Estrogen alters hippocampal dendritic spine shape and enhances synaptic protein immunoreactivity and spatial memory in female mice

Abstract

Estrogen (E) treatment induces axospinous synapses in rat hippocampus in vivo and in cultured hippocampal neurons in vitro. To better explore the molecular mechanisms underlying this phenomenon, we have established a mouse model for E action in the hippocampus by using Golgi impregnation to examine hippocampal dendritic spine morphology, radioimmunocytochemistry (RICC) and silver-enhanced immunocytochemistry to examine expression levels of synaptic protein markers, and hippocampal-dependent object-placement memory as a behavioral readout for the actions of E. In ovariectomized mice of several strains and F(1) hybrids, the total dendritic spine density on neurons in the CA1 region was not enhanced by E treatment, a finding that differs from that in the female rat. E treatment of ovariectomized C57BL/6J mice, however, caused an increase in the number of spines with mushroom shapes. By RICC and silver-enhanced immunocytochemistry, we found that the immunoreactivity of postsynaptic markers (PSD95 and spinophilin) and a presynaptic marker (syntaxin) were enhanced by E treatment throughout all fields of the dorsal hippocampus. In the object-placement tests, E treatment enhanced performance of object placement, a spatial episodic memory task. Taken together, the morphology and RICC results suggest a previously uncharacterized role of E in synaptic structural plasticity that may be interpreted as a facilitation of the spine-maturation process and may be associated with enhancement of hippocampal-dependent memory.

Fig. 1.

Effects of EB administration on the dendritic spine density on hippocampal CA1 pyramidal neurons. (a) The total spine density does not change with EB treatment (P < 0.31). (b and c) Dendrites of CA1 pyramidal neurons from oil-treated (b) and EB-treated (c) mice. (d and e) Spines have varying morphologies. We counted spines with a well formed mushroom-shaped head and a narrow neck (M) vs. thin (T) or filopodia-like (I) protrusions. Note that spines were visualized for counting by focusing up and down (which is not possible to show in the photo). (f) EB treatment significantly increases the number of mushroom-type spines (P < 0.01). ^*, statistically significant change. For each of the experimental and control groups, 16 animals were used; spines on 1,200 tertiary dendrites were counted.

Fig. 2.

Autoradiograms depicting presynaptic and postsynaptic protein IR detected by RICC in the mouse hippocampus. (a) Control represents the binding of ³⁵S-labeled anti-rabbit secondary antibody in the absence of primary antibody. (b-d) RICC of spinophilin (b), PSD95 (c), and syntaxin (d).

Fig. 3.

The enhancing effect of EB treatment on IR of the postsynaptic protein spinophilin. (a) IR (mean ± SEM) detected by RICC for spinophilin in different subregions of the hippocampus. ^*, statistically significant changes (P < 0.001; Scheffé's test). ROD, relative OD; DG, dentate gyrus; SO, stratum oriens; MO, molecular layer; Slu, stratum lucidum.

Fig. 5.

The enhancing effect of EB treatment on IR of the postsynaptic proteins spinophilin and PSD95 and the presynaptic protein syntaxin in the whole dorsal hippocampal region of oil-treated (Oil) and EB-treated (EB) mice. ^*, Statistically significant changes. IR (mean ± SEM) was detected by RICC for spinophilin (A; P < 0.001), PSD95 (B; P < 0.035), and syntaxin (C; P < 0.0017).

Fig. 4.

The enhancing effect of EB treatment on IR of the spinophilin, as measured by SEI. (A and B) SEI micrographs of spinophilin in CA1 region of brains from oil-treated (Oil) and EB-treated (EB) mice, respectively. (Scale bar, 5 μm.) (C) Number of silver grain counts (mean ± SEM) detected by SEI. ^*, Significant changes (P < 0.05, t = 2.657; unpaired t test).

Fig. 6.

Enhancing effects of EB administration to OVX mice on performance of the object-placement task. Each experimental and control group consisted of 10 OVX mice. All tests were conducted 24 h after injection of the last EB. EB was administered 1 μg/day for 5 days in the object-placement test. The delay between sampling the objects and the memory tests was 30 min. ^*, Statistically significant differences (ANOVA with paired t test). Oil-treated mice (OVX+Oil) could no longer discriminate old vs. new locations (P > 0.05), whereas EB-treated mice (OVX+EB) spent significantly more time with the new locations (P < 0.01), indicating that they still remembered the spatial locations.