Hippocampal synapses depend on hippocampal estrogen synthesis

Abstract

Estrogens have been described to induce synaptogenesis in principal neurons of the hippocampus and have been shown to be synthesized and released by exactly these neurons. Here, we have focused on the significance of local estrogen synthesis on spine synapse formation and the synthesis of synaptic proteins. To this end, we reduced hippocampal estrogen synthesis in vitro with letrozole, a reversible nonsteroidal aromatase inhibitor. In hippocampal slice cultures, letrozole treatment resulted in a dose-dependent decrease of 17beta-estradiol as quantified by RIA. This was accompanied by a significant decrease in the density of spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a downregulation of spinophilin, a marker of dendritic spines, and synaptophysin, a protein of presynaptic vesicles, in response to letrozole. Surprisingly, no increase in the density of spines, boutons, and synapses and in spinophilin expression was seen after application of estradiol to the medium of cultures that had not been treated with letrozole. However, synaptophysin expression was upregulated under these conditions. Our results point to an essential role of endogenous hippocampal estrogen synthesis in the maintenance of hippocampal spine synapses.

Figure 1.

Letrozole inhibits estrogen synthesis. A dose-dependent decrease in 17β-estradiol release into the medium was found after treatment of slice cultures with various doses of letrozole (▪; mean ± SD; n = 3 cultures per group; ★p < 0.05 to control). Estradiol synthesis was significantly reduced at a dose of 10^-7 m letrozole in dispersion cultures (▦; mean ± SD; n = 3 cultures per group; ★p < 0.05 to control).

Figure 2.

Electron micrographs of the stratum radiatum of the CA1 region after treatment with letrozole or estradiol. A-C, No differences between different treatments were observed in the neuropil. D-F, Framed areas at higher magnification. In all groups, morphologically intact synapses were found (arrows). G, Quantitative evaluation of synapses in the stratum radiatum of the CA1 region in slice cultures. No differences in the number of shaft synapses were found between treatments. A significant decrease in spine synapses and in bouton number was seen after treatment of the slices with letrozole, whereas spine synapse and bouton numbers did not increase in response to estradiol (n = 5; mean ± SD; ★p < 0.05 to control).

Figure 3.

Letrozole reduces spine density in cultured hippocampal neurons. In hippocampal dispersion cultures, letrozole significantly reduces spine density on apical dendrites of Lucifer yellow-stained and photoconverted pyramidal neurons (n = 16 cells per group; mean ± SD; ★p < 0.01 to control). No significant effect was seen after estradiol treatment.

Figure 4.

Estradiol does not increase spine density in hippocampal slice cultures. A, Biocytin-filled pyramidal cell of the CA1 region in a control slice culture. Spines of various shapes are recognizable along the dendrite (inset). B, Spine density on apical stem dendrites of hippocampal CA1 pyramidal cells in organotypic slice cultures. No differences in spine density between the groups were seen (n = 8 cells per group). This holds true for any dendritic segment analyzed and for slices from male and female animals. C, Spine density on third-order dendrites of hippocampal CA1 pyramidal cells in organotypic slice cultures. No difference in spine density was found, neither between control and estrogen-treated cultures nor between gender (n = 8 cells per group; mean ± SD).

Figure 5.

Immunostaining for spinophilin. A-C, Stratum radiatum of CA1 in hippocampal slice cultures. Immunoreactivity for spinophilin in an estradiol-treated slice culture (A), control slice culture (B), and letrozole-treated slice culture (C). D, Punctate spinophilin immunoreactivity on dendrites of a single pyramidal cell in dispersion culture (control). E, Image analysis of spinophilin immunolabeling in the stratum radiatum of CA1 after estradiol or letrozole treatment. Letrozole treatment resulted in a significant downregulation of spinophilin. An upregulation by estradiol was not found (n = 5 slices per group; mean ± SD; ★p < 0.05). F, Image analysis of spinophilin immunolabeling in single pyramidal cells after estradiol and letrozole treatment, respectively. Letrozole led to a significant downregulation of spinophilin immunoreactivity (n = 20 cells per group; mean ± SD; ★p < 0.05 to control).

Figure 6.

Immunoreactivity for synaptophysin. A-C, Stratum radiatum of CA1 in hippocampal slice cultures. Immunoreactivity of synaptophysin in estradiol-treated slice culture (A), control slice culture (B), and letrozole-treated slice culture (C). D, Synaptophysin immunoreactivity in a dispersion culture. Red puncta outline dendrites. Cell nuclei are counterstained with DAPI (blue). E, Image analysis of synaptophysin immunostaining in the stratum radiatum of CA1. Estradiol significantly upregulates and letrozole downregulates immunostaining for synaptophysin (n = 5 slices per group; mean ± SD; ★p < 0.05 to control). F, Image analysis of synaptophysin in single pyramidal cells after estradiol and letrozole treatment, respectively. Letrozole treatment resulted in a significant downregulation of synaptophysin immunoreactivity. Conversely, intensity of immunolabeling was increased after treatment with estradiol (n = 20 cells per group; mean ± SD; ★p < 0.05).

Figure 7.

“Rescue” of synaptophysin labeling after treatment with letrozole. After 24 hr of treatment with 10^-7 m letrozole, a significant downregulation of synaptophysin immunolabeling was found. This effect could be restored if the medium was supplemented with estradiol, together with letrozole (n = 20; mean ± SD; ★p < 0.05 to control).

Figure 8.

Synaptophysin expression in dispersion cultures from male (▪) and female (▦) animals. The physiological doses of 10^-12 m estradiol for male and 10^-10 m estradiol for female animals had no effect on immunolabeling, quantified by image analysis, and the significant downregulation of synaptophysin by letrozole could not be restored by these physiological doses (n = 20 cells per group; mean ± SD; ★p < 0.05)