oil-injected (nonhypertensive) mice, AngII decreased the total number of GluN1 in ERß-containing PVN dendrites. In contrast, AngII resulted in a reapportionment of GluN1 from the cytoplasm to the plasma membrane of ERß-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, AngII increased total GluN1, dendritic size and radical production in ERß-containing neurons. These results indicate that unique patterns of hypothalamic glutamate receptor plasticity and dendritic structure accompany the elevated blood pressure in peri- and post-AOF time points. Our findings suggest the possibility that distinct neurobiological processes are associated with the increased blood pressure during perimenopausal and postmenopausal periods.

G-protein-coupled estrogen receptor 1 is anatomically positioned to modulate synaptic plasticity in the mouse hippocampus.

Both estrous cycle and sex affect the numbers and types of neuronal and glial profiles containing the classical estrogen receptors α and ß, and synaptic levels in the rodent dorsal hippocampus. Here, we examined whether the membrane estrogen receptor, G-protein-coupled estrogen receptor 1 (GPER1), is anatomically positioned in the dorsal hippocampus of mice to regulate synaptic plasticity. By light microscopy, GPER1-immunoreactivity (IR) was most noticeable in the pyramidal cell layer and interspersed interneurons, especially those in the hilus of the dentate gyrus. Diffuse GPER1-IR was found in all lamina but was most dense in stratum lucidum of CA3. Ultrastructural analysis revealed discrete extranuclear GPER1-IR affiliated with the plasma membrane and endoplasmic reticulum of neuronal perikarya and dendritic shafts, synaptic specializations in dendritic spines, and clusters of vesicles in axon terminals. Moreover, GPER1-IR was found in unmyelinated axons and glial profiles. Overall, the types and amounts of GPER1-labeled profiles were similar between males and females; however, in females elevated estrogen levels generally increased axonal labeling. Some estradiol-induced changes observed in previous studies were replicated by the GPER agonist G1: G1 increased PSD95-IR in strata oriens, lucidum, and radiatum of CA3 in ovariectomized mice 6 h after administration. In contrast, estradiol but not G1 increased Akt phosphorylation levels. Instead, GPER1 actions in the synapse may be due to interactions with synaptic scaffolding proteins, such as SAP97. These results suggest that although estrogen's actions via GPER1 may converge on the same synaptic elements, different pathways are used to achieve these actions.

Female protection from slow-pressor effects of angiotensin II involves prevention of ROS production independent of NMDA receptor trafficking in hypothalamic neurons expressing angiotensin 1A receptors.

Renin­angiotensin system overactivity, upregulation of postsynaptic NMDA receptor function, and increased reactive oxygen species (ROS) production in the hypothalamic paraventricular nucleus (PVN) are hallmarks of angiotensin II (AngII)-induced hypertension, which is far more common in young males than in young females. We hypothesize that the sex differences in hypertension are related to differential AngII-induced changes in postsynaptic trafficking of the essential NMDA receptor GluN1 subunit and ROS production in PVN cells expressing angiotensin Type 1a receptor (AT1aR). We tested this hypothesis using slow-pressor (14-day) infusion of AngII (600 ng/kg/min) in mice, which elicits hypertension in males but not in young females. Two-month-old male and female transgenic mice expressing enhanced green fluorescent protein (EGFP) in AT1aR-containing cells were used. In males, but not in females, AngII increased blood pressure and ROS production in AT1aR­EGFP PVN cells at baseline and following NMDA treatment. Electron microscopy showed that AngII increased cytoplasmic and total GluN1­silver-intensified immunogold (SIG) densities and induced a trend toward an increase in near plasmalemmal GluN1­SIG density in AT1aR­EGFP dendrites of males and females. Moreover, AngII decreased dendritic area and diameter in males, but increased dendritic area of small (<1 µm) dendrites and decreased diameter of large (>1 µm) dendrites in females. Fluorescence microscopy revealed that AT1aR and estrogen receptor ß do not colocalize, suggesting that if estrogen is involved, its effect is indirect. These data suggest that the sexual dimorphism in AngII-induced hypertension is associated with sex differences in ROS production in AT1aR-containing PVN cells but not with postsynaptic NMDA receptor trafficking.

Membrane trafficking of NADPH oxidase p47(phox) in paraventricular hypothalamic neurons parallels local free radical production in angiotensin II slow-pressor hypertension.

