Sex steroid hormones, the estrous cycle, and rapid modulation of glutamatergic synapse properties in the striatal brain regions with a focus on 17ß-estradiol and the nucleus accumbens.

The striatal brain regions encompassing the nucleus accumbens core (NAcc), shell (NAcs) and caudate-putamen (CPu) regulate cognitive functions including motivated behaviors, habit, learning, and sensorimotor action, among others. Sex steroid hormone sensitivity and sex differences have been documented in all of these functions in both normative and pathological contexts, including anxiety, depression and addiction. The neurotransmitter glutamate has been implicated in regulating these behaviors as well as striatal physiology, and there are likewise documented sex differences in glutamate action upon the striatal output neurons, the medium spiny neurons (MSNs). Here we review the available data regarding the role of steroid sex hormones such as 17ß-estradiol (estradiol), progesterone, and testosterone in rapidly modulating MSN glutamatergic synapse properties, presented in the context of the estrous cycle as appropriate. Estradiol action upon glutamatergic synapse properties in female NAcc MSNs is most comprehensively discussed. In the female NAcc, MSNs exhibit development period-specific sex differences and estrous cycle variations in glutamatergic synapse properties as shown by multiple analyses, including that of miniature excitatory postsynaptic currents (mEPSCs). Estrous cycle-differences in NAcc MSN mEPSCs can be mimicked by acute exposure to estradiol or an ERα agonist. The available evidence, or lack thereof, is also discussed concerning estrogen action upon MSN glutamatergic synapse in the other striatal regions as well as the underexplored roles of progesterone and testosterone. We conclude that there is strong evidence regarding estradiol action upon glutamatergic synapse function in female NAcs MSNs and call for more research regarding other hormones and striatal regions.

ERα Stimulation Rapidly Modulates Excitatory Synapse Properties in Female Rat Nucleus Accumbens Core.

INTRODUCTION: The nucleus accumbens core (NAcc) is a sexually differentiated brain region that is modulated by steroid hormones such as 17ß-estradiol (estradiol), with consequential impacts on relevant motivated behaviors and disorders such as addiction, anxiety, and depression. NAcc estradiol levels naturally fluctuate, including during the estrous cycle in adult female rats, which is analogous to the menstrual cycle in adult humans. Across the estrous cycle, excitatory synapse properties of medium spiny neurons rapidly change, as indicated by analysis of miniature excitatory postsynaptic currents (mEPSCs). mEPSC frequency decreases during estrous cycle phases associated with high estradiol levels. This decrease in mEPSC frequency is mimicked by acute topical exposure to estradiol. The identity of the estrogen receptor (ER) underlying this estradiol action is unknown. Adult rat NAcc expresses three ERs, all extranuclear: membrane ERα, membrane ERß, and GPER1. METHODS: In this brief report, we take a first step toward addressing this challenge by testing whether activation of ERs via acute topical agonist application is sufficient for inducing changes in mEPSC properties recorded via whole-cell patch clamp. RESULTS: An agonist of ERα induced large decreases in mEPSC frequency, while agonists of ERß and GPER1 did not robustly modulate mEPSC properties. CONCLUSIONS: These data provide evidence that activation of ERα is sufficient for inducing changes in mEPSC frequency and is a likely candidate underlying the estradiol-induced changes observed during the estrous cycle. Overall, these findings extend our understanding of the neuroendocrinology of the NAcc and implicate ERα as a primary target for future studies.

The estrous cycle and 17ß-estradiol modulate the electrophysiological properties of rat nucleus accumbens core medium spiny neurons.

The nucleus accumbens core is a key nexus within the mammalian brain for integrating the premotor and limbic systems and regulating important cognitive functions such as motivated behaviors. Nucleus accumbens core functions show sex differences and are sensitive to the presence of hormones such as 17ß-estradiol (estradiol) in normal and pathological contexts. The primary neuron type of the nucleus accumbens core, the medium spiny neuron (MSN), exhibits sex differences in both intrinsic excitability and glutamatergic excitatory synapse electrophysiological properties. Here, we provide a review of recent literature showing how estradiol modulates rat nucleus accumbens core MSN electrophysiology within the context of the estrous cycle. We review the changes in MSN electrophysiological properties across the estrous cycle and how these changes can be mimicked in response to exogenous estradiol exposure. We discuss in detail recent findings regarding how acute estradiol exposure rapidly modulates excitatory synapse properties in nucleus accumbens core but not caudate-putamen MSNs, which mirror the natural changes seen across estrous cycle phases. These recent insights demonstrate the strong impact of sex-specific estradiol action upon nucleus accumbens core neuron electrophysiology.

