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Published in final edited form as: Neurobiol Aging. 2018 Sep 25;73:200–210. doi: 10.1016/j.neurobiolaging.2018.09.025

EFFECTS OF ESTROGEN AND AGING ON SYNAPTIC MORPHOLOGY AND DISTRIBUTION OF PHOSPHORYLATED Tyr1472 NR2B IN THE FEMALE RAT HIPPOCAMPUS

Elizabeth M Waters 1, Sanoara Mazid 1, Mariana Dodos 2, Rishi Puri 3, William G Janssen 3, John H Morrison 4, Bruce S McEwen 1,*, Teresa A Milner 1,2,*
PMCID: PMC11548941  NIHMSID: NIHMS2031515  PMID: 30384123

Abstract

Age and estrogens may impact the mobility of NMDA receptors 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 NMDA receptors, is altered in the dorsal hippocampus of young (3–4 month old) and aged (~24 month old) ovariectomized rats treated with 17β-estradiol or vehicle for 2 days. The number of gold particles labeling pY1472 was higher in pre- and post-synaptic 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, 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.

Keywords: Estrogen deprivation, hormone replacement, presynaptic glutamate receptors, CA1 region, aging

1. INTRODUCTION

The ability of estrogen levels to regulate hippocampal function and structural characteristics has been studied for more than three decades in female rodents. In both rodents and non-human primates, hippocampal synapse density increases on CA1 pyramidal cell basal dendrites when estrogen levels are highest (Woolley and McEwen, 1992; Hao et al., 2003). However, this estrogen effect is limited in rodents to young adult females as estrogen fails to increase CA1 synapse density in aged female rats (Adams et al., 2001c), although spinophilin labeling, a key marker of spines, is maintained throughout adult life in non-human primates (Hao et al., 2003). Changes in estrogen’s ability to maintain synapses and promote synapse formation may be due to changes in estrogen receptor (ER) expression as synaptic ERα decreases but ERβ persists in CA1 synaptic profiles of aged rats (Adams et al., 2002; Waters et al., 2011; Foster, 2012). Since ERs have been shown to regulate synaptic protein levels in CA1, CA3, and the hilus of the dentate gyrus (Brake et al., 2001; Waters et al., 2009) and second messenger pathways are implicated in synapse formation and maintenance (Akama and McEwen, 2003; Znamensky et al., 2003; Foster, 2012; Kramar et al., 2013; Bean et al., 2014; Hasegawa et al., 2015; Tuscher et al., 2016), the loss of ERs and the concomitant reduction in estrogen sensitivity of the brain has far ranging implications for synaptic health during aging.

Estrogen-mediated synapse formation coincides with improved learning and memory in adult females and disruption of this effect through inhibition via estrogen activation of signaling pathways or interactions with N-methyl-D-Aspartate receptors (NMDARs) prevents synaptic plasticity and enhanced cognitive performance (Woolley and McEwen, 1994; Smith and McMahon, 2006; Lewis et al., 2008; Vedder et al., 2013; Bean et al., 2014). In adults, NMDARs are comprised of two obligatory NR1 subunits and two regulatory subunits, mainly NR2A and NR2B (Cull-Candy and Leszkiewicz, 2004; MacDonald et al., 2006). NMDAR subunit expression and localization within synapses is altered by estrogen in young and aged female rats (Adams et al., 2001a; Adams et al., 2004). Expression levels of NMDAR subunits along with post-translational modifications ultimately regulate the ability of NMDAR to detect glutamate and affect synaptic responsiveness (Hunt and Castillo, 2012). NMDA receptor subunits contain many serine/threonine as well as tyrosine phosphorylation sites that are substrates for several kinases including cAMP-dependent protein kinase A and CAMKII; interactions between NMDA receptors and these kinases underlie many forms of synaptic plasticity (Chen and Roche, 2007).

Within the post-synaptic density, the NR2B subunit of the NMDA receptor is the predominant tyrosine-phosphorylated protein (Moon et al., 1994). Try1472 (Y1472) is the major phosphorylation site within NR2B (Nakazawa et al., 2001; Takasu et al., 2002). Phosphorylation of NR2B at Y1472 is low at baseline but acutely and rapidly increases after long-term potentiation (LTP); this is important for surface expression of NMDARs, and contributes to NMDAR activation (Nakazawa et al., 2001; Nong et al., 2004; Prybylowski et al., 2005; Lu et al., 2015), while inhibition of tyrosine phosphorylation reduces LTP facilitation (Chen et al., 2014). In the hippocampus, altered tyrosine phosphorylation of NMDA receptors would presumably be involved in the cognitive decline associated with normal aging and neurodegenerative diseases such as Alzheimer’s (Guidi et al., 2015; Foster et al., 2017).