NADPH oxidase-generated reactive oxygen species (ROS) are highly implicated in the development of angiotensin II (AngII)-dependent hypertension mediated in part through the hypothalamic paraventricular nucleus (PVN). This region contains vasopressin and non-vasopressin neurons that are responsive to cardiovascular dysregulation, but it is not known whether ROS is generated by one or both cell types in response to "slow-pressor" infusion of AngII. We addressed this question using ROS imaging and electron microscopic dual labeling for vasopressin and p47(phox), a cytoplasmic NADPH oxidase subunit requiring mobilization to membranes for the initiation of ROS production. C57BL/6 mice or vasopressin-enhanced green fluorescent protein (VP-eGFP) mice were infused systemically with saline or AngII (600 ng · kg(-1) · min(-1), s.c.) for 2 weeks, during which they slowly developed hypertension. Ultrastructural analysis of the PVN demonstrated p47(phox) immunolabeling in many glial and neuronal profiles, most of which were postsynaptic dendrites. Compared with saline, AngII recipient mice had a significant increase in p47(phox) immunolabeling on endomembranes just beneath the plasmalemmal surface (+42.1 ± 11.3%; p < 0.05) in non-vasopressin dendrites. In contrast, AngII infusion decreased p47(phox) immunolabeling on the plasma membrane (-35.5 ± 16.5%; p < 0.05) in vasopressin dendrites. Isolated non-VP-eGFP neurons from the PVN of AngII-infused mice also showed an increase in baseline ROS production not seen in VP-eGFP neurons. Our results suggest that chronic low-dose AngII may offset the homeostatic control of blood pressure by differentially affecting membrane assembly of NADPH oxidase and ROS production in vasopressin and non-vasopressin neurons located within the PVN.

Stress differentially alters mu opioid receptor density and trafficking in parvalbumin-containing interneurons in the female and male rat hippocampus.

Stress differentially affects hippocampal-dependent learning relevant to addiction and morphology in male and female rats. Mu opioid receptors (MORs), which are located in parvalbumin (PARV)-containing GABAergic interneurons and are trafficked in response to changes in the hormonal environment, play a critical role in promoting principal cell excitability and long-term potentiation. Here, we compared the effects of acute and chronic immobilization stress (AIS and CIS) on MOR trafficking in PARV-containing neurons in the hilus of the dentate gyrus in female and male rats using dual label immunoelectron microscopy. Following AIS, the density of MOR silver-intensified gold particles (SIGs) in the cytoplasm of PARV-labeled dendrites was significantly reduced in females (estrus stage). Conversely, AIS significantly increased the proportion of cytoplasmic MOR SIGs in PARV-labeled dendrites in male rats. CIS significantly reduced the number of PARV-labeled neurons in the dentate hilus of males but not females. However, MOR/PARV-labeled dendrites and terminals were significantly smaller in CIS females, but not males, compared with controls. Following CIS, the density of cytoplasmic MOR SIGs increased in PARV-labeled dendrites and terminals in females. Moreover, the proportion of near-plasmalemmal MOR SIGs relative to total decreased in large PARV-labeled dendrites in females. After CIS, no changes in the density or trafficking of MOR SIGs were seen in PARV-labeled dendrites or terminals in males. These data show that AIS and CIS differentially affect available MOR pools in PARV-containing interneurons in female and male rats. Furthermore, they suggest that CIS could affect principal cell excitability in a manner that maintains learning processes in females but not males.

BDNF variant Val66Met interacts with estrous cycle in the control of hippocampal function.

Natural fluctuations in circulating estradiol are associated with behavioral changes, including severe disturbances in mood and cognition in some women. Common genetic variation in some of the molecular mediators of estradiol effects on these behaviors, in brain regions such as the hippocampus, may explain individual variation in estradiol effects on behavior. We tested whether the common human variant BDNF Val66Met interacts with estradiol in the control of hippocampal function in cycling female mice homozygous for the wild-type Val or BDNF Met variant. BDNF Met increased anxiety behavior, impaired memory, and increased expression of BDNF and its receptor TrkB in the hippocampal formation. BDNF Met also dramatically altered the fluctuation of spatial memory, hippocampal Akt phosphorylation, and PSD-95 protein expression across the estrous cycle. The variant BDNF Val66Met should therefore be considered as a strong candidate for mediating genetic differences in ovarian steroid-related behavioral changes and disorders.

Both Nuclear and Membrane Estrogen Receptor Alpha Impact the Expression of Estrogen Receptors and Plasticity Markers in the Mouse Hypothalamus and Hippocampus.