FireMaster® 550 (FM 550) exposure during the perinatal period impacts partner preference behavior and nucleus accumbens core medium spiny neuron electrophysiology in adult male and female prairie voles, Microtus ochrogaster.

One of the most widely used flame retardant (FR) mixtures in household products is Firemaster 550 (FM 550). FM 550 leaches from items such as foam-based furniture and infant products, resulting in contamination of the household environment and biota. Previous studies indicate sex-specific behavioral deficits in rodents and zebrafish in response to developmental FM 550 exposure. These deficits include impacts on social and attachment behaviors in a prosocial rodent: the prairie vole (Microtus ochrogaster). The prairie vole is a laboratory-acclimated rodent that exhibits spontaneous attachment behaviors including pair bonding. Here we extend previous work by addressing how developmental exposure to FM 550 impacts pair bonding strength via an extended-time partner preference test, as well as neuron electrophysiological properties in a region implicated in pair bond behavior, the nucleus accumbens (NAcc) core. Dams were exposed to vehicle or 1000 µg of FM 550 via subcutaneous injections throughout gestation, and female and male pups were directly exposed beginning the day after birth until weaning. Pair bond behavior of adult female and male offspring was assessed using a three hour-long partner preference test. Afterwards, acute brain slices of the NAcc core were produced and medium spiny neuron electrophysiological attributes recorded via whole cell patch-clamp. Behavioral impacts were sex-specific. Partner preference behavior was increased in exposed females but decreased in exposed males. Electrophysiological impacts were similar between sexes and specific to attributes related to input resistance. Input resistance was decreased in neurons recorded from both sexes exposed to FM 550 compared to vehicle. This study supports the hypothesis that developmental exposure to FM 550 impacts attachment behaviors and demonstrates a novel FM 550 effect on neural electrophysiology.

Estrogen receptor alpha, G-protein coupled estrogen receptor 1, and aromatase: Developmental, sex, and region-specific differences across the rat caudate-putamen, nucleus accumbens core and shell.

Sex steroid hormones such as 17ß-estradiol (estradiol) regulate neuronal function by binding to estrogen receptors (ERs), including ERα and GPER1, and through differential production via the enzyme aromatase. ERs and aromatase are expressed across the nervous system, including in the striatal brain regions. These regions, comprising the nucleus accumbens core, shell, and caudate-putamen, are instrumental for a wide-range of functions and disorders that show sex differences in phenotype and/or incidence. Sex-specific estrogen action is an integral component for generating these sex differences. A distinctive feature of the striatal regions is that in adulthood neurons exclusively express membrane but not nuclear ERs. This long-standing finding dominates models of estrogen action in striatal regions. However, the developmental etiology of ER and aromatase cellular expression in female and male striatum is unknown. This omission in knowledge is important to address, as developmental stage influences cellular estrogenic mechanisms. Thus, ERα, GPER1, and aromatase cellular immunoreactivity was assessed in perinatal, prepubertal, and adult female and male rats. We tested the hypothesis that ERα, GPER1, and aromatase exhibits sex, region, and age-specific differences, including nuclear expression. ERα exhibits nuclear expression in all three striatal regions before adulthood and disappears in a region- and sex-specific time-course. Cellular GPER1 expression decreases during development in a region- but not sex-specific time-course, resulting in extranuclear expression by adulthood. Somatic aromatase expression presents at prepuberty and increases by adulthood in a region- but not sex-specific time-course. These data indicate that developmental period exerts critical sex-specific influences on striatal cellular estrogenic mechanisms.

Differential and synergistic roles of 17ß-estradiol and progesterone in modulating adult female rat nucleus accumbens core medium spiny neuron electrophysiology.

