Sex differences in training-induced activity of the ubiquitin proteasome system in the dorsal hippocampus and medial prefrontal cortex of male and female mice.

The ubiquitin proteasome system (UPS) is a primary mechanism through which proteins are degraded in cells. UPS activity in the dorsal hippocampus (DH) is necessary for multiple types of memory, including object memory, in male rodents. However, sex differences in DH UPS activation after fear conditioning suggest that other forms of learning may also differentially regulate DH UPS activity in males and females. Here, we examined markers of UPS activity in the synaptic and cytoplasmic fractions of DH and medial prefrontal cortex (mPFC) tissue collected 1 h following object training. In males, training increased phosphorylation of proteasomal subunit Rpt6, 20S proteasome activity, and the amount of PSD-95 in the DH synaptic fraction, as well as proteasome activity in the mPFC synaptic fraction. In females, training did not affect measures of UPS or synaptic activity in the DH synaptic fraction or in either mPFC fraction but increased Rpt6 phosphorylation in the DH cytoplasmic fraction. Overall, training-induced UPS activity was greater in males than in females, greater in the DH than in the mPFC, and greater in synaptic fractions than in cytosol. These data suggest that object training drives sex-specific alterations in UPS activity across brain regions and subcellular compartments important for memory.

Extracellular matrix metalloproteinase-9 (MMP-9) is required in female mice for 17ß-estradiol enhancement of hippocampal memory consolidation.

Hippocampal plasticity and memory are modulated by the potent estrogen 17ß-estradiol (E2). Research on the molecular mechanisms of hippocampal E2 signaling has uncovered multiple intracellular pathways that contribute to these effects, but few have questioned the role that extracellular signaling processes may play in E2 action. Modification of the extracellular matrix (ECM) by proteases like matrix metalloproteinase-9 (MMP-9) is critical for activity-dependent remodeling of synapses, and MMP-9 activity is required for hippocampal learning and memory. Yet little is known about the extent to which E2 regulates MMP-9 in the hippocampus, and the influence this interaction may have on hippocampal memory. Here, we examined the effects of hippocampal MMP-9 activity on E2-induced enhancement of spatial and object recognition memory consolidation. Post-training bilateral infusion of an MMP-9 inhibitor into the dorsal hippocampus of ovariectomized female mice blocked the enhancing effects of E2 on object placement and object recognition memory, supporting a role for MMP-9 in estrogenic regulation of memory consolidation. E2 also rapidly increased the activity of dorsal hippocampal MMP-9 without influencing its protein expression, providing further insight into hippocampal E2/MMP-9 interactions. Together, these results provide the first evidence that E2 regulates MMP-9 to modulate hippocampal memory and highlight the need to further study estrogenic regulation of extracellular modification.

17ß-estradiol activation of dorsal hippocampal TrkB is independent of increased mature BDNF expression and is required for enhanced memory consolidation in female mice.

The potent estrogen 17ß-estradiol (E2) is known to enhance hippocampal memory and plasticity, however the molecular mechanisms underlying these effects remain unclear. Brain derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB) are regulated by E2, but the potential mechanistic roles of neurotrophic signaling in E2-induced enhancement of memory are not well understood. Here, we examined the effects of hippocampal TrkB signaling on E2-induced enhancement of memory consolidation in the object placement and recognition tasks. Bilateral infusion of the TrkB antagonist ANA-12 into the dorsal hippocampus of ovariectomized female mice blocked E2-induced enhancement of memory consolidation, supporting a role for TrkB-mediated signaling in estrogenic regulation of memory. Although dorsal hippocampal E2 infusion increased levels of phospho-TrkB and mature BDNF (mBDNF) in the dorsal hippocampus within 4-6 h, E2-induced increases in hippocampal mBDNF expression were not required for hippocampal TrkB activation and were not inhibited by TrkB antagonism. Thus, E2 regulates TrkB signaling to facilitate memory consolidation in a manner independent of mBDNF expression. Together these results provide the first direct evidence that E2 modulation of hippocampal TrkB signaling is required for its beneficial effects on memory consolidation and provide additional characterization of the ways in which TrkB/BDNF signaling is regulated by E2 in the hippocampus.

Oestradiol as a neuromodulator of learning and memory.