Estrogens via estrogen receptor alpha (ERα) genomic and nongenomic signaling can influence plasticity processes in numerous brain regions. Using mice that express nuclear only ERα (NOER) or membrane only ERα (MOER), this study examined the effect of receptor compartmentalization on the paraventricular nucleus of the hypothalamus (PVN) and the hippocampus. The absence of nuclear and membrane ERα expression impacted females but not males in these two brain areas. In the PVN, quantitative immunohistochemistry showed that the absence of nuclear ERα increased nuclear ERß. Moreover, in the hippocampus CA1, immuno-electron microscopy revealed that the absence of either nuclear or membrane ERα decreased extranuclear ERα and pTrkB in synapses. In contrast, in the dentate gyrus, the absence of nuclear ERα increased pTrkB in synapses, whereas the absence of membrane ERα decreased pTrkB in axons. However, the absence of membrane only ERα decreased the sprouting of mossy fibers in CA3 as reflected by changes in zinc transporter immunolabeling. Altogether these findings support the idea that both membrane and nuclear ERα contribute overlapping and unique actions of estrogen that are tissue- and cellular-specific.

Rapid estrogen signaling in the brain: implications for the fine-tuning of neuronal circuitry.

Rapid actions of estrogens were first described >40 years ago. However, the importance of rapid estrogen-mediated actions in the CNS is only now becoming apparent. Several lines of evidence demonstrate that rapid estrogen-mediated signaling elicits potent effects on molecular and cellular events, resulting in the "fine-tuning" of neuronal circuitry. At an ultrastructural level, the details of estrogen receptor localization and how these are regulated by the circulating hormone and age are now becoming evident. Furthermore, the mechanisms that allow membrane-associated estrogen receptors to couple with intracellular signaling pathways are also now being revealed. Elucidation of complex actions of rapid estrogen-mediated signaling on synaptic proteins, connectivity, and synaptic function in pyramidal neurons has demonstrated that this neurosteroid engages specific mechanisms in different areas of the brain. The regulation of synaptic properties most likely underlies the fine-tuning of neuronal circuitry. This in turn may influence how learned behaviors are encoded by different circuitry in male and female subjects. Importantly, as estrogens have been suggested as potential treatments of a number of disorders of the CNS, advancements in our understanding of rapid estrogen signaling in the brain will serve to aid in the development of potential novel estrogen-based treatments.

Distribution of phosphorylated TrkB receptor in the mouse hippocampal formation depends on sex and estrous cycle stage.

Tropomyosin-related kinase B receptor (TrkB) is a neurotrophin receptor important for the synaptic plasticity underlying hippocampal-dependent learning and memory. Because this receptor is widely expressed in hippocampal neurons, the precise location of TrkB activation is likely important for its specific actions. The goal of this study was to identify the precise sites of TrkB activation in the mouse hippocampal formation and to determine any changes in the distribution of activated TrkB under conditions of enhanced brain-derived neurotrophic factor (BDNF) expression and hippocampal excitability. Using electron microscopy, we localized TrkB phosphorylated at tyrosine 816 (pTrkB) in the hippocampal formation of male and female mice under conditions of naturally low circulating estradiol and naturally high circulating estradiol, when BDNF expression, TrkB signaling, and synaptic plasticity are enhanced. To compare relative amounts of pTrkB in each group, we counted profiles containing pTrkB-immunoreactivity (pTrkB-ir) in all hippocampal subregions. pTrkB-ir was in axons, axon terminals, dendrites, and dendritic spines of neurons in the hippocampal formation, but the majority of pTrkB-ir localized to presynaptic profiles. pTrkB-ir also was abundant in glial profiles, which were further identified as microglia using immunofluorescence and confocal microscopy. Axonal and glial pTrkB-ir and pTrkB-ir in the CA1 stratum radiatum were more abundant in high-estradiol states (proestrus females) than low-estradiol states (estrus and diestrus females and males). These findings suggest that presynaptic TrkB is positioned to modulate estradiol-mediated and BDNF-dependent synaptic plasticity. Furthermore, they suggest a novel role for TrkB in microglial function in the neuroimmune system.

Sex and age influence gonadal steroid hormone receptor distributions relative to estrogen receptor ß-containing neurons in the mouse hypothalamic paraventricular nucleus.