The current experiment hypothesized that 17β-Estradiol (E2) treatment and age would interact in the regulation of synaptic morphology and composition, including mitochondria and phosphorylated Y1742 NR2B (pNR2B) levels and distribution. Together with our previous findings that age altered ER expression and regulation within synapses (Adams et al., 2002; Waters et al., 2011) and estrogen’s ability to modulate NR2B mobility within the synapse regardless of age (Adams et al., 2004), the current question had important implications for estrogen replacement in the context of age when estrogen levels and hippocampal dependent functions decline. The hippocampus of young and aged female rats that were ovariectomized (OVXed) and treated with vehicle or E2 was analyzed by quantitative three-dimensional electron microscopy to identify the characteristics of synapses in the CA1 stratum radiatum and the distribution of phosphorylated Try1472 NR2B-immunoreactivity within the synapses.

2. MATERIALS AND METHODS

2.1. Animals

All experiments were approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Young (3–4 month; 225 grams; N = 11) and aged (23–24 month; 350 grams; N = 11) adult female Sprague-Dawley rats from Harlan (Indianapolis, IN) were used in these studies. The young females were virgins and the aged females were either virgins or retired breeders (Adams et al., 2002). Animals were housed in a temperature-controlled room (12-h light/dark cycle; lights on at 0700). Food and water were available ad libitum. The tissue samples used in the electron microscopic experiments were from the same rats as those used in previous studies (Adams et al., 2001c; Adams et al., 2002; Adams et al., 2004; Yildirim et al., 2008; Waters et al., 2011).

2.2. Experimental Groups

Prior to bilateral ovariectomy, young rats exhibited regular estrous cycles and aged rats were acyclic (i.e., constant estrous or diestrus) (Adams et al., 2001a), thus the time from the last estrogen exposure to the onset of estradiol treatment after OVX varied for the young and aged females. Estradiol replacement began seven days after OVX, when a silastic capsule (outer diameter 3.18 mm; inner diameter 1.96 mm) filled with either E2 (10% in cholesterol) or cholesterol (Veh) was implanted subcutaneously. To account for differences in body weight, implants in young rats were 1 cm in length and implants in aged rats were 2 cm in length (Lauber et al., 1990; Funabashi et al., 1998). In previous studies using this E2 replacement paradigm in long-term OVXed aged rats, a similar uterine response was observed and the circulating estradiol levels were within the physiological range (Adams et al., 2001b).

2.3. Tissue preparation and embedding

Two days after the capsules were implanted, rats were anesthetized with 30% chloral hydrate (i.p.) and perfused transcardially with 2% dextran in 0.1 M phosphate buffer (PB) (pH 7.4, 50 ml/min) for 1 min, followed by 4% paraformaldehyde and 0.125% glutaraldehyde in PB for 10–15 min (Adams et al., 2001c). The brains were removed and postfixed overnight in the last fixative. One block from the dorsal hippocampus (1 mm thick) was randomly selected from 4 rats per group to be processed for embedding.

Freeze substitution and low-temperature embedding of the specimens were performed as described previously (Van Lookeren Campagne et al., 1991; Adams et al., 2002; Janssen et al., 2005). Initial cryoprotection consisted of immersing sample hippocampal blocks in increasing concentrations (10, 20, and 30%) of glycerol in PB and then rapidly plunging in liquid propane cooled by liquid nitrogen (−190°C) in a Universal Cryofixation System KF80 (Reichert-Jung, Vienna). For en bloc fixation, samples were immersed in 1.5% uranyl acetate in anhydrous methanol (−90°C, 24 h) in a cryosubstitution Automatic Freeze-Substitution System unit (Leica, Vienna) while the temperature was increased in steps of 4°C/h from −90 to −45°C. After a wash with anhydrous methanol, samples were infiltrated with Lowicryl HM20 resin [Electron Microscopy Sciences (EMS)] at −45°C with a progressive increase in the ratio of resin to methanol for 1 h each, followed by pure Lowicryl overnight. Polymerization was performed with UV light (360 nm) at −45°C for 48 h, followed by 24 h at room temperature.

A rectangular area of the dorsal hippocampal CA1 (~400–500 μm long × 200–250 μm wide) that contained the pyramidal cell layer and stratum radiatum was trimmed with a Mesa knife (EMS) on a UTC Leica Ultratome. Five-six consecutive ultrathin sections then were cut at 80 nm thick using a Diatome diamond knife (EMS) and mounted on formvar/carbon-coated 2×1 mm oval slot nickel grids (EMS).

2.4. Phosphorylated Tyr1472 NR2B antibody

A polyclonal rabbit antibody to the phosphopeptide corresponding to the amino acid residues surrounding the phopho-Tyr1472 of NMDA NR2B was used in these studies (pNR2B; Phosphosolutions p1516–1472). In Western blots of rat hippocampal lysates, this antibody immunolabels the ~180k NR2B subunit of the NMDAR phosphorylated at tyr1472 (Hicklin et al., 2011; Gladding et al., 2012). Immunolabeling for the antibody is blocked by treatment of hippocampal lysates with lamda phosphatase and by preadsorption with the phosphopeptide used as the antigen but not the corresponding dephosphopeptide (manufacturer’s data sheet).

