Sex steroid hormones matter for learning and memory: estrogenic regulation of hippocampal function in male and female rodents

Abstract

Ample evidence has demonstrated that sex steroid hormones, such as the potent estrogen 17β-estradiol (E2), affect hippocampal morphology, plasticity, and memory in male and female rodents. Yet relatively few investigators who work with male subjects consider the effects of these hormones on learning and memory. This review describes the effects of E2 on hippocampal spinogenesis, neurogenesis, physiology, and memory, with particular attention paid to the effects of E2 in male rodents. The estrogen receptors, cell-signaling pathways, and epigenetic processes necessary for E2 to enhance memory in female rodents are also discussed in detail. Finally, practical considerations for working with female rodents are described for those investigators thinking of adding females to their experimental designs.

Figure 1.

Illustration of serum E₂ and progesterone fluctuations during the 4–5 d rodent estrous cycle. Each stage lasts ∼24 h. (M) metestrus (also called diestrus I), (D) diestrus (also called diestrus II), (P) proestrus, (E) estrus.

Figure 2.

Simplified schematic of steroid hormone biosynthesis. A cholesterol precursor is cleaved to generate the progestins pregnenolone and progesterone. These hormones are essential precursors for all other steroid hormones. Progesterone can then be converted into several other hormones, including corticoids and pregnane neurosteroids (shown in gray). Progesterone can also be further metabolized to produce androgens. The enzyme aromatase cleaves an additional carbon from androgens to yield estrogens.

Figure 3.

Classical and nonclassical mechanisms of E₂ action. In the classical mechanism (left), E₂ binds to ERα and ERβ in the cytoplasm, and then the E₂–ER complex translocates into the nucleus and binds to an estrogen response element on the DNA. Together with histone acetyltransferases and other co-regulators, the E₂–ER complex binds to the estrogen response element on DNA to facilitate gene transcription and protein synthesis. Nonclassical mechanisms (right) involve action at or near the plasma membrane that activates cell-signaling cascades. Within the dorsal hippocampus, cell-signaling enzymes associated with estrogenic regulation of hippocampal memory are phosphorylated by several membrane receptors including: (1) metabotropic glutamate receptor 1a (mGluR) and its interactions with ERα and ERβ, (2) NMDA receptors, and (3) G-protein-coupled estrogen receptor (GPER). The resulting activation of cell-signaling pathways (e.g., ERK) triggers epigenetic alterations (e.g., histone acetylation) that regulate gene transcription and protein synthesis. Cell-signaling alterations may also influence local protein levels in cellular compartments such as dendritic spines. (Adapted from Frick 2015 with permission from Elsevier © 2015.)

Figure 4.

Post-training systemic E₂ injection enhances spatial memory consolidation in the Morris water maze and object recognition memory consolidation in ovariectomized mice. (A) Ovariectomized mice received eight hidden-platform training trials prior to E₂ administration. Immediately after the final training trial (arrow), mice were injected with cyclodextrin vehicle (Control) or one of three doses (0.1, 0.2, or 0.4 mg/kg) of cyclodextrin-encapsulated E₂. When memory for the platform location was tested 24 h later, only mice injected with 0.2 mg/kg E₂ remembered the platform location as indicated by the fact that mice in all other groups swam significantly longer distances on day 2 compared with mice in the 0.2 mg/kg group (*P < 0.05). (B) Ovariectomized mice accumulated 30 sec exploring two identical objects and then were immediately injected with cyclodextrin vehicle (Control) or one of three doses (0.1, 0.2, or 0.4 mg/kg) of cyclodextrin-encapsulated E₂. During testing 48 h later, mice receiving 0.2 or 0.4 mg/kg E₂ spent significantly more time with the novel object than chance (dashed line at 15 sec), indicating that these doses enhanced object recognition memory consolidation (*P < 0.05 relative to chance). In contrast, the control and 0.1 mg/kg groups did not spend more time than chance with the novel object. Error bars in both panels represent the mean ± SEM. (Adapted from Gresack and Frick 2006 with permission from Elsevier © 2015.)

Figure 5.

Schematic illustration of the molecular mechanisms required for E₂ and ERs to enhance hippocampal memory consolidation. Phosphorylation of the p42 isoform of ERK is necessary for E₂ to enhance object recognition memory consolidation. This phosphorylation is triggered by numerous upstream events including interactions between mGluR1a and the canonical ERs (ERα and ERβ), and activation of NMDA receptors, protein kinase A (PKA), and phosphatidylinositol-3-kinase (PI3K). E₂-induced phosphorylation of ERK, PI3K, and Akt elicits mTOR signaling, promoting local protein synthesis. E₂-activated ERK also transduces into the nucleus to phosphorylate the transcription factor CREB. Activation of ERK and histone acetyltransferases (HAT) is also necessary for E₂ to increase histone H3 acetylation (Ac); E₂ increases H3 acetylation at the pII and pIV promoters of the Bdnf gene. DNA methylation is also essential for E₂ to enhance memory consolidation, although the specific cytosine residues methylated are unknown. Finally, GPER enhances memory consolidation by activating c-Jun N-terminal kinase (JNK), which facilitates gene expression via transcription factors such as ATF2. (Reprinted from Frick 2015 with permission from Elsevier © 2015.)