Within the hypothalamic paraventricular nucleus (PVN), estrogen receptor (ER) ß and other gonadal hormone receptors play a role in central cardiovascular processes. However, the influence of sex and age on the cellular and subcellular relationships of ERß with ERα, G-protein ER (GPER1), as well as progestin and androgen receptors (PR and AR) in the PVN is uncertain. In young (2- to 3-month-old) females and males, ERß-enhanced green fluorescent protein (EGFP) containing neurons were approximately four times greater than ERα-labeled and PR-labeled nuclei in the PVN. In subdivisions of the PVN, young females, compared to males, had: (1) more ERß-EGFP neurons in neuroendocrine rostral regions; (2) fewer ERα-labeled nuclei in neuroendocrine and autonomic projecting medial subregions; and (3) more ERα-labeled nuclei in an autonomic projecting caudal region. In contrast, young males, compared to females, had approximately 20 times more AR-labeled nuclei, which often colocalized with ERß-EGFP in neuroendocrine (approximately 70%) and autonomic (approximately 50%) projecting subregions. Ultrastructurally, in soma and dendrites, PVN ERß-EGFP colocalized primarily with extranuclear AR (approximately 85% soma) and GPER1 (approximately 70% soma). Aged (12- to 24-month-old) males had more ERß-EGFP neurons in a rostral neuroendocrine subregion compared to aged females and females with accelerated ovarian failure (AOF) and in a caudal autonomic subregion compared to post-AOF females. Late-aged (18- to 24-month-old) females compared to early-aged (12- to 14-month-old) females and AOF females had fewer AR-labeled nuclei in neuroendrocrine and autonomic projecting subregions. These findings indicate that gonadal steroids may directly and indirectly influence PVN neurons via nuclear and extranuclear gonadal hormone receptors in a sex-specific manner.

Estrogen induces caspase-dependent cell death during hypothalamic development.

The sexually dimorphic population of dopamine neurons in the anteroventral periventricular nucleus of the preoptic region of the hypothalamus (AVPV) develops postnatally under the influence of testosterone, which is aromatized to estrogen. There are fewer dopaminergic neurons labeled with tyrosine hydroxylase (TH) in the male AVPV than the female, and sex steroids determine this sex difference, yet the role of cell death in specifying numbers of dopaminergic neurons in the AVPV is unknown. Estradiol treatment of the AVPV, in vivo and in vitro, was used to manipulate TH-ir cell number. In vitro, concurrent treatment with the estrogen receptor antagonist ICI 182,780 rescued TH-ir cells. Cyclosporin A, an inhibitor of cell death dependent on the opening of a mitochondrial permeability transition pore also blocked TH-ir cell loss. In vivo, estradiol increased the number of apoptotic profiles, both TUNEL and Hoechst labeled nuclei, in the AVPV. This increased apoptosis was also dependent on the presence of the alpha form of the estrogen receptor. To test for caspase dependent TH-ir cell loss, the pancaspase inhibitor ZVAD (N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone) was used to rescue TH-ir cells from estradiol-mediated reduction in number. Together, these data suggest that an intrinsic cell death pathway is activated by estrogen to regulate TH-ir cell number. Thus, in contrast to the more widespread neuroprotective actions of sex steroids in the mammalian nervous system, in the AVPV estrogen regulates dopaminergic neuron number through a caspase-dependent mechanism of apoptotic cell death.

Sex and chronic stress alter delta opioid receptor distribution within rat hippocampal CA1 pyramidal cells following behavioral challenges.

Following oxycodone (Oxy) conditioned place preference (CPP), delta opioid receptors (DORs) differentially redistribute in hippocampal CA3 pyramidal cells in female and male rats in a manner that would promote plasticity and opioid-associative learning processes. However, following chronic immobilization stress (CIS), males do not acquire Oxy-CPP and the trafficking of DORs in CA3 pyramidal neurons is attenuated. Here, we examined the subcellular distribution of DORs in CA1 pyramidal cells using electron microscopy in these same cohorts. CPP: Saline (Sal)-females compared to Sal-males have more cytoplasmic and total DORs in dendrites and more DOR-labeled spines. Following Oxy-CPP, DORs redistribute from near-plasmalemma pools in dendrites to spines in males. CIS: Control females compared to control males have more near-plasmalemmal dendritic DORs. Following CIS, dendritic DORs are elevated in the cytoplasm in females and near-plasmalemma in males. CIS PLUS CPP: CIS Sal-females compared to CIS Sal-males have more DORs on the plasmalemma of dendrites and in spines. After Oxy, the distribution of DORs does not change in either females or males. CONCLUSION: Following Oxy-CPP, DORs within CA1 pyramidal cells remain positioned in naïve female rats to enhance sensitivity to DOR agonists and traffic to dendritic spines in naïve males where they can promote plasticity processes. Following CIS plus behavioral enrichment, DORs are redistributed within CA1 pyramidal cells in females in a manner that could enhance sensitivity to DOR agonists. Conversely, CIS plus behavioral enrichment does not alter DORs in CA1 pyramidal cells in males, which may contribute to their diminished capacity to acquire Oxy-CPP.