2.5. Post-embedding immunogold electron microscopic labeling

For immunolabeling, the grids with ultrathin sections were treated with a saturated solution of NaOH in absolute ethanol, rinsed, and incubated at room temperature in 0.1% sodium borohydride and 50 mM glycine, followed by Tris-buffered saline (TBS) containing 2% human serum albumin (HSA). The grids then were incubated overnight with the pNR2B antibody (1:50 dilution) in the TBS/HSA solution. The next day, the grids were washed, and incubated in secondary 10 nm gold-tagged goat-anti-rabbit IgG F(ab′)2 antibody (EMS) diluted 1:75 in TBS with 2% HSA and 5 mg/ml polyethyleneglycol 20,000 Da. The dilutions of the primary and secondary antibodies first were determined empirically. Sections were washed and dried, then counterstained with 1% uranyl acetate and Reynolds lead citrate.

2.6. Electron microscopic analysis

2.6.1. Data collection.

A person blind to the group identity performed all data collection and analyses. The primary goal of the present study was to provide a detailed characterization of the patterns of pNR2B immunolabeling within synaptic complexes for each experimental group. For this, protein distribution for pNR2B in synaptic profiles of different sizes, rather than number of gold particles or synaptic profiles per animal, is analyzed. Similar to previous studies from our group (Hara et al., 2014; Crimins et al., 2017), the sections were collected using a systematic-random approach on a HF2000 transmission electron microscope (Hitachi High Technologies America, Inc.). To insure that images were taken from stratum radiatum, photographs were captured in the middle of a region 100–200 μm below the bottom of the pyramidal cell layer. Images were captured at a direct magnification of 17,000 with a digital camera system (v3.2 Advanced Microscopy Techniques, Danvers, MA) using Photoshop to assist with aligning the serial sections.

Four blocks from each experimental group (N = 4/group) were photographed and analyzed: 1) Young OVX + vehicle (Veh); 2) Young OVX + E; 3) Aged OVX + Veh; and 4) Aged OVX + E. For each block, 5 serial images from 10 random fields (total 50 μm2/field) were collected in stratum radiatum located 50 – 150 μm below the pyramidal cell layer. The middle section was considered section 0 and then sections above this section were labeled +1 and +2 whereas section below this section were labeled −1 and −2. Prior to analysis, synaptic complexes that consisted of a terminal, synaptic specialization and dendritic spine were identified and numbered in section 0. Synapses that lacked clear visualization and delineation of classic synaptic structures such as presynaptic and postsynaptic membranes, a synaptic cleft, and postsynaptic density (PSD) were excluded from the quantitative analysis. Using this approach, approximately 100 synaptic complexes were collected for each block.

2.6.2. PSD measurement.

The size and area of PSD formed between pNR2B-containing terminals and/or pNR2B-containing dendritic spines were measured using the ruler tool in Adobe Photoshop Cs5 (version 12.0 ×64, Adobe systems incorporated). For this, PSD length was taken on the one section with the longest length and the area was determined by combining the PSD length from all sections with the spine multiplied by section thickness.

2.6.3. Mitochondrial distribution and volume:

The number and volume of the mitochondria in profiles labeled for pNR2B were measured using the ruler tool in Adobe Photoshop. The volume was calculated using the measured diameter of the mitochondria in each section it appeared and the section thickness.

2.6.4. Analysis of synaptic distribution of pNR2B gold particles:

Six zones were defined for each synaptic complex: (1) the presynaptic terminal zone across from the PSD extending 0–60 nm from the inner border of the presynaptic membrane; (2) the presynaptic terminal zone > 60 nm from the inner border of the presynaptic membrane; (3) the synaptic cleft; (4) on the PSD; (5) near PSD from 30–60 nm of the postsynaptic membrane; (6) the dendritic spine cytoplasm which included regions > 60 nm from the PSD. A dendritic spine or presynaptic terminal was considered labeled if one or more gold particles were present anywhere within it or in the synaptic cleft. Immunogold particles were summed in all planes of a section that contained a given synaptic complex (Fig. 1).

Fig. 1. Schematic illustrating the serial section analysis used to determine pNR2B gold particle location within synaptic complexes.

Fig. 1.

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Synaptic complexes were analyzed in five serial sections: middle section = section 0; sections above middle section = +1 and +2; sections below middle section = −1 and −2. Six zones (center) were analyzed: (1) the presynaptic terminal zone across from the PSD extending 0–60 nm from the inner border of the presynaptic membrane; (2) the presynaptic terminal zone > 60 nm from the inner border of the presynaptic membrane; (3) the synaptic cleft; (4) on the PSD in the postsynaptic dendritic spine; (5) near PSD from 30–60 nm of the postsynaptic membrane; (6) the cytoplasm of the dendritic spine which included regions > 60 nm from the PSD.

2.7. Statistical analysis

Statistical analyses were performed using JMP 12 (SAS Institute Inc., Cary NC). The coefficients of variation (CV = standard deviation/mean) and coefficient error (CE) were calculated. Potential group differences in percentage of labeled synapses and number of gold particles per synaptic compartment between young OVX vehicle- and estrogen-treated rats, as well as aged OVX vehicle- and estrogen-treated rats, were evaluated by two-way analysis of variance (ANOVA). The grouped data also were compared by two-way ANOVA, and multiple comparisons were made by Tukey-Kramer adjustments. Significance was set at p < 0.05. All values are given as means ± SEM.

