G protein-coupled estrogen receptor (GPER) in the dorsal hippocampus regulates memory consolidation in gonadectomized male mice, likely via different signaling mechanisms than in female mice

Abstract

Studies in ovariectomized (OVX) female rodents suggest that G protein-coupled estrogen receptor (GPER) is a key regulator of memory, yet little is known about its importance to memory in males or the cellular mechanisms underlying its mnemonic effects in either sex. In OVX mice, bilateral infusion of the GPER agonist G-1 into the dorsal hippocampus (DH) enhances object recognition and spatial memory consolidation in a manner dependent on rapid activation of c-Jun N-terminal kinase (JNK) signaling, cofilin phosphorylation, and actin polymerization in the DH. However, the effects of GPER on memory consolidation and DH cell signaling in males are unknown. Thus, the present study first assessed effects of DH infusion of G-1 or the GPER antagonist G-15 on object recognition and spatial memory consolidation in gonadectomized (GDX) male mice. As in OVX mice, immediate post-training bilateral DH infusion of G-1 enhanced, whereas G-15 impaired, memory consolidation in the object recognition and object placement tasks. However, G-1 did not increase levels of phosphorylated JNK (p46, p54) or cofilin in the DH 5, 15, or 30 min after infusion, nor did it affect phosphorylation of ERK (p42, p44), PI3K, or Akt. Levels of phospho-cAMP-responsive element binding protein (CREB) were elevated in the DH 30 min following G-1 infusion, indicating that GPER in males activates a yet unknown signaling mechanism that triggers CREB-mediated gene transcription. Our findings show for the first time that GPER in the DH regulates memory consolidation in males and suggests sex differences in underlying signaling mechanisms.

Keywords: CREB; Cofilin; JNK; Mouse; Object placement; Object recognition; Spatial memory.

Fig. 1.

Schematic diagram illustrating the general experimental design. Male C57BL/6 mice (~8 weeks old) underwent bilateral orchiectomy and implantation of dorsal hippocampus (DH) cannulae, and were then given at least 7 days to recover before the start of behavioral testing. All mice were then trained in the object recognition (OR) and object placement (OP) tasks, infused with vehicle (4% or 16% DMSO), G-1 (4 or 8 ng/hemisphere), or G-15 (1.85 or 7.4 ng/hemisphere) immediately after training (upwards arrow), and then tested at the delays indicated in the figure (see text for additional detail). Two weeks separated testing in each task and the sequence of OR and OP testing was counterbalanced within each group. Two weeks after the final behavioral test, mice were infused again and DH tissue was collected bilaterally 5, 15, or 30 min later for homogenization and Western blotting to assess levels of phosphorylated c-Jun N-terminal kinase (JNK), cofilin, extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and cyclic-AMP binding protein (CREB). Figure created with BioRender.com.

Fig. 2.

The GPER agonist G-1 enhanced memory consolidation in male mice. A) During OR testing, GDX mice receiving DH infusion of G-1 (4 or 8 ng/h) spent significantly more time with the novel object than chance (dashed line at 15 s). Both G-1 groups also spent significantly more time with the novel object than the vehicle group. B) During OP testing, mice receiving DH infusion of 8 ng/h G-1 spent significantly more time than chance with the moved object, whereas those treated with vehicle or 4 ng/h G-1 did not. The 8 ng/h G-1 group also spent more time with the moved object than the vehicle and 4 ng/h groups. C, D) The groups did not differ significantly in time to accumulate 30 s of exploration during testing in OP or OR tasks. Circles represent individual mice and each error bar represents the mean ± standard error of the mean (SEM) time (s) spent with the novel (OR) or moved (OP) object. (*p < 0.05, **p < 0.005, ****p < 0.0001 relative to chance; ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001 relative to vehicle or 4 ng/h group) (n = 12–13 mice/group).

Fig. 3.

The GPER antagonist G-15 impaired memory consolidation in male mice. A) In the OR task, GDX mice receiving DH infusion of 7.4 ng/h G-15 spent significantly less time than chance (dashed line) or than the vehicle group with the novel object during testing. B) During OP testing, mice receiving DH infusion of G-15 (1.85 or 7.4 ng/h) spent significantly less time than chance with the moved object, but only the group that received 7.4 ng/h of G-15 spent significantly less time with the moved object than vehicle. C, D) There were no significant differences among groups in time to accumulate 30 s of exploration during OP or OR testing. Circles represent individual mice and each error bar represents the mean ± SEM time (s) spent with the novel (OR) or moved (OP) object. (*p < 0.05, **p < 0.01, ***p < 0.001 relative to chance; ^#p < 0.05, ^##p < 0.01 relative to vehicle) (n = 12–14 mice/group).

Fig. 4.

G-1 did not affect JNK or cofilin phosphorylation in the DH of male mice within 30 min of infusion. A, B) Infusion of 8 ng/h G-1 did not alter levels of phospho-p46 or phospho-p54 JNK at the 5, 15, or 30 min timepoints. C) Levels of phospho-cofilin were significantly higher 30 min after either vehicle or G-1 infusion, suggesting non-specific effects of infusion rather than a specific increase by GPER activation. Circles represent individual mice and each error bar represents the mean ± SEM % change from vehicle controls ($ represents a significant main effect of Time. Significant simple main effects of Time within treatment are represented by *p < 0.05, **p < 0.01, ***p < 0.001) (n = 6–18 mice/group). D) Images of representative blots at each time point for phosphorylated and total forms of each protein.

Fig. 5.

G-1 did not activate ERK/PI3K/Akt pathways in the DH of male mice within 30 min of infusion. A, B) Infusion of 8 ng/h G-1 did not alter levels of p42 ERK or p44 ERK relative to vehicle 5, 15, or 30 min after infusion. C, D) G-1 also did not affect PI3K or Akt phosphorylation relative to vehicle at any time point. Circles represent individual mice; each error bar represents the mean ± SEM % change from vehicle controls (n = 5–19 mice/group). E) Images of representative blots at each time point for phosphorylated and total forms of each protein.

Fig. 6.

G-1 increased phospho-CREB levels in the DH 30 min after infusion. A) Images of representative blots at each time point for phosphorylated and total forms of each protein. B) Infusion of either vehicle or 8 ng/h G-1 increased CREB phosphorylation in the male DH 30 min later, although the significant Treatment effects suggests that the increase in the G-1 group was greater than that of vehicle. Circles represent individual mice; each error bar represents the mean ± SEM % change from vehicle controls (& represents a significant main effect of Treatment; $ represents a significant main effect of Time. Significant simple main effects of Time within treatment are represented by *p < 0.05, **p < 0.01, ****p < 0.0001) (n = 6–19 mice/group).