Although hormones such as glucocorticoids have been broadly accepted in recent decades as general neuromodulators of memory processes, sex steroid hormones such as the potent oestrogen 17ß-oestradiol have been less well recognized by the scientific community in this capacity. The predominance of females in studies of oestradiol and memory and the general (but erroneous) perception that oestrogens are 'female' hormones have probably prevented oestradiol from being more widely considered as a key memory modulator in both sexes. Indeed, although considerable evidence supports a crucial role for oestradiol in regulating learning and memory in females, a growing body of literature indicates a similar role in males. This Review discusses the mechanisms of oestradiol signalling and provides an overview of the effects of oestradiol on spatial, object recognition, social and fear memories. Although the primary focus is on data collected in females, effects of oestradiol on memory in males will be discussed, as will sex differences in the molecular mechanisms that regulate oestrogenic modulation of memory, which may have important implications for the development of future cognitive therapeutics.

Estrogen receptor signaling through metabotropic glutamate receptors.

As the non-nuclear initiated effects of steroid hormone signaling have become more widely accepted, there has been a need to define the novel mechanisms of hormone receptor action that account for these outcomes. One mechanism that has emerged is the coupling of classical estrogen receptors (ERα and ERß) with metabotropic glutamate receptors (mGluRs) to initiate G protein signaling cascades that ultimately influence neuronal physiology, structure, and behavior. Since its initial discovery in hippocampal neurons, evidence of ER/mGluR associations have been found throughout the nervous system, and the heterogeneity of possible receptor pairings afforded by multiple ER and mGluR subtypes appears to drive diverse molecular outcomes that can impact processes like cognition, motivation, movement, and pain. Recent evidence also suggests that the role of mGluRs in steroid hormone signaling may not be unique to ERs, but rather a conserved mechanism of membrane-initiated hormone receptor action.

Dorsal Hippocampal Actin Polymerization Is Necessary for Activation of G-Protein-Coupled Estrogen Receptor (GPER) to Increase CA1 Dendritic Spine Density and Enhance Memory Consolidation.

Activation of the membrane estrogen receptor G-protein-coupled estrogen receptor (GPER) in ovariectomized mice via the GPER agonist G-1 mimics the beneficial effects of 17ß-estradiol (E2) on hippocampal CA1 spine density and memory consolidation, yet the cell-signaling mechanisms mediating these effects remain unclear. The present study examined the role of actin polymerization and c-Jun N-terminal kinase (JNK) phosphorylation in mediating effects of dorsal hippocampally infused G-1 on CA1 dendritic spine density and consolidation of object recognition and spatial memories in ovariectomized mice. We first showed that object learning increased apical CA1 spine density in the dorsal hippocampus (DH) within 40 min. We then found that DH infusion of G-1 increased both CA1 spine density and phosphorylation of the actin polymerization regulator cofilin, suggesting that activation of GPER may increase spine morphogenesis through actin polymerization. As with memory consolidation in our previous work (Kim et al., 2016), effects of G-1 on CA1 spine density and cofilin phosphorylation depended on JNK phosphorylation in the DH. Also consistent with our previous findings, E2-induced cofilin phosphorylation was not dependent on GPER activation. Finally, we found that infusion of the actin polymerization inhibitor, latrunculin A, into the DH prevented G-1 from increasing apical CA1 spine density and enhancing both object recognition and spatial memory consolidation. Collectively, these data demonstrate that GPER-mediated hippocampal spinogenesis and memory consolidation depend on JNK and cofilin signaling, supporting a critical role for actin polymerization in the GPER-induced regulation of hippocampal function in female mice.SIGNIFICANCE STATEMENT Emerging evidence suggests that G-protein-coupled estrogen receptor (GPER) activation mimics effects of 17ß-estradiol on hippocampal memory consolidation. Unlike canonical estrogen receptors, GPER activation is associated with reduced cancer cell proliferation; thus, understanding the molecular mechanisms through which GPER regulates hippocampal function may provide new avenues for the development of drugs that provide the cognitive benefits of estrogens without harmful side effects. Here, we demonstrate that GPER increases CA1 dendritic spine density and hippocampal memory consolidation in a manner dependent on actin polymerization and c-Jun N-terminal kinase phosphorylation. These findings provide novel insights into the role of GPER in mediating hippocampal morphology and memory consolidation, and may suggest first steps toward new therapeutics that more safely and effectively reduce memory decline in menopausal women.

mGluR5 Mediates Dihydrotestosterone-Induced Nucleus Accumbens Structural Plasticity, but Not Conditioned Reward.