All images were captured and further processed by using the Adobe Photoshop Cs5 program. Only minor adjustments of brightness, contrast and sharpening were made, which in no case altered the appearance of the original material.

3. RESULTS

3.1. E2 regulated spine morphology in young but not aged females.

In the hippocampus, several types of dendritic spines, including thin, stubby, mushroom and branched (Bourne and Harris, 2008; Lee et al., 2012), are found opposed by axon terminals of similar size. For each CA1 stratum radiatum pNR2B labeled synapse, the spine and presynaptic terminal diameter were analyzed from the section (−2, −1.0, +1, +2) with the largest diameter compared to all sections where the same synaptic complex was identifiable.

For diameter of pNR2B-containing axon terminals, there were main effects of E2 treatment [F(1,1521) = 9.77, p = 0.0018] as well as age x E2 treatment [F(1,1521) = 4.44, p = 0.0353]. Post-hoc tests showed that E2 treatment significantly increased pNR2B-containing terminal diameter in young females (p = 0.0015) and in aged females (p = 0.0154) compared to the Veh treated Young females (Fig. 2A).

Fig. 2. Morphological changes in pNR2B labeled synaptic complexes related to age and E2 treatment.

Fig. 2.

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A. The diameter of pNR2B-containing terminals was increased in Young + E2 females and in both aged female groups compared with Young + Veh. There was no difference in pNR2B-containing diameter between aged females with E2 or Veh. B. Compared to Young + Veh females, the diameter of pNR2B-containing dendritic spines increased in aged females regardless of treatment. C, D. The length and area of PSDs formed between pNR2B-containing pre- and/or post-synaptic profiles were not altered by age or treatment. N = 4 rats/group; n ~ 100 synaptic profiles/rat; *P < 0.05

The diameter of pNR2B-containing dendritic spines differed between treatment groups; there were significant main effects of age [F(1,1455) = 12.20, p = 0.0005] and E2 treatment [F(1,1455) = 5.03, p = 0.0251; Fig. 2B]. Post-hoc analyses indicated that, compared to Young + Veh, mean spine diameter increased in Aged + Veh (p = 0.0496) and Aged + E2 (p = 0.0003) groups (Fig. 2B).

Neither the PSD length (Fig. 2C) nor the PSD area (Fig. 2D) formed between pNR2B-containing terminals and/or pNR2B-containing dendritic spines was significantly different between any of the experimental groups.

3.2. E2 restored mitochondria in aged female axon terminals.

In all four experimental groups, mitochondria were found presynaptically (Fig. 3A) and postsynaptically (Fig. 3B) in pNR2B-containing synaptic complexes. Examination of mitochondria number in presynaptic pNR2B-containing profiles showed significant main effects of age [F(1,1203) = 6.80, p = 0.0092] and E2 treatment [F(1,1203) = 5.19, p = 0.0229]. Moreover, the interaction of age and treatment factors with regard to mitochondria number also was significant [F(1,1203) = 5.25, p = 0.0221]. In postsynaptic pNR2B-containing profiles, there were no significant main effects or interactions in the number of mitochondria.

Fig. 3. Mitochondrial counts, but not morphology, in pNR2B-labeled presynaptic profiles differed with aged and estradiol treatment.

Fig. 3.

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A, B. Representative electron micrographs show mitochondria in presynaptic (A) and postsynaptic (B) profiles. m, mitochondria, sp, dendritic spine, T, axon terminal. Scale bar, 500 nm. C. The number of mitochondria in presynaptic NR2B-labeled profiles is significantly decreased in Aged + Veh females compared to both groups of young females but significantly increased after E2. There was no significant difference in the number of mitochondria in postsynaptic profiles in all 4 groups. D. Mitochondrial volume was not altered by age or treatment in pre- or post-synaptic compartments of pNR2B labeled profiles. N = 4 rats/group; n ~100 synaptic profiles/rat; *P < 0.05

In the pNR2B labeled terminals, post-hoc analysis showed that Aged + Veh females had significantly fewer mitochondria than Aged + E2 (p = 0.0081) and both Young + Veh and Young + E2 treated groups (p = 0.0026, p = 0.0029, respectively; Fig 3C left). In contrast, the number of mitochondria was not significantly different in Young + Veh females compared to Young + E2 females. Although the number of mitochondria decreased in Aged + Veh females compared to the other three groups, total mitochondrial volume did not change overall in pNR2B containing presynaptic profiles (Fig 3D). Moreover, postsynaptic mitochondria pNR2B profiles were not changed after E2 in young or aged females (Fig, 3C right).

Mitochondria change their shapes through the combined actions of fission, fusion and motility (Youle and van der Bliek, 2012). Fission generates new organelles, which are usually small and round; fusion allows functional mitochondria to combine with dysfunctional mitochondria and share components, generally resulting in larger mitochondria (Youle and van der Bliek, 2012). To address the possibility that mitochondrial fission or fusion was affected by aged and/or treatment, the ratio of the form factor to total mitochondria volume in pNR2B-containing profiles was examined in each group. No changes in mitochondrial forms were detected in pre- or post-synaptic profiles of any group (data not shown).

