Estrogen mobilizes a subset of estrogen receptor-alpha-immunoreactive vesicles in inhibitory presynaptic boutons in hippocampal CA1 - PubMed (original) (raw)

Estrogen mobilizes a subset of estrogen receptor-alpha-immunoreactive vesicles in inhibitory presynaptic boutons in hippocampal CA1

Sharron A Hart et al. J Neurosci. 2007.

Abstract

Although the classical mechanism of estrogen action involves activation of nuclear transcription factor receptors, estrogen also has acute effects on neuronal signaling that occur too rapidly to involve gene expression. These rapid effects are likely to be mediated by extranuclear estrogen receptors associated with the plasma membrane and/or cytoplasmic organelles. Here we used a combination of serial-section electron microscopic immunocytochemistry, immunofluorescence, and Western blotting to show that estrogen receptor-alpha is associated with clusters of vesicles in perisomatic inhibitory boutons in hippocampal CA1 and that estrogen treatment mobilizes these vesicle clusters toward synapses. Estrogen receptor-alpha is present in approximately one-third of perisomatic inhibitory boutons, and specifically in those that express cholecystokinin, not parvalbumin. We also found a high degree of extranuclear estrogen receptor-alpha colocalization with neuropeptide Y. Our results suggest a novel mode of estrogen action in which a subset of vesicles within a specific population of inhibitory boutons responds directly to estrogen by moving toward synapses. The mobilization of these vesicles may influence acute effects of estrogen mediated by estrogen receptor-alpha signaling at inhibitory synapses.

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Figures

Figure 1.

Figure 1.

Extranuclear ERα-IR in the dorsal CA1 cell body layer. A, ERα-IR visualized with bright-field microscopy. The dashed line delineates the boundary of the cell body layer (above) and stratum radiatum (below). Extranuclear ERα-immunoreactive puncta (small dark dots, some of which are indicated by arrowheads) are numerous and occasionally appear to ring unlabeled somata (asterisk). A cell with interneuron-like morphology located in the proximal stratum radiatum contains nuclear ERα-IR (arrow). B, High-magnification view of the same area from tissue processed with the primary antiserum omitted. No labeling is observed. C, Western blot probed with MC-20, the antiserum used for quantification of ERα-IR, showing a single band at ∼67 kDa. D, Electron micrograph of one of two consecutive sections showing MC-20 labeling for ERα on a portion of vesicles (arrowhead) located in a presynaptic bouton forming a symmetric synapse (arrow) with a CA1 pyramidal cell soma. Note that ERα-immunoreactive vesicles appear to be clustered. E, Higher magnification of the same section as in D shows ERα-IR associated with vesicles (large arrowhead) and nearby on the bouton plasma membrane (small arrowheads). F, The section adjacent to that in D shows the same ERα-immunoreactive vesicle cluster. G, ERα labeling with 6F11 is qualitatively similar to labeling with MC-20, with ERα-IR associated with vesicles and the bouton plasma membrane (arrowhead). Scale bars: A, B, 10 μm; D, F, G, 200 nm; E, 200 nm.

Figure 2.

Figure 2.

Immunogold and Western blot confirmation of ERα-IR association with synaptic vesicles. A, Electron micrograph of ERα-IR visualized with immunogold (arrow) in a presynaptic bouton in the CA1 cell body layer. Scale bar, 500 nm. B, A Western blot of cytoplasmic, synaptosomal, and synaptic vesicle proteins probed for synaptophysin (Syn) and ERα shows that ERα is present in both cytoplasmic and synaptic vesicle fractions. Cytoplasmic fractions show a single band at ∼67 kDa, whereas synaptic vesicle fractions show a major band at ∼67 kDa and two additional minor bands (see Results).

Figure 3.

Figure 3.

