The extracellular signal-regulated kinase (ERK) pathway is critical for various forms of learning and memory, and is activated by the potent estrogen 17␤-estradiol (E 2 ). Here, we asked whether E 2 modulates memory via ERK activation and putative membrane-bound estrogen receptors (ERs). Using ovariectomized mice, we first demonstrate that intraperitoneal injection of 0.2 mg/kg E 2 significantly increases dorsal hippocampal levels of phosphorylated ERK protein 1 h after injection. Second, we show that E 2 administered intraperitoneally (0.2 mg/kg) or via intrahippocampal infusion (5.0 g/side) immediately after training in an object recognition task significantly enhances memory retention, and that the beneficial effect of intraperitoneal E 2 is blocked by dorsal hippocampal inhibition of ERK activation. Third, using bovine serum albumin-conjugated 17␤-estradiol (BSA-E 2 ), we demonstrate that E 2 binding at membrane-bound ERs can increase dorsal hippocampal ERK activation and enhance object memory consolidation in an ERK-dependent manner. Fourth, we show that this effect is independent of nuclear ERs, but is dependent on the dorsal hippocampus. By demonstrating that E 2 enhances memory consolidation via dorsal hippocampal ERK activation, this study is the first to identify a specific molecular pathway by which E 2 modulates memory and to demonstrate a novel role for membrane-bound ERs in mediating E 2 -induced improvements in hippocampal memory consolidation.

The involvement of epigenetic alterations in mediating effects of estrogens on memory is unknown. The present study determined whether histone acetylation and DNA methylation are critical for the potent estrogen 17β-estradiol (E 2 ) to enhance object recognition memory. We show that dorsal hippocampal E 2 infusion increases acetylation of dorsal hippocampal histone H3, but not H4-an effect blocked by dorsal hippocampal inhibition of ERK activation. Further, intrahippocampal inhibition of ERK activation or DNA methyltransferase (DNMT) activity blocked the memory-enhancing effects of E 2 . Consistent with these effects, E 2 decreased levels of HDAC2 protein and increased DNMT expression in the dorsal hippocampus. These findings provide evidence that the beneficial effects of E 2 on memory consolidation are associated with epigenetic alterations, and suggest these can be triggered by dorsal hippocampal ERK signaling.T he specific molecular mechanisms underlying the mnemonic effects of estrogens remain largely unknown. We recently showed that activation of the extracellular signal-regulated kinase/ mitogen-activated protein kinase (ERK/MAPK) signaling cascade in the dorsal hippocampus is necessary for the potent estrogen 17β-estradiol (E 2 ) to enhance novel object recognition in young female mice (1). In this study, i.p. injection of E 2 immediately after object recognition training significantly enhanced long-term memory and increased p42 ERK phosphorylation in the dorsal hippocampus; both effects were blocked by inhibiting MAPK kinase (MEK), the exclusive upstream activator of ERK (1).Activated ERK can promote the expression of genes associated with learning and memory, in part, by activating transcription factors such as CREB (2, 3). ERK may also increase gene expression by regulating epigenetic mechanisms necessary for memory formation, such as histone acetylation and DNA methylation; recent findings suggest that ERK activation influences both processes (4-7). Thus, these mechanisms may also play a role in estrogenic modulation of memory. DNA is wound around a core of eight histone proteins, two each of histones H2A, H2B, H3, and H4. Acetylation of lysine residues on histone tails relaxes the bond between histones and DNA, allowing transcriptional access. Histone acetylation is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) (8). Genetic disruption of HAT activity impairs hippocampal memory, and these deficits are rescued by HDAC inhibitors (9). HDAC inhibitors such as trichostatin A (TSA) prevent deacetylation of histones H3 and H4, and enhance induction of hippocampal long-term potentiation (LTP), long-term contextual fear conditioning (CFC), and novel object recognition (4, 10-12). Acetylation of hippocampal histone H3, but not H4, is increased following ERK activation or CFC (4, 5), and ERK activation is necessary for other protein kinases (e.g., protein kinase C, PKC) to increase hippocampal H3 acetylation (4). Collectively, these data suggest that histone acetylation regul...