3.3. Overall, age increased and E2 decreased pNR2B gold particle labeling.

In the CA1 stratum radiatum, pNR2B gold labeling was found in synaptic complexes consisting of presynaptic axon terminals (Fig. 4A, top) and postsynaptic dendritic spines (Fig. 4A, center). These labeled terminals and spines opposed unlabeled profiles as well as pNR2B labeled profiles (Fig. 4A, bottom). Total pNR2B gold particle number in terminals and spines showed significant main effects of age [F(1,1632) = 7.12, p = 0.0077] and E2 treatment [(F1,1632) = 11.36, p = 0.0008], but there were no significant interactions between age and E2. Post-hoc analysis showed Aged + Veh females had increased pNR2B gold particles compared to Young + E2 females (p = 0.0001), however, there was no significant increase compared to Aged + E2 females (p = 0.0678; Fig. 4B).

Fig. 4. Age and E2 treatment affected the number of pNR2B gold particles in pre- and post-synaptic profiles.

Fig. 4.

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A. Electron micrographs show examples of synaptic complexes with pNR2B-gold particles in only presynaptic profiles (top), only postsynaptic profiles (middle) or both pre- and post-synaptic profiles (bottom). sp = dendritic spine, T = axon terminal. Scale bar = 500 nm. B. Aged + Veh females had significantly more total pNR2B gold particles in synaptic complexes than Young + E2 females. C. In synaptic complexes with only pre-synaptic pNR2B-labeled profiles, Aged + Veh females had significantly more pNR2B gold particles compared to Young + E2 females and Aged + E2 females. In synaptic complexes with only post-synaptic pNR2B-labeled profiles, there were no significant differences in the number of gold particles between any of the groups. In synaptic complexes with pNR2B labeling in both pre- and post-synaptic profiles, Aged + Veh females had significantly more gold particles than both young female groups. D. When synaptic complexes with pNR2B labeling in both pre- and post-synaptic profiles was further subdivided, Aged + Veh females had significantly more pNR2B gold particles in terminal profiles compared to Young + Veh and Young + E2 females. N = 4 rats/group; n ~ 100 synaptic profiles/rat; *P < 0.05

In the synaptic complexes in which pNR2B gold particles were only in terminals, there was a significant main effect of E2 treatment [F(1,303) = 5.62, p = 0.0184] due to decreased labeling in young and aged E2 treated rats. The post-hoc analysis showed that Young + E2 and Aged + E2 had significantly less pNR2B gold particles in synaptic complexes than Aged + Veh (p = 0.0265 and p = 0.0339, respectively; Fig. 4C left). Synaptic complexes containing pNR2B labeling only in the postsynaptic spine profiles had no main effects or interactions of age or E2 treatment (Fig 4C center). In synaptic complexes in which both pre- and post-synaptic profiles were labeled, there were significant main effects of age [F(1,480) = 6.99, p = 0.0084] and E2 treatment [F(1,480) = 5.26, p = 0.0222], but no interaction between age and treatment. Post-hoc analyses showed that Aged + Veh females had significantly more synaptic complexes with pNR2B gold particles in both pre- and post-synaptic profiles compared to the Young + Veh females (p = 0.0382) and Young + E2 females (p = 0.0042; Fig. 4C right).

Within synaptic complexes where both the terminal and spine were labeled, there was a main effect of age in the presynaptic compartment [F(1,480) = 6.00, p = 0.0146]. Post-hoc analysis showed that the number of pNR2B gold particles in the presynaptic compartment increased in the Aged + Veh group compared to both Young + Veh and Young + E2 (p = 0.0112, 0.0432) but there was no difference compared to Aged + E2 (p = 0.1090). Additionally, there were no differences between Aged + E2 and both groups. The postsynaptic compartment showed a main effect of E2 treatment associated with pNR2B labeling [F(1,480) = 4.19, p = 0.0412]. Post-hoc analysis showed no differences in pNR2B gold particles between any groups (Fig. 4D). However, the number of pNR2B gold particles in the postsynaptic compartment of the Aged + Veh group tended (p = 0.0643) to be less than the Young + E2 group.

3.4. E2 treatment decreased pNR2B gold particles in a compartment specific manner.

Synaptic and extrasynaptic pNR2B labeling was compared by assigning gold particles to bins determined by their distance from the postsynaptic density or synaptic cleft (Fig. 1; see detailed description in methods). Presynaptically, there was a significant main effect of age on the number of pNR2B gold particles both in the near terminal bin [F(1,1632) = 4.17, p = 0.0413] and in the synaptic cleft [F(1,1632) = 11.84, p = 0.0006]. Moreover there was a trend [F(1,1632) = 3.57, p = 0.0589] for a main effect of age on the number of pNR2B gold particles in the far terminal bin. Post-hoc analyses indicated that Aged + Veh had significantly more pNR2B gold particles in the near terminal bin compared to Young + Veh (p = 0.0273), Young + E2 (p = 0.0333), and Aged + E2 (p = 0.0479). In the synaptic cleft, post-hoc analysis showed that Aged + E2 had significantly more pNR2B gold particles compared to Young + E2 (p = 0.0028; Fig. 5A, right) and Young + Veh (p = 0.0453). Post-hoc analyses showed a trend (p = 0.0512) for increased pNR2B gold particles in Aged + Veh compared to the Young + E2 group. In young females, E2 had no significant effect on pNR2B gold particles in the presynaptic terminal or synaptic cleft (Fig. 5A).