Naturally occurring cyclical changes in sex steroid hormones such as 17ß-estradiol and progesterone can modulate neuron function and behavior in female mammals. One example is the estrous cycle in rats, which is composed of multiple phases. We previously reported evidence of differences between estrous cycle phases in excitatory synapse and intrinsic electrophysiological properties of rat nucleus accumbens core (AcbC) medium spiny neurons (MSNs). The AcbC is a nexus between the limbic and premotor systems and is integral for controlling motivated and reward-associated behaviors and disorders, which are sensitive to the estrous cycle and hormones. The present study expands our prior findings by testing whether circulating levels of estradiol and progesterone correlate with changes in MSN electrophysiology across estrous cycle phases. As part of this project, the excitatory synapse and intrinsic excitability properties of MSNs in late proestrus of adult female rats were assessed. Circulating levels of estradiol correlate with resting membrane potential, the time constant of the membrane, and rheobase. Circulating levels of progesterone correlate with miniature excitatory postsynaptic current (mEPSC) frequency and amplitude. Circulating levels of estradiol and progesterone together correlate with mEPSC amplitude, resting membrane potential, and input resistance. The late proestrus phase features a prominent and unique decrease in mEPSC frequency. These data indicate that circulating levels of estradiol and progesterone alone or in combination interact with specific MSN electrophysiological properties, indicating differential and synergistic roles of these hormones. Broadly, these findings illustrate the underlying endocrine actions regarding how the estrous cycle modulates MSN electrophysiology.NEW & NOTEWORTHY This research indicates that estradiol and progesterone act both differentially and synergistically to modulate neuron physiology in the nucleus accumbens core. These actions by specific hormones provide key data indicating the endocrine mechanisms underlying how the estrous cycle modulates neuron physiology in this region. Overall, these data reinforce that hormones are an important influence on neural physiology.

Temporal and bidirectional influences of estradiol on voluntary wheel running in adult female and male rats.

The sex steroid hormone 17ß-estradiol (estradiol) regulates animal behavior as both a non-rapid hormone signal and as a rapid-acting neuromodulator. By practical necessity, estradiol's divergent temporal actions on rodent behavior are typically studied singularly and in one sex. We hypothesized that estradiol simultaneously acts through both temporal mechanisms to sex-specifically modulate a single behavior; and furthermore, that estradiol action in one temporal domain may regulate action in another. To test this hypothesis, we utilized one of the most robust rat behaviors exhibiting sex differences and estradiol-responsiveness, voluntary wheel running. Adult female and male rats were gonadectomized and exposed to daily repeated estradiol benzoate (EB) injections. Estradiol-sensitive running behavior was continually assessed in both the rapid and non-rapid temporal domains. We found that in female rats, estradiol rapidly decreased voluntary wheel running, but only after prior daily EB injections, supporting the hypothesis that non-rapid estradiol action influences rapid estradiol actions. Males exhibited a similar but less robust response, demonstrating sex-responsiveness. This rapid estradiol-induced decrease in running contrasted to non-rapid estradiol action which overall increased running in both sexes, revealing a bidirectional nature of estradiol's temporal influence. Non-rapid estradiol action also demonstrated sex-responsiveness, as a higher dose of EB was required to induce increased running in males compared to females. These findings indicate that estradiol rapidly, non-rapidly, and bidirectionally modulates wheel running in a sex-responsive manner, and that rapid estradiol action is modulated by non-rapid estradiol action. Overall, these data illustrate estradiol as a pleiotropic sex-responsive neuromodulator of a single behavior across temporal domains.

Acute neuroestrogen blockade attenuates song-induced immediate early gene expression in auditory regions of male and female zebra finches.