Colocalization of GAD-immunoreactive varicosities and ERα-immunoreactive puncta. A, A 2-μm-thick image stack shows GAD-immunoreactive varicosities surrounding unlabeled somata (asterisks), presumably of pyramidal cells. B, Higher-magnification views of the boxed area in A through 0.4 μm of tissue. Portions of three GAD-immunoreactive varicosities (green) are shown, one of which contains punctate ERα-IR (red). C, Quantification of GAD-immunoreactive and ERα-immunoreactive colocalization. The entire height of each bar represents the average total number of labeled varicosities (GAD-IR) or puncta (ERα-IR) per 4 μm stack of optical sections (21,600 μm3 volume); the colored portion of each bar represents the average number and percentage of varicosities or puncta that are double labeled. Error bars indicate SEM. Scale bars: A, 10 μm; B, 1 μm.

Figure 4.

Figure 4.

Three-dimensional reconstruction of an axon segment containing ERα-immunoreactive vesicles. An axon segment from an estradiol-treated animal reconstructed through 80 serial sections shows two boutons that form perisomatic inhibitory synapses (yellow) with a CA1 pyramidal cell. Only one of the boutons contains a cluster of ERα-immunoreactive vesicles (red). Green, Non-ERα-immunoreactive vesicles; gray, bouton plasma membrane.

Figure 5.

Figure 5.

Three-dimensional reconstructions and vesicle distances in perisomatic inhibitory boutons containing ERα-immunoreactive vesicle clusters. A, B, Side and end views (rotated by 90°) of reconstructed boutons from an oil-treated (A) and an estradiol-treated (B) animal showing that ERα-immunoreactive vesicle clusters (red) are located closer to the synapse (yellow) in the bouton from an estrogen-treated animal (green, non-ERα-immunoreactive vesicles; gray, bouton plasma membrane). C, D, Histograms showing the relative distance from the synapse of vesicles in reconstructed boutons containing ERα-immunoreactive clusters in oil-treated (C) and estradiol-treated (D) animals. Distributions of ERα-immunoreactive vesicles are represented with filled bars (gray, oil; black, estradiol), whereas the distribution of non-ERα-immunoreactive vesicles are represented by open bars. The inset in D shows cumulative histograms of the relative distances of ERα-immunoreactive vesicles in oil (gray) and estradiol (black) animals demonstrating that labeled vesicles are located significantly closer to synapses after estrogen treatment (K-S test, p < 0.01).

Figure 6.

Figure 6.

Colocalization of PV-, CCK-, and NPY-IR with extranuclear ERα-IR. A, ERα-immunoreactive puncta (red) did not colocalize with PV-immunoreactive structures (green). B, Some ERα-immunoreactive puncta (red) did colocalize (yellow) with CCK-immunoreactive puncta (green). C, Some ERα-immunoreactive puncta (red) also colocalized (yellow) with NPY-IR (green). D, The majority (∼73%) of NPY-immunoreactive puncta (red) are colocalized (yellow) with GAD-IR (green). E, Quantification of CCK- and NPY-IR with ERα-IR. The entire height of each bar represents the average total number of labeled puncta per 2 μm stack of optical sections (10,800 μm3 volume); the colored portion of each bar represents the average number and percentage of puncta that were double labeled. Error bars indicate SEM. Scale bar, 1 μm.

References

    1. Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V, Trentalance A, Visca P, Marino M. Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. Mol Biol Cell. 2005;16:231–237. - PMC - PubMed
    1. Adams MM, Shah RA, Janssen WG, Morrison JH. Different modes of hippocampal plasticity in response to estrogen in young and aged female rats. Proc Natl Acad Sci USA. 2001;98:8071–8076. - PMC - PubMed
    1. Azcoitia I, Sierra A, Garcia-Segura LM. Localization of estrogen receptor β-immunoreactivity in astrocytes of the adult rat brain. Glia. 1999;26:260–267. - PubMed
    1. Bevitt DJ, Milton ID, Piggot N, Henry L, Carter MJ, Toms GL, Lennard TW, Westley B, Angus B, Horne CH. New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry. J Pathol. 1997;183:228–232. - PubMed
    1. Bi R, Broutman G, Foy M, Thompson RF, Baudry M. The tyrosine kinase and MAP kinase pathways mediate multiple effects of estrogen in hippocampus. Proc Natl Acad Sci USA. 2000;97:3602–3607. - PMC - PubMed

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