Fig. 5. The distribution of pNR2B gold particles within pre- and post-synaptic compartments varied by age and E2 treatment.

Fig. 5.

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A. Presynaptic compartments. On the portion of the terminal near the synapse (bin 1), the number of pNR2B gold particles was significantly greater in Aged + Veh females compared to other groups. In the portion of the terminal distal to the synapse (bin 2) the number of pNR2B gold particles tended to be greater in Aged + Veh females compared to Young + E2 females. In the synaptic cleft (bin 3) the number of pNR2B gold particles in Aged + E2 females was significantly greater than both of the young female groups. B. Postsynaptic compartments. On the PSD of dendritic spines (bin 4) the number of pNR2B gold particles in Young and Aged + Veh females was significantly more than Young + E2 females. Moreover, the number pNR2B gold particles in Young + Veh females was significantly more than Aged + E2 females on the PSD. In the region of the dendritic spine near the PSD (bin 5), Young + Veh females had significantly less pNR2B gold particles than Aged + E2 females. In the region of the dendritic spine distal to the PSD regions (bin 6), Young + Veh females tended to have more pNR2B gold particles than Aged + E2 females. N = 4 rats/group; n ~ 100 synaptic profiles/rat; *P < 0.05; ^P = 0.05–0.06

Postsynaptically, the number of pNR2B gold particles in bins showed significant main effects of E2 treatment in the PSD [F(1,1632) = 17.54, p < 0.0001] and spine [F(1,1632) = 6.56, p = 0.0105]. A main effect of age was present in the near PSD bin [F(1,1632) = 10.12, p = 0.0015]. No other main effects or interactions were detected. Post-hoc analysis indicated that the numbers of pNR2B gold particles on the PSD compartment were elevated in Young + Veh females compared to Young + E2 (p = 0.0011) and Aged + E2 treated females (p = 0.0266). There was a significant difference in pNR2B gold particles on the PSD compartment of Aged + Veh compared to Young + E2 (p = 0.0098) but not to Aged + E2 (p = 0.1321). In the near PSD, Aged + E2 females had significantly more pNR2B gold particles (p = 0.0385) than Young + Veh females. In the cytoplasm of dendritic spines, the number of pNR2B gold particles tended (p = 0.0689) to be less in Aged + E2 females compared to Young + Veh females (Fig. 5C). In these compartments, there were no significant differences between Aged + E2 females.

3.5. Three-dimensional reconstruction of synaptic complexes depicts changes in terminal size and pNR2B gold particle distribution related to E2 status and age.

The relation of pNR2B gold particles within one representative synaptic complex from each group is shown by three-dimensional reconstruction (Fig 6). In all four groups, mitochondria are found primarily in presynaptic terminals. The terminal diameter of the Young + Veh group was less than that in the Young and Aged + E2 group, whereas the PSD length was similar in all four groups. Overall, pNR2B gold particles were found in all portions of both presynaptic and postsynaptic compartments in all experimental groups. No abnormalities in overall synapse morphology or mitochondrial distribution were noted. The total amount of pNR2B gold particles in synaptic complexes was greater in the Aged + Veh group compared to all other groups. In the PSD, E2 treatment in the young animals decreased the number of pNR2B gold particles. In the presynaptic terminal, elevated levels of pNR2B particles were found in the Aged + Veh group compared to the other three groups. In the postsynaptic compartment, more pNR2B gold particles were observed in the Young + Veh and Aged + Veh groups compared to their E2 counterparts.

Fig. 6. Three-dimensional reconstruction of representative synaptic complexes from the four groups shows the morphology and subcellular distribution of pNR2B gold particles within the pre- and post-synaptic compartment.

Fig. 6.

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A. Representative serial electron micrographs for Young + E2 rats (see figure 1 for section numbering scheme). Scale bar = 500 nm. B. Three-dimensional reconstruction of a representative synaptic complex for each treatment group highlights: 1) prominence of mitochondria in the presynaptic terminal; 2) smaller terminal diameter in the Young + Veh group compared to Young and Aged + E2 groups; 3) decreased pNR2B gold particles in the PSD of Young animals after E2 exposure; and 4) elevated pNR2B in the presynaptic profile in the Aged + Veh groups.

4. DISCUSSION

The aged brain is fundamentally different from the young brain in its capacity for plasticity, particularly in response to estrogen (Miranda et al., 1999; Adams et al., 2001b; Bean et al., 2014; Milner et al., 2014). Moreover, estrogens also affect synaptic communication in brain regions involved in learning and memory, such as the hippocampus, which is of particular importance in the context of human aging when circulating estrogen levels decline and a wide variety of adverse memory related outcomes are reported (Sherwin, 2000; Kritz-Silverstein and Barrett-Connor, 2002; Khoo et al., 2010; McCarrey and Resnick, 2015). Here, quantitative analysis of the distribution of pNR2B Y1472 in stratum radiatum of CA1 in young and aged females using post-embedding immunocytochemistry and serial section reconstruction suggest that aged synapses as well as mitochondria in synaptic complexes can exhibit youthful like responses to E2.