Neuron-derived estrogens are synthesized by aromatase and act through membrane receptors to modulate neuronal physiology. In many systems, long-lasting hormone treatments can alter sensory-evoked neuronal activation. However, the significance of acute neuroestrogen production is less understood. Both sexes of zebra finches can synthesize estrogens rapidly in the auditory cortex, yet it is unclear how this modulates neuronal cell signaling. We examined whether acute estrogen synthesis blockade attenuates auditory-induced expression of early growth response 1 (Egr-1) in the auditory cortex of both sexes. cAMP response element-binding protein phosphorylation (pCREB) induction by song stimuli and acute estrogen synthesis was also examined. We administered the aromatase inhibitor fadrozole prior to song exposure and measured Egr-1 across several auditory regions. Fadrozole attenuated Egr-1 in the auditory cortex greater in males than females. Females had greater expression and clustering of aromatase cells than males in high vocal center (HVC) shelf. Auditory-induced Egr-1 expression exhibited a large sex difference following fadrozole treatment. We did not observe changes in pCREB expression with song presentation or aromatase blockade. These findings are consistent with the hypothesis that acute neuroestrogen synthesis can drive downstream transcriptional responses in several cortical auditory regions, and that this mechanism is more prominent in males.

Metabotropic glutamate receptor subtype 5 (mGlu5) is necessary for estradiol mitigation of light-induced anxiety behavior in female rats.

Anxiety-related behaviors are influenced by steroid hormones such as 17ß-estradiol and environmental stimuli such as acute stressors. For example, rats exhibit increased anxiety-related behaviors in the presence, but not the absence, of light. In females, estradiol potentially mitigates these effects. Experiments across behavioral paradigms and brain regions indicate that estradiol action can be mediated via activation of metabotropic glutamate receptors, including Group I subtype five (mGlu5). mGlu5 has been implicated in mediating estradiol's effects upon psychostimulant-induced behaviors, dopamine release and neuron phenotype in striatal regions. Whether estradiol activation of mGlu5 modulates anxiety or locomotor behavior in the absence of psychostimulants is unknown. Here we test if mGlu5 is necessary for estradiol mitigation of light-induced acute anxiety and locomotor behaviors. Ovariectomized adult female rats were pre-treated with either the mGlu5 antagonist MPEP or saline before estradiol or oil treatment. Anxiety and locomotor behaviors were assessed in the presence or absence of white light to induce high and low acute anxiety behavior phenotypes, respectively. In the presence of white light, estradiol treatment mitigated light-induced anxiety-related behaviors but not overall locomotor activity. MPEP treatment blocked estradiol effects upon light-induced anxiety-related behaviors but did not affect overall locomotor activity. In the absence of white light, estradiol or MPEP treatment did not influence anxiety-related behaviors or locomotor activity, consistent with a low anxiety phenotype. These novel findings indicate that mGlu5 activation is necessary for estradiol mitigation of anxiety-related behaviors induced by an acute stressor.

Estradiol rapidly modulates excitatory synapse properties in a sex- and region-specific manner in rat nucleus accumbens core and caudate-putamen.

Estradiol acutely facilitates sex differences in striatum-dependent behaviors. However, little is understood regarding the underlying mechanism. In striatal regions in adult rodents, estrogen receptors feature exclusively extranuclear expression, suggesting that estradiol rapidly modulates striatal neurons. We tested the hypothesis that estradiol rapidly modulates excitatory synapse properties onto medium spiny neurons (MSNs) of two striatal regions, the nucleus accumbens core and caudate-putamen in adult female and male rats. We predicted there would be sex-specific differences in pre- and postsynaptic locus and sensitivity. We further analyzed whether MSN intrinsic properties are predictive of estrogen sensitivity. Estradiol exhibited sex-specific acute effects in the nucleus accumbens core: miniature excitatory postsynaptic current (mEPSC) frequency robustly decreased in response to estradiol in female MSNs, and mEPSC amplitude moderately increased in response to estradiol in both male and female MSNs. This increase in mEPSC amplitude is associated with MSNs featuring increased intrinsic excitability. No MSN intrinsic electrical property associated with changes in mEPSC frequency. Estradiol did not acutely modulate mEPSC properties in the caudate-putamen of either sex. This is the first demonstration of acute estradiol action on MSN excitatory synapse function. This demonstration of sex and striatal region-specific acute estradiol neuromodulation revises our understanding of sex hormone action on striatal physiology and resulting behaviors.NEW & NOTEWORTHY This study is the first to demonstrate rapid estradiol neuromodulation of glutamatergic signaling on medium spiny neurons (MSNs), the major output neuron of the striatum. These findings emphasize that sex is a significant biological variable both in MSN sensitivity to estradiol and in pre- and postsynaptic mechanisms of glutamatergic signaling. MSNs in different regions exhibit diverse responses to estradiol. Sex- and region-specific estradiol-induced changes to excitatory signaling on MSNs explain sex differences partially underlying striatum-mediated behaviors and diseases.