Uncovering the mechanisms of estrogen effects on hippocampal function.

Estrogens have direct effects on the brain areas controlling cognition. One of the most studied of these regions is the dorsal hippocampal formation, which governs the formation of spatial and episodic memories. In laboratory animals, most investigators report that estrogen enhances synaptic plasticity and improves performance on hippocampal-dependent cognitive behaviors. This review summarizes work conducted in our laboratory and others toward identifying estrogen's actions in the hippocampal formation, and the mechanisms for these actions. Physiologic and pharmacologic estrogen affects cognitive behavior in mammals, which may be applicable to human health and disease. The effects of estrogen in the hippocampal formation that lead to modulation of hippocampal function include effects on cell morphology, synapse formation, signaling, and excitability that have been studied in laboratory mice, rats, and primates. Finally, estrogen may signal through both nuclear and extranuclear hippocampal estrogen receptors to achieve its downstream effects.

Plasma Membrane Affiliated AMPA GluA1 in Estrogen Receptor ß-containing Paraventricular Hypothalamic Neurons Increases Following Hypertension in a Mouse Model of Post-menopause.

Sex and ovarian function contribute to hypertension susceptibility, however, the mechanisms are not well understood. Prior studies show that estrogens and neurogenic factors, including hypothalamic glutamatergic NMDA receptor plasticity, play significant roles in rodent hypertension. Here, we investigated the role of sex and ovarian failure on AMPA receptor plasticity in estrogen-sensitive paraventricular nucleus (PVN) neurons in naïve and angiotensin II (AngII) infused male and female mice and female mice at early and late stages of accelerated ovarian failure (AOF). High-resolution electron microscopy was used to assess the subcellular distribution of AMPA GluA1 in age-matched male and female estrogen receptor beta (ERß) enhanced green fluorescent protein (EGFP) reporter mice as well as female ERß-EGFP mice treated with 4-vinylcyclohexene diepoxide. In the absence of AngII, female mice at a late stage of AOF displayed higher levels of GluA1 on the plasma membrane, indicative of functional protein, in ERß-expressing PVN dendrites when compared to male, naïve female and early stage AOF mice. Following slow-pressor AngII infusion, males, as well as early and late stage AOF females had elevated blood pressure. Significantly, only late stage-AOF female mice infused with AngII had an increase in GluA1 near the plasma membrane in dendrites of ERß-expressing PVN neurons. In contrast, prior studies reported that plasmalemmal NMDA GluN1 increased in ERß-expressing PVN dendrites in males and early, but not late stage AOF females. Together, these findings reveal that early and late stage AOF female mice display unique molecular signatures of long-lasting synaptic strength prior to, and following hypertension.

Effects of estrogen and aging on synaptic morphology and distribution of phosphorylated Tyr1472 NR2B in the female rat hippocampus.

Age and estrogens may impact the mobility of N-methyl-D-aspartate receptors (NMDARs) in hippocampal synapses. Here, we used serial section immunogold electron microscopy to examine whether phosphorylated tyrosine 1472 NR2B (pY1472), which is involved in the surface expression of NMDARs, is altered in the dorsal hippocampus of young (3-4 months old) and aged (∼24 months old) ovariectomized rats treated with 17ß-estradiol or vehicle for 2 days. The number of gold particles labeling pY1472 was higher in presynaptic and postsynaptic compartments of aged rats with low estradiol (vehicle-treated) compared to other groups. In terminals, pY1472 levels were elevated in aged rats but reduced by estradiol treatment to levels seen in young rats. Conversely, the mitochondria number was lower in aged females but was restored to young levels by estradiol. In the postsynaptic density and dendritic spines, estradiol reduced pY1472 in young and aged rats. As phosphorylation at Y1472 blocks NR2B endocytosis, reduction of pY1472 by estradiol suggests another mechanism through which estrogen enhances synaptic plasticity by altering localization of NMDAR subunits within synapses.

Ovarian steroids modulate leu-enkephalin levels and target leu-enkephalinergic profiles in the female hippocampal mossy fiber pathway.