4.1. CA1 synapses in young and aged female hippocampus benefited from E2 effects.

Overall, E2 replacement had beneficial effects in young and aged females. In axon terminals, the diameter after E2 treatment was increased in both young and aged females compared to Young + Veh females. Also, mitochondria numbers that had decreased by 50% in aged females were restored after E2 to the same levels as young females. In contrast, age rather than E2 had the greater influence on spine diameter. Age related increase in spine diameter could reflect the shift in spine morphology from a mixture of all spine types in young female hippocampus to fewer thin spines in aged hippocampus [see review: (Hara et al., 2015)]. Differential effects of age and E2 on pNR2B Y1472 depended on its location within the synapse.

Previous examination of NR2A and NR2B demonstrated that young and aged females have similar synaptic levels of both receptor subtypes; however, there is a specific loss of NR2B near the PSD in aged females that is reversed by E2 (Adams et al., 2004). The current examination of pNR2B Y1472 suggests that in the estrogen deprived-state, aged females faired worse than young females as reflected by increased pNR2B levels. However, E2 treatment in aged females reversed elevated pNR2B Y1472 levels throughout the axon terminal and in the PSD to the same levels found in young females. The area of the synapse near the PSD is highly dynamic but here, pNR2B did not show significant changes with age or E2 treatment.

Duration of estrogen deprivation could have had a bigger effect on synapses in the aged females compared to the young females since the aged females were acyclic prior to the start of the study. E2 effects on synaptic parameters are affected more by the duration of estrogen deprivation than chronological age (Smith et al., 2010; Vedder et al., 2014), and growing evidence suggests that the beneficial effects of E2 replacement decline rapidly as the length of the deprivation grows (McLaughlin et al., 2008; Smith et al., 2010; Vedder et al., 2014; Hara et al., 2015). Additionally, as some of the aged rats were retired breeders, this could have influences on the effects of estrogen at the synapses (Barha et al., 2015).

ERs are found in both pre- and post-synaptic compartments in the hippocampal neurons and are strongly linked to NMDAR regulation and dependent LTP (Romeo et al., 2005; Morissette et al., 2008; Vedder et al., 2013; Hasegawa et al., 2015; Oberlander and Woolley, 2016; Smith et al., 2016). ER dependent actions increase both rapid and NMDAR dependent synaptic transmission in young females along with enhancing memory (Woolley et al., 1997; Smith and McMahon, 2005; Snyder et al., 2011; Vedder et al., 2013; Kumar et al., 2015). Increasing ER expression in aged females restores estrogen effects on memory and NMDAR dependent LTP (Bean et al., 2015).

Age related changes in estrogen’s ability to maintain synapses, promote synapse formation, and regulate synapse function may be due to changes in ER expression as synaptic ERα decreases but ERβ persists in CA1 synaptic profiles of aged rats (Adams et al., 2002; Waters et al., 2011). Moreover, age-related shifts in estrogen synaptic responses may involve shifts in ERα and ERβ ratios as well as other components of the rapid signaling cascade, including Ca++ dysregulation and alterations in kinase/phosphate activity (Foster, 2012). This, along with ER’s ability to connect with direct and indirect modulators of NMDAR potentiation and regulation (Woolley and McEwen, 1994; Collingridge et al., 2013; Hasegawa et al., 2015), suggests that loss of ERs results not only in reductions in estrogen signaling but also alterations in the estrogen signaling pathways that could have far ranging implications for synaptic health during aging.

4.2. Role of NR2B phosphorylation in reported actions of E2

Estrogen’s ability to intersect with the cellular mechanisms that regulate NMDA receptor phosphorylation continues to shed new light on synaptic function and plasticity and animal behavior. Estrogen does not regulate detectable overall levels of NR2B or pNR2B at either the tyrosine (1472 and 1336) or serine (1387, 1303 and 1480) sites (Snyder et al., 2011). However, estrogen enhancement of LTP is NR2B dependent LTP (Smith and McMahon, 2006; Smith et al., 2016) and others have suggested that estrogens effects on excitatory synapses likely results from recruitment of NR2B-containing NMDA receptors to synapses (Snyder et al., 2011). The movement of NR2B containing NMDARs to synapses is increased by Y1472 phosphorylation of NR2B (Zhang et al., 2008); phosphorylation of Y1472 NR2B is enhanced at the initiation of LTP in hippocampal CA1 neurons (Nakazawa et al., 2001). However, multiple phosphorylation sites regulate the subcellular localization of NR2B containing NMDARs through relocation to the surface, stabilization at the surface and lateral diffusion to extrasynaptic sites (Lussier et al., 2015). Thus, changes in Y1472 pNR2B are likely concurrent with phosphorylation of NR2B at other sites, e.g., ser1303 that is regulated by CAMKII phosphorylation (Chen and Roche, 2007), another estrogen sensitive protein important for induction and maintenance of synaptic plasticity (Logan et al., 2011).