A Membrane G-Protein-Coupled Estrogen Receptor Is Necessary but Not Sufficient for Sex Differences in Zebra Finch Auditory Coding.

Estradiol acts as a neuromodulator in brain regions important for cognition and sensory processing. Estradiol also shapes brain sex differences but rarely have these concepts been considered simultaneously. In male and female songbirds, estradiol rapidly increases within the auditory forebrain during song exposure and enhances local auditory processing. We tested whether G-protein-coupled estrogen receptor 1 (GPER1), a membrane-bound estrogen receptor, is necessary and sufficient for neuroestrogen regulation of forebrain auditory processing in male and female zebra finches (Taeniopygia guttata). At baseline, we observed that females had elevated single-neuron responses to songs vs males. In males, narrow-spiking (NS) neurons were more responsive to conspecific songs than broad-spiking (BS) neurons, yet cell types were similarly auditory responsive in females. Following acute inactivation of GPER1, auditory responsiveness and coding were suppressed in male NS yet unchanged in female NS and in BS of both sexes. By contrast, GPER1 activation did not mimic previously established estradiol actions in either sex. Lastly, the expression of GPER1 and its coexpression with an inhibitory neuron marker were similarly abundant in males and females, confirming anatomical similarity in the auditory forebrain. In this study, we found: (1) a role for GPER1 in regulating sensory processing and (2) a sex difference in auditory processing of complex vocalizations in a cell type-specific manner. These results reveal sex specificity of a rapid estrogen signaling mechanism in which neuromodulation accounts and/or compensates for brain sex differences, dependent on cell type, in brain regions that are anatomically similar in both sexes.

Local Estrogen Synthesis Regulates Parallel Fiber-Purkinje Cell Neurotransmission Within the Cerebellar Cortex.

Estrogens affect cerebellar activity and cerebellum-based behaviors. Within the adult rodent cerebellum, the best-characterized action of estradiol is to enhance glutamatergic signaling. However, the mechanisms by which estradiol promotes glutamatergic neurotransmission remain unknown. Within the mouse cerebellum, we found that estrogen receptor activation of metabotropic glutamate receptor type 1a strongly enhances neurotransmission at the parallel fiber-Purkinje cell synapse. The blockade of local estrogen synthesis within the cerebellum results in a diminution of glutamatergic neurotransmission. Correspondingly, decreased estrogen availability via gonadectomy or blockade of aromatase activity negatively affects locomotor performance. These data indicate that locally derived, and not just gonad-derived, estrogens affect cerebellar physiology and function. In addition, estrogens were found to facilitate parallel fiber-Purkinje cell synaptic transmission in both sexes. As such, the actions of estradiol to support cerebellar neurotransmission and cerebellum-based behaviors might be fundamental to understanding the normal processing of activity within the cerebellar cortex.

Biological Sex, Estradiol and Striatal Medium Spiny Neuron Physiology: A Mini-Review.

The caudate-putamen, nucleus accumbens core and shell are important striatal brain regions for premotor, limbic, habit formation, reward, and other critical cognitive functions. Striatal-relevant behaviors such as anxiety, motor coordination, locomotion, and sensitivity to reward, all change with fluctuations of the menstrual cycle in humans and the estrous cycle in rodents. These fluctuations implicate sex steroid hormones, such as 17ß-estradiol, as potent neuromodulatory signals for striatal neuron activity. The medium spiny neuron (MSN), the primary neuron subtype of the striatal regions, expresses membrane estrogen receptors and exhibits sex differences both in intrinsic and synaptic electrophysiological properties. In this mini-review, we first describe sex differences in the electrophysiological properties of the MSNs in prepubertal rats. We then discuss specific examples of how the human menstrual and rat estrous cycles induce differences in striatal-relevant behaviors and neural substrate, including how female rat MSN electrophysiology is influenced by the estrous cycle. We then conclude the mini-review by discussing avenues for future investigation, including possible roles of striatal-localized membrane estrogen receptors and estradiol.

