Elsevier

Brain Research

Volume 1379, 16 March 2011, Pages 86-97
Brain Research

Research Report
Estrogen and aging affect the synaptic distribution of estrogen receptor beta-immunoreactivity in the CA1 region of female rat hippocampus

https://doi.org/10.1016/j.brainres.2010.09.069Get rights and content

Abstract

Estradiol (E) mediates increased synaptogenesis in the hippocampal CA1 stratum radiatum (sr) and enhances memory in young and some aged female rats, depending on dose and age. Young female rats express more estrogen receptor α (ERα) immunolabeling in CA1sr spine synapse complexes than aged rats and ERα regulation is E sensitive in young but not aged rats. The current study examined whether estrogen receptor β (ERβ) expression in spine synapse complexes may be altered by age or E treatment. Young (3–4 months) and aged (22–23 months) female rats were ovariectomized 7 days prior to implantation of silastic capsules containing either vehicle (cholesterol) or E (10% in cholesterol) for 2 days. ERβ immunoreactivity (ir) in CA1sr was quantitatively analyzed using post-embedding electron microscopy. ERβ-ir was more prominent post-synaptically than pre-synaptically and both age and E treatment affected its synaptic distribution. While age decreased the spine synaptic complex localization of ERβ-ir (i.e., within 60 nm of the pre- and post-synaptic membranes), E treatment increased synaptic ERβ in both young and aged rats. In addition, the E treatment, but not age, increased dendritic shaft labeling. This data demonstrates that like ERα the levels of ERβ-ir decrease in CA1 axospinous synapses with age, however, unlike ERα the levels of ERβ-ir increase in these synapses in both young and aged rats in response to E. This suggests that synaptic ERβ may be a more responsive target to E, particularly in aged females.

Introduction

In young rats, elevated levels of estrogens (either in the proestrus phase of the estrous cycle and with experimental replacement) increase axospinous synapse density on CA1 pyramidal cells (Gould et al., 1990, Woolley et al., 1990, Woolley & McEwen, 1992, Woolley & McEwen, 1993). Parallel with changes in spine/synapse densities, estrogen treatment also has been shown to change levels of synaptic proteins in CA1, CA3 and the dentate gyrus (Brake et al., 2001, Waters et al., 2009) and activate second messenger pathways (Kelly and Ronnekleiv, 2009). In contrast, estrogen fails to increase CA1 synapse density in aged female rats (Adams et al., 2001c), which may be due in part to decreased synaptic ERα-immunoreactivity (ir) in CA1 synaptic profiles in aged rats as well as less down-regulation after estradiol (E) treatment (Adams et al., 2002). Interestingly, these axospinous synapses do display estrogen-induced alterations in synaptic glutamatergic N-methyl-d-aspartate (NMDA) receptors (Adams et al., 2001a,b).

There are two known forms of classical estrogen receptors (ERs), ERα and ERβ, and they have a high and nearly equal affinity for estrogens (Levin, 2001). In the CNS, genomic actions of estrogen are mediated through nuclear receptors (McEwen and Alves, 1999). Moreover, rapid, non-genomic actions of estrogen that may be mediated through extranuclear ERα and/or ERβ (Levin, 2001, McEwen et al., 2001). Electron microscopic studies have revealed that, like ERα (Milner et al., 2001, Towart et al., 2003), ERβ-ir is located at extranuclear sites: dendritic spines, axons, terminals, and glia (Milner et al., 2005). These findings strongly suggest that ERα and ERβ can mediate estrogen's classical transcriptional/genomic actions as well as more rapid signaling in the rat hippocampal formation.

Although ERβ-ir has been analyzed in young and aged animals (Mehra et al., 2005, Milner et al., 2005), estrogen regulation of the synaptic distribution has not been studied. While the regulation of spine formation in CA1 pyramidal neurons by E may involve contributions from both ERβ and ERα, the role of each receptor plays during the decline in estrogen sensitivity with age is unclear. With increasing age, altered ER distribution would have important implications for estrogen signaling and hippocampal dependent function. Indeed, age-related alterations in ERβ distribution may lead to a decreased impact of E on aged synapses. To address this hypothesis, the subcellular distribution of ERβ-ir in the stratum radiatum (sr) of the CA1 region of the dorsal hippocampus was analyzed by quantitative electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle (Veh) or E.

Section snippets

Results

Post-embedding immunocytochemistry was performed to quantitatively examine whether the levels or subcellular distribution of ERβ-ir in the CA1sr of the dorsal hippocampus are affected by age and/or E. Young and aged female rats were ovariectomized (OVX) for 7 days prior implanting silastic capsules containing either vehicle (Veh; cholesterol) or E (10% in cholesterol). Two days after the implant, the animals' brains were perfusion-fixed with paraformaldehyde and glutaraldehyde and the dorsal

Discussion

The present study demonstrates that like ERα the levels of ERβ-ir decrease in CA1 axospinous synapses with age, however, unlike ERα the levels of ERβ-ir increase in these synapses in both young and aged rats in response to E (Fig. 7). This suggests that synaptic ERβ may be a more responsive target to E, particularly in aged females.

Animals

Female Sprague–Dawley rats, including 11 young (3–4 month; 225 g) and 11 aged (23–24 month; 350 g), were obtained from Harlan (Indianapolis, IN) for use in post-embedding electron microscopic studies. These animals have been used in previous studies examining the interaction of estrogen replacement and aging on markers of synaptic plasticity (Adams et al., 2001a, Adams et al., 2001b, Adams et al., 2002). Animals were housed in a temperature-controlled room (12-h light/dark cycle; lights on at 0700)

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    Supported by: NIH Grants: NIA PO1-AG16765 (JHM), NS07080 (BSM and TAM) and DA08259 (TAM).

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    Current address: Department of Pharmacology, Hacettepe University, Faculty of Medicine, Ankara, Turkey 06100.

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