As a result of age or estrogen deprivation, increased or prolonged phosphorylation of Y1472 NR2B could reduce the presynaptic pools of NMDARs. Estrogen deprivation increased presynaptic pNR2B; this effect was reversed by E2 treatment in young females, but not in aged female synapses, suggesting that persistent phosphorylation of NR2B has the potential to alter presynaptic function in aged females due to NMDARs ability to modulate both short- and long-term responses to activity (Duguid and Smart, 2009). Age effects on phosphorylation of NR2B at Y1472 result in reduced receptor diffusion to extrasynaptic sites and blocked internalization (Chen et al., 2012; Gladding et al., 2012; Lussier et al., 2015), ultimately increasing presynaptic pNR2B. Elevated pNR2B may contribute to behavior outcomes as aged mice demonstrate increased phosphorylation of NR2B Y1472 in prefrontal cortex and no age related deficits in spatial memory (Zamzow et al., 2016). Prolonged surface expression of NMDAR is likely regulated by a number of mechanisms; thus far, Fyn kinase is implicated (Lu et al., 2015), though interactions of estrogen signaling pathways with Fyn kinase remain unexplored. Likewise, aged related changes in STriatal-Enriched protein tyrosine Phosphatase (STEP) could contribute to the alterations in synaptic Y1472 phosphorylation of NR2B and memory performance (Cases et al., 2018; Castonguay et al., 2018).

4.3. What this tells us about estrogen, aging and hippocampal function: Potential Functional Implications

The influences of estrogen on hippocampal-dependent learning and synaptic plasticity are complex, tapping into many cellular processes as well as exhibiting rapid, short term and long term effects (Sandstrom and Williams, 2004; MacLusky et al., 2005; Kumar et al., 2015; Kim et al., 2016; Tuscher et al., 2016), including NMDAR trafficking (Potier et al., 2016). In the amygdala, phosphorylation of N2B at Tyr-1472 is linked to both chemical and contextual fear learning (Lu et al., 2015). Changes in memory and synapse phenotypes with increased age and estrogen loss (Daniel, 2013; Hara et al., 2015) as well as altered mitochondrial function (Gaignard et al., 2017), together with these findings, further implicate an altered synapse function. With this tissue set, we demonstrated that synaptic protein levels are altered by age and, while aged females may be sensitive to estradiol, the changes may not be consistent with that detected in young, estradiol-treated females (Fig. 7). For pNR2B, aged females did retain most aspects of E2 sensitive responses, although they were not similar to young females across the board. While cellular and molecular composition of the aged brain diverges from the young brain, the likelihood of beneficial effects of estrogen persists, however, with unique outcomes in aged females and the functional consequences of these differences yet to be explored.

Fig. 7. Schematic diagram summarizing synaptic distribution of pNR2B relative to ERα, ERβ and pAKT as demonstrated in the same tissue set.

Fig. 7.

Open in a new tab

In young and aged females, ERβ and pAKT immunoreactivity within CA1 excitatory synapses generally respond to estradiol by changing in the same direction, either increase or decrease, although not always to the same degree so that differences in the overall level do occur. In contrast, in aged females ERα appears to lose estradiol responsiveness such that the decrease in level seen in young females does not occur. Presynaptically, a decrease in ERα may be undetectable in aged females because the levels are already low. Age results in a new pattern of estrogen regulation for the pre- and post-synaptic ion channel NR2B. Phosphorylation of NR2B at Y1427 is impacted by age and sensitive to estradiol replacement. Aged vehicle-treated females had higher levels of pN2B throughout the synaptic terminal and the dendritic spine compared to young females, which were decreased after estradiol replacement. In young females pNR2B levels was only affected by estradiol in the postsynaptic compartment closest to the synaptic cleft. Every synapse included in the analysis for all four endpoints contained immunoreactivity for the protein being examined. Expression levels of each are relative only to the protein being represented, direction of change rather than amount is represented. This representation compares proteins closer to the synaptic cleft (<60 nm) to those farther away (>60 nm).

Highlights:

  1. Phosphorylated Y1472 NR2B (pNR2B) is pre- & post-synaptic in rat hippocampus CA1.

  2. Aged vs. young ovariectomized females had more presynaptic pNR2B & less mitochondria.

  3. Age effects on pNR2B and mitochondria in synapses are reversed by estradiol (E2).

  4. E2 reduced pY1472 could promote synaptic plasticity by altering NR2B localization.

Funding:

This work was supported by NIH grants AG16765 (J.H.M, B.S.M., E.M.W., T.A.M.), AG059850 (E.M.W., T.A.M.), DA08259 and HL096571 (T.A.M.),

Abbreviations:

E2

17β-Estradiol

ER

estrogen receptor

Fyn kinase

Tyrosine-protein kinase Fyn

LTP

Long-term potentiation

m

mitochondria

NMDAR

N-methyl-D-Aspartate receptors

OVX

ovariectomy

pNR2B

phosphorylated Y1472 NMDAR subunit GluN2B

PSD

postsynaptic density

Sp

dendritic spine

STEP

STriatal-Enriched protein tyrosine Phosphatase

T

axon terminal

Veh

vehicle

Footnotes

Conflict of Interest: The authors declare no competing financial interests.

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