Our laboratory has demonstrated that 17␤-estradiol (E 2 ) enhances hippocampal memory consolidation via rapid activation of multiple intracellular signaling cascades, including the ERK/MAPK cascade (Fernandez et al., 2008; Fan et al., 2010). However, the receptor mechanisms responsible for these effects of E 2 remain unclear. In vitro, estrogen receptor ␣ (ER␣) signaling through metabotropic glutamate receptor 1a (mGluR1a) leads to ERK-dependent CREB phosphorylation (Boulware et al., 2005), suggesting that interactions between ERs and mGluR1a may be vital to the memory-enhancing effects of E 2 . As such, the present study tested the roles of classical estrogen receptors (ER␣ and ER␤) and mGluR1a in mediating the effects of E 2 on hippocampal memory consolidation. Dorsal hippocampal (DH) infusion of ER␣ (PPT) or ER␤ (DPN) agonists enhanced novel object recognition and object placement memory in ovariectomized female mice in an ERK-dependent manner, suggesting that these receptors influence memory by rapidly activating hippocampal cell signaling. Next, DH infusion of the mGluR1a antagonist LY367385 blocked the object and spatial memory facilitation induced by E 2 , PPT, and DPN, demonstrating that ER/mGluR1a signaling is critical for the memory-enhancing effects of E 2 . Finally, we show that ER␣, ER␤, mGluR1, and ERK all reside within specialized membrane microdomains of the DH, and that ER␣ and ER␤ physically interact with mGluR1, providing a means through which ERs may activate mGluRs and downstream signaling. Together, these findings provide the first in vivo evidence demonstrating that ER/mGluR signaling can mediate the beneficial effects of E 2 on hippocampal memory consolidation.

The mammalian target of rapamycin (mTOR) signaling pathway is an important regulator of protein synthesis and is essential for various forms of hippocampal memory. Here, we asked whether the enhancement of object recognition memory consolidation produced by dorsal hippocampal infusion of 17b-estradiol (E 2 ) is dependent on mTOR signaling in the dorsal hippocampus, and whether E 2 -induced mTOR signaling is dependent on dorsal hippocampal phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) activation. We first demonstrated that the enhancement of object recognition induced by E 2 was blocked by dorsal hippocampal inhibition of ERK, PI3K, or mTOR activation. We then showed that an increase in dorsal hippocampal ERK phosphorylation 5 min after intracerebroventricular (ICV) E 2 infusion was also blocked by dorsal hippocampal infusion of the three cell signaling inhibitors. Next, we found that ICV infusion of E 2 increased phosphorylation of the downstream mTOR targets S6K (Thr-421) and 4E-BP1 in the dorsal hippocampus 5 min after infusion, and that this phosphorylation was blocked by dorsal hippocampal infusion of inhibitors of ERK, PI3K, and mTOR. Collectively, these data demonstrate for the first time that activation of the dorsal hippocampal mTOR signaling pathway is necessary for E 2 to enhance object recognition memory consolidation and that E 2 -induced mTOR activation is dependent on upstream activation of ERK and PI3K signaling.The potent estrogen 17b-estradiol (E 2 ) is a critical regulator of hippocampal synaptic morphology. In rodents and nonhuman primates, E 2 increases hippocampal levels of synaptic proteins, including the presynaptic proteins synaptophysin and syntaxin, and the postsynaptic proteins spinophilin and PSD-95 (Brake et al.

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We previously demonstrated that dorsal hippocampal extracellular signal-regulated kinase (ERK) activation is necessary for 17␤-estradiol (E 2 ) to enhance novel object recognition in young ovariectomized mice (Fernandez et al., 2008). Here, we asked whether E 2 has similar memory-enhancing effects in middle-aged and aged ovariectomized mice, and whether these effects depend on ERK and phosphatidylinositol 3-kinase (PI3K)/Akt activation. We first demonstrated that intracerebroventricular or intrahippocampal E 2 infusion immediately after object recognition training enhanced memory consolidation in middle-aged, but not aged, females. The E 2 -induced enhancement in middle-aged females was blocked by intrahippocampal inhibition of ERK or PI3K activation. Intrahippocampal or intracerebroventricular E 2 infusion in middle-aged females increased phosphorylation of p42 ERK in the dorsal hippocampus 15 min, but not 5 min, after infusion, an effect that was blocked by intrahippocampal inhibition of ERK or PI3K activation. Dorsal hippocampal PI3K and Akt phosphorylation was increased 5 min after intrahippocampal or intracerebroventricular E 2 infusion in middle-aged, but not aged, females. Intracerebroventricular E 2 infusion also increased PI3K phosphorylation after 15 min, and this effect was blocked by intrahippocampal PI3K, but not ERK, inhibition. These data demonstrate for the first time that activation of dorsal hippocampal PI3K/ Akt and ERK signaling pathways is necessary for E 2 to enhance object recognition memory in middle-aged females. They also reveal that similar dorsal hippocampal signaling pathways mediate E 2 -induced object recognition memory enhancement in young and middle-aged females and that the inability of E 2 to activate these pathways may underlie its failure to enhance object recognition in aged females.

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