In the hippocampal formation (HF), the enkephalin opioids and estrogen are each known to modulate learning and cognitive performance relevant to drug abuse. Within the HF, leu-enkephalin (LENK) is most prominent in the mossy fiber (MF) pathway formed by the axons of dentate gyrus (DG) granule cells. To examine the influence of ovarian steroids on MF pathway LENK levels, we used quantitative light microscopic immunocytochemistry to evaluate LENK levels in normal cycling rats and in estrogen-treated ovariectomized rats. Rats in estrus had increased levels of LENK-immunoreactivity (ir) in the DG hilus compared to rats in diestrus or proestrus. Rats in estrus and proestrus had higher levels of LENK-ir in CA3a-c compared to rats in diestrus. Ovariectomized (OVX) rats 24 h (but not 6 or 72 h) after estradiol benzoate (EB; 10 microg) administration had increased LENK-ir in the DG hilus and CA3c. Electron microscopy showed a larger proportion of LENK-labeled small terminals and axons in the DG hilus compared to CA3 which may have contributed to region-specific changes in LENK-ir densities. Next we evaluated the subcellular relationships of estrogen receptor (ER) alpha, ERbeta and progestin receptor (PR) with LENK-labeled MF pathway profiles using dual-labeling electron microscopy. ERbeta-ir colocalized in some LENK-labeled MF terminals and smaller terminals while PR-ir was mostly in CA3 axons, some of which also showed colocalization with LENK. ERalpha-ir was in dendritic spines, but no colocalization with LENK-labeled profiles was observed. The present studies indicate that estrogen can modulate LENK in subregions of the MF pathway in a dose-and time-dependent manner. These effects might be triggered by direct activation of ERbeta or PR in LENK-containing terminals.

Ultrastructural localization of extranuclear progestin receptors relative to C1 neurons in the rostral ventrolateral medulla.

To better understand the role of progestins in the C1 area of the rostral ventrolateral medulla (RVLM), immunocytochemical localization of progestin receptors (PRs) was combined with tyrosine hydroxylase (TH) in single sections of RVLM from proestrus rat brains prepared for light and electron microscopy. By light microscopy, PR-immunoreactivity (-ir) was detected in a few nuclei that were interspersed between TH-labeled perikarya and dendrites. Electron microscopy revealed that PR-ir was in several extranuclear locations. The majority of PR-labeling was in non-TH immunoreactive axons (51+/-9%) near the plasma membrane. Additional dual labeling studies revealed that PR-immunoreactive axons could give rise to terminals containing the GABAergic marker GAD65. PR-ir also was found in non-neuronal processes (29+/-9%), some resembling astrocytes. Occasionally, PR-ir was in non-TH-labeled terminals (10+/-3%) affiliated with clusters of small synaptic vesicles, or in patches contained in the cytoplasm of dendrites (10+/-1%). These findings suggest that progestins can primarily modulate neurons in the C1 area of the RVLM by presynaptic mechanisms involving GABAergic transmission. Moreover, they suggest that PR activation may contribute to progestin's effects on arterial blood pressure during pregnancy as well as to sex differences in central cardiovascular regulation.

Progesterone receptor expression in cajal-retzius cells of the developing rat dentate gyrus: Potential role in hippocampus-dependent memory.

The development of medial temporal lobe circuits is critical for subsequent learning and memory functions later in life. The present study reports the expression of progesterone receptor (PR), a powerful transcription factor of the nuclear steroid receptor superfamily, in Cajal-Retzius cells of the molecular layer of the dentate gyrus of rats. PR was transiently expressed from the day of birth through postnatal day 21, but was absent thereafter. Although PR immunoreactive (PR-ir) cells did not clearly express typical markers of mature neurons, they possessed an ultrastructural morphology consistent with neurons. PRir cells did not express markers for GABAergic neurons, neuronal precursor cells, nor radial glia. However, virtually all PR cells co-expressed the calcium binding protein, calretinin, and the glycoprotein, reelin, both reliable markers for Cajal-Retzius neurons, a transient population of developmentally critical pioneer neurons that guide synaptogenesis of perforant path afferents and histogenesis of the dentate gyrus. Indeed, inhibition of PR activity during the first two weeks of life impaired adult performance on both the novel object recognition and object placement memory tasks, two behavioral tasks hypothesized to describe facets of episodic-like memory in rodents. These findings suggest that PR plays an unexplored and important role in the development of hippocampal circuitry and adult memory function.