Research ReportEstrogen receptor KO mice study on rapid modulation of spines and long-term depression in the hippocampus
Introduction
In addition to slow/genomic (within days) functions of 17β-estradiol, rapid (within 2 h) modulation of synaptic plasticity by estradiol has been attracted much attention in relation to memory performance (Bi et al., 2000, Foy et al., 1999, Ooishi et al., 2012, Vouimba et al., 2000). Estradiol plays an essential role not only in the female but also in the male hippocampus, because the male rat hippocampus endogenously synthesizes several-fold more estradiol than the female hippocampus does (Higo et al., 2009, Hojo et al., 2004, Hojo et al., 2008, Kato et al., 2013, Kawato et al., 2002, Kretz et al., 2004). To clarify rapid synaptic modulation, the molecular mechanisms of estrogen signaling should be identified. Analysis of rapid spinogenesis suggests participation of synaptic estrogen receptors such as estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Estradiol rapidly increased the spine density of male rat hippocampal glutamatergic neurons in CA1 (Mukai et al., 2007, Murakami et al., 2006). ERα agonist, propyl-pyrazole triol (PPT), but not ERβ agonist, diarylpropionitrile (DPN), showed the increase in the spine density. Synaptic localization of ERα was demonstrated in rat hippocampal glutamatergic neurons by immunoelectron microscopic analysis (Mukai et al., 2007). Localization of ERβ at synapses is also observed in rat hippocampal glutamatergic neurons by immunoelectron microscopic analysis (Milner et al., 2005). These synaptic ERα and ERβ are good candidates which mediate rapid modulation by estrogen. Different from rats, however, only a few studies have successfully showed the estradiol effect on spinogenesis in mouse slices, partly due to difficulty in the staining of mouse spines with a suitable dye (Li et al., 2004).
Electrophysiological investigation is another sensitive method to examine rapid effects of estradiol at synapses within 2 h. Estradiol exerts a rapid influence on hippocampal long-term potentiation (LTP) in CA1 (Bi et al., 2000, Foy et al., 1999, Ooishi et al., 2012), long-term depression (LTD) in CA1 (Mukai et al., 2007, Vouimba et al., 2000) or kainate current in CA1 (Gu and Moss, 1996, Gu et al., 1999). ERα agonist but not ERβ agonist induces rapid enhancement of the NMDA-induced LTD, indicating that ERα drives the enhancement of LTD (Mukai et al., 2007).
To directly examine different roles of ERα and ERβ in modulating synaptic plasticity, we here use ERαKO and ERβKO mice. Two representative and different types of synaptic modulation (spinogenesis and LTD) by estradiol are investigated. Selective ERKO mice are key tools to identify the role of each estrogen receptor, because a widely used inhibitor ICI is not selective for ERα or ERβ, and ICI cannot suppress estradiol-induced rapid modulation of LTD or kainate currents (Gu et al., 1999, Mukai et al., 2007). Using male ERKO mice, we successfully demonstrate that both spinogenesis and LTD are enhanced through ERα (not through ERβ). Time-lapse imaging enables us to confirm the estradiol-induced increase in spine density in the live hippocampus by using mice which express yellow fluorescent protein (YFP). This analysis also reveals dynamic nature of spinogenesis.
Section snippets
Rapid effect of estradiol on spinogenesis in hippocampal slices from estrogen receptor KO mice
We analyzed the mechanisms of estradiol for modulation of spine density and morphology. Following a 2 h treatment with 10 nM estradiol, we compared spines with those in control samples (i.e. with no estradiol). To investigate different contributions of ERα and ERβ to spinogenesis, spines of glutamatergic neurons in CA1 of hippocampal slices prepared from ERαKO and ERβKO mice were compared with those in wild type mice. From dose-dependent and time-dependent analysis of spines, a 2 h treatment with
Discussion
The current study clearly demonstrates different roles of ERα and ERβ in rapid modulation of spinogenesis and LTD by estradiol.
Estradiol-induced rapid spinogenesis: mice and rats study
In earlier study, we demonstrated that the application of the ERα selective agonist PPT, but not the ERβ selective agonist DPN, increased spine density in the CA1 region of the rat hippocampus. Furthermore, the ERα/ERβ antagonist ICI-182780 inhibited estradiol-induced spinogenesis (Mukai et al., 2007). These
Animals
ERα knockout mice [ERα (–/–)] were obtained by inbreeding ERα (–/+) mice. The development of ERαKO mice was accomplished by deleting the whole exon 2 of the mouse ERα gene (Dupont et al., 2000). Note that nomenclature of ERα exon changed recently, and the current exon 1 and exon 2 (Kos et al., 2002, Pendaries et al., 2002) correspond to the previous exon 2 and exon 3, respectively. ERβ knockout mice [ERβ (–/–)] were obtained by inbreeding ERβ (–/+) with ERβ (–/–) mice. The development of ERβKO
Acknowledgments
We thank Dr. Shigeaki Kato, Dr. Matsumoto at The University of Tokyo, and Dr. A. Krust at Universite Louis Pasteur for technical assistance of ERα and ERβ knockout mice.
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