Clustered organization and region-specific identities of estrogen-producing neurons in the forebrain of Zebra Finches (Taeniopygia guttata).

A fast, neuromodulatory role for estrogen signaling has been reported in many regions of the vertebrate brain. Regional differences in the cellular distribution of aromatase (estrogen synthase) in several species suggest that mechanisms for neuroestrogen signaling differ between and even within brain regions. A more comprehensive understanding of neuroestrogen signaling depends on characterizing the cellular identities of neurons that express aromatase. Calcium-binding proteins such as parvalbumin and calbindin are molecular markers for interneuron subtypes, and are co-expressed with aromatase in human temporal cortex. Songbirds like the zebra finch have become important models to understand the brain synthesis of steroids like estrogens and the implications for neurobiology and behavior. Here, we investigated the regional differences in cytoarchitecture and cellular identities of aromatase-expressing neurons in the auditory and sensorimotor forebrain of zebra finches. Aromatase was co-expressed with parvalbumin in the caudomedial nidopallium (NCM) and HVC shelf (proper name) but not in the caudolateral nidopallium (NCL) or hippocampus. By contrast, calbindin was not co-expressed with aromatase in any region investigated. Notably, aromatase-expressing neurons were found in dense somato-somatic clusters, suggesting a coordinated release of local neuroestrogens from clustered neurons. Aromatase clusters were also more abundant and tightly packed in the NCM of males as compared to females. Overall, this study provides new insights into neuroestrogen regulation at the network level, and extends previous findings from human cortex by identifying a subset of aromatase neurons as putative inhibitory interneurons.

Inhibition of local estrogen synthesis in the hippocampus impairs hippocampal memory consolidation in ovariectomized female mice.

The potent estrogen 17ß-Estradiol (E2) plays a critical role in mediating hippocampal function, yet the precise mechanisms through which E2 enhances hippocampal memory remain unclear. In young adult female rodents, the beneficial effects of E2 on memory are generally attributed to ovarian-synthesized E2. However, E2 is also synthesized in the adult brain in numerous species, where it regulates synaptic plasticity and is synthesized in response to experiences such as exposure to females or conspecific song. Although de novo E2 synthesis has been demonstrated in rodent hippocampal cultures, little is known about the functional role of local E2 synthesis in mediating hippocampal memory function. Therefore, the present study examined the role of hippocampal E2 synthesis in hippocampal memory consolidation. Using bilateral dorsal hippocampal infusions of the aromatase inhibitor letrozole, we first found that blockade of dorsal hippocampal E2 synthesis impaired hippocampal memory consolidation. We next found that elevated levels of E2 in the dorsal hippocampus observed 30min after object training were blocked by dorsal hippocampal infusion of letrozole, suggesting that behavioral experience increases acute and local E2 synthesis. Finally, aromatase inhibition did not prevent exogenous E2 from enhancing hippocampal memory consolidation, indicating that hippocampal E2 synthesis is not necessary for exogenous E2 to enhance hippocampal memory. Combined, these data are consistent with the hypothesis that hippocampally-synthesized E2 is necessary for hippocampus-dependent memory consolidation in rodents.

Sex differences and rapid estrogen signaling: A look at songbird audition.

The actions of estrogens have been associated with brain differentiation and sexual dimorphism in a wide range of vertebrates. Here we consider the actions of brain-derived 'neuroestrogens' in the forebrain and the accompanying differences and similarities observed between males and females in a variety of species. We summarize recent evidence showing that baseline and fluctuating levels of neuroestrogens within the auditory forebrain of male and female zebra finches are largely similar, and that neuroestrogens enhance auditory representations in both sexes. With a comparative perspective we review evidence that non-genomic mechanisms of neuroestrogen actions are sexually differentiated, and we propose a working model for nonclassical estrogen signaling via the MAPK intracellular signaling cascade in the songbird auditory forebrain that is informed by the way sex differences may be compensated. This view may lead to a more comprehensive understanding of how sex influences estradiol-dependent modulation of sensorimotor representations.