Gonadal hormones play a vital role in driving motivated behavior. They not only modulate responses to naturally rewarding stimuli, but also influence responses to drugs of abuse. A commonality between gonadal hormones and drugs of abuse is that they both impact the neurocircuitry of reward, including the regulation of structural plasticity in the nucleus accumbens (NAc). Previous hormonal studies have focused on the mechanisms and behavioral correlates of estradiol-induced dendritic spine changes in the female NAc. Here we sought to determine the effects of androgens on medium spiny neuron (MSN) spine plasticity in the male NAc. Following treatment with the androgen receptor agonist dihydrotestosterone (DHT), MSNs in castrated male rats exhibited a significant decrease in dendritic spine density. This effect was isolated to the shell subregion of the NAc. The effect of DHT was dependent on mGluR5 activity, and local mGluR5 activation and subsequent endocannabinoid signaling produce an analogous NAc shell spine decrease. Somewhat surprisingly, DHT-induced conditioned place preference remained intact following systemic inhibition of mGluR5. These findings indicate that androgens can utilize mGluR signaling, similar to estrogens, to mediate changes in NAc dendritic structure. In addition, there are notable differences in the direction of spine changes, and site specificity of estrogen and androgen action, suggesting sex differences in the hormonal regulation of motivated behaviors.

Interactions between estrogen receptors and metabotropic glutamate receptors and their impact on drug addiction in females.

Contribution to Special Issue on Fast effects of steroids. Estrogen receptors α and ß (ERα and ERß) have a unique relationship with metabotropic glutamate receptors (mGluRs) in the female rodent brain such that estradiol is able to recruit intracellular G-protein signaling cascades to influence neuronal physiology, structure, and ultimately behavior. While this association between ERs and mGluRs exists in many cell types and brain regions, its effects are perhaps most striking in the nucleus accumbens (NAc). This review will discuss the original characterization of ER/mGluR signaling and how estradiol activity in the NAc confers increased sensitivity to drugs of abuse in females through this mechanism.

Estradiol Facilitation of Cocaine Self-Administration in Female Rats Requires Activation of mGluR5.

In comparison to men, women initiate drug use at earlier ages and progress from initial use to addiction more rapidly. This heightened intake and vulnerability to drugs of abuse is regulated in part by estradiol, although the signaling mechanisms by which this occurs are not well understood. Recent findings indicate that within the nucleus accumbens core, estradiol induces structural plasticity via membrane-localized estrogen receptor α, functionally coupled to metabotropic glutamate receptor subtype 5 (mGluR5). Hence, we sought to determine whether mGluR5 activation was essential for estradiol-mediated enhancement of cocaine self-administration. Ovariectomized (OVX) female rats were allowed to freely self-administer cocaine under extended access conditions (6 h/d) for 10 consecutive days. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) or vehicle was administered before estradiol (or oil), on a 2 d on/2 d off schedule throughout the extended access period. MPEP treatment prevented the estradiol-dependent enhancement of cocaine self-administration in OVX females. In a separate experiment, potentiation of mGluR5 function with the positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (in the absence of estradiol treatment) failed to increase cocaine self-administration. These data suggest that mGluR5 activation is necessary for estradiol-mediated enhancement of responses to cocaine, but that direct mGluR5 activation is insufficient to mimic the female response to estradiol. Building on previous studies in male animals, these findings further highlight the therapeutic potential of mGluR5 antagonism in the treatment of addiction and suggest that there may be added therapeutic benefit in females.

Opposite Effects of mGluR1a and mGluR5 Activation on Nucleus Accumbens Medium Spiny Neuron Dendritic Spine Density.

The group I metabotropic glutamate receptors (mGluR1a and mGluR5) are important modulators of neuronal structure and function. Although these receptors share common signaling pathways, they are capable of having distinct effects on cellular plasticity. We investigated the individual effects of mGluR1a or mGluR5 activation on dendritic spine density in medium spiny neurons in the nucleus accumbens (NAc), which has become relevant with the potential use of group I mGluR based therapeutics in the treatment of drug addiction. We found that systemic administration of mGluR subtype-specific positive allosteric modulators had opposite effects on dendritic spine densities. Specifically, mGluR5 positive modulation decreased dendritic spine densities in the NAc shell and core, but was without effect in the dorsal striatum, whereas increased spine densities in the NAc were observed with mGluR1a positive modulation. Additionally, direct activation of mGluR5 via CHPG administration into the NAc also decreased the density of dendritic spines. These data provide insight on the ability of group I mGluRs to induce structural plasticity in the NAc and demonstrate that the group I mGluRs are capable of producing not just distinct, but opposing, effects on dendritic spine density.