Figure 6.

Dorsal hippocampal ERK, PI3K, and mTOR activation are involved in E₂-induced enhancement of object recognition memory consolidation in ovariectomized mice. (A) The phosphorylation of p42 ERK was significantly increased in young ovariectomized mice 5 min after bilateral dorsal hippocampal infusion of 5 μg/hemisphere E₂ (*P < 0.05 relative to vehicle). This effect was blocked by the ERK inhibitor U0126 (0.5 μg/hemisphere), the PI3K inhibitor LY298002 (0.005 μg/hemisphere), or the mTOR inhibitor rapamycin (0.25 μg/hemisphere). (B) All three inhibitors also prevented E₂ from enhancing object recognition memory consolidation, as indicated by the fact that only mice infused with E₂ + vehicle spent more time than chance (15 sec) with the novel object (*P < 0.05). Error bars in both panels represent the mean ± SEM. Phosphorylated ERK levels were normalized to total ERK. Insets are representative Western blots of phosphorylated and total protein.

Figure 7.

Dorsal hippocampal mGluR1a activation is essential in ovariectomized mice for ERα and ERβ to enhance object recognition and object placement memory consolidation, and to increase dorsal hippocampal ERK phosphorylation. (A,B) Immediate post-training infusion of PPT (0.2 pg) or DPN (20 pg) into the dorsal third ventricle significantly increased the time spent with the novel object (A) and moved object (B) relative to chance (dashed line at 15 sec, **P < 0.01); these effects were blocked by dorsal hippocampal infusion of the mGluR1a antagonist LY367385 (10 pg/hemisphere). Bars represent the mean ± SEM time spent with each object. (C) Five min after dorsal third ventricle infusion, PPT and DPN significantly increased phospho-p42 ERK levels (*P < 0.05; **P < 0.01 relative to vehicle); this effect was completely abolished by dorsal hippocampal infusion of LY367385. Bars represent mean ± SEM % change from vehicle. Phosphorylated ERK levels were normalized to total ERK. Insets are representative Western blots of phosphorylated and total protein. (Reprinted from Boulware et al. 2013).

Figure 8.

Histone acetylation and DNA methylation are critical for E₂ to enhance object recognition memory consolidation in ovariectomized mice. (A) Dorsal hippocampal infusion of 5 μg/hemisphere E₂ significantly increased acetyl H3 protein levels in the dorsal hippocampus within 30 min (*P < 0.05 relative to vehicle). This increase was blocked by the ERK activation inhibitor U0126, suggesting that the E₂-induced increase in H3 acetylation is dependent on ERK phosphorylation. (Adapted from Zhao et al. 2012.) (B) Dorsal hippocampal infusion of 5 μg/hemisphere E₂ significantly decreased HDAC2 protein levels in the dorsal hippocampus within 4 h relative to vehicle (*P < 0.05). (Adapted from Zhao et al. 2012.) (C) Chromatin immunoprecipitation analysis showed that dorsal hippocampal E₂ infusion increased acetylation of Bdnf promoters pII and pIV in both young and middle-aged ovariectomized mice (*P < 0.05 relative to age-matched controls). Among vehicle-infused controls, acetylation of pI and pIV was significantly lower in middle-aged females than in young females (#P < 0.05 relative to young vehicle-infused controls), indicating an age-related reduction in acetylation of these promoters. Data were normalized to LINE1 for each sample and then normalized to young vehicle-infused mice for each promoter region and represented as fold of control. (D) Dorsal hippocampal infusion of 5 μg/hemisphere E₂ significantly increased DNMT3B protein levels in the dorsal hippocampus of young ovariectomized mice within 4 h relative to vehicle (*P < 0.05). (Adapted from Zhao et al. 2010.) (E) Dorsal hippocampal infusion of 5-AZA prevented E₂ from enhancing object recognition memory consolidation. E₂ or 5-AZA alone enhanced memory consolidation (*P < 0.05 relative to chance). (Adapted from Zhao et al. 2010.) For all panels, each bar represents the mean ± SEM. Acetyl H3 was normalized to total H3. HDAC2 and DNMT3B were normalized to β-actin. Insets are representative Western blots of phosphorylated and total protein.