Harmonic resonance and entrainment of propagating chemical waves by external mechanical stimulation in BZ self-oscillating hydrogels.

Smart polymer materials that are nonliving yet exhibit complex "life-like" or biomimetic behaviors have been the focus of intensive research over the past decades, in the quest to broaden our understanding of how living systems function under nonequilibrium conditions. Identification of how chemical and mechanical coupling can generate resonance and entrainment with other cells or external environment is an important research question. We prepared Belousov-Zhabotinsky (BZ) self-oscillating hydrogels which convert chemical energy to mechanical oscillation. By cyclically applying external mechanical stimulation to the BZ hydrogels, we found that when the oscillation of a gel sample entered into harmonic resonance with the applied oscillation during stimulation, the system kept a "memory" of the resonant oscillation period and maintained it post stimulation, demonstrating an entrainment effect. More surprisingly, by systematically varying the cycle length of the external stimulation, we revealed the discrete nature of the stimulation-induced resonance and entrainment behaviors in chemical oscillations of BZ hydrogels, i.e., the hydrogels slow down their oscillation periods to the harmonics of the cycle length of the external mechanical stimulation. Our theoretical model calculations suggest the important roles of the delayed mechanical response caused by reactant diffusion and solvent migration in affecting the chemomechanical coupling in active hydrogels and consequently synchronizing their chemical oscillations with external mechanical oscillations.

Is the membrane estrogen receptor, GPER1, a promiscuous receptor that modulates nuclear estrogen receptor-mediated functions in the brain?

Contribution to Special Issue on Fast effects of steroids. Estrogen signals both slowly to regulate transcription and rapidly to activate kinases and regulate calcium levels. Both rapid, non-genomic signaling as well as genomic transcriptional signaling via intracellular estrogen receptors (ER)s can change behavior. Rapid non-genomic signaling is initiated from the plasma membrane by a G-protein coupled receptor called GPER1 that binds 17ß-estradiol. GPER1 or GPR30 is one of the candidates for a membrane ER (mER) that is not only highly expressed in pathology i.e. cancers but also in several behaviorally-relevant brain regions. In the brain, GPER1 signaling, in response to estrogen, facilitates neuroprotection, social behaviors and cognition. In this review, we describe several notable characteristics of GPER1 such as the ability of several endogenous steroids as well as artificially synthesized molecules to bind the GPER1. In addition, GPER1 is localized to the plasma membrane in breast cancer cell lines but may be present in the endoplasmic reticulum or the Golgi apparatus in the hippocampus. Unusually, GPER1 can also translocate to the perinuclear space from the plasma membrane. We explore the idea that subcellular localization and ligand promiscuity may determine the varied downstream signaling cascades of the activated GPER1. Lastly, we suggest that GPER1 can act as a modulator of ERα-mediated action on a convergent target, spinogenesis, in neurons that in turn drives female social behaviors such as lordosis and social learning.

Thyroid hormones regulate anxiety in the male mouse.

Thyroid hormone levels are implicated in mood disorders in the adult human but the mechanisms remain unclear partly because, in rodent models, more attention has been paid to the consequences of perinatal hypo and hyperthyroidism. Thyroid hormones act via the thyroid hormone receptor (TR) α and ß isoforms, both of which are expressed in the limbic system. TR's modulate gene expression via both unliganded and liganded actions. Though the thyroid hormone receptor (TR) knockouts and a transgenic TRα1 knock-in mouse have provided us valuable insight into behavioral phenotypes such as anxiety and depression, it is not clear if this is because of the loss of unliganded actions or liganded actions of the receptor or due to locomotor deficits. We used a hypothyroid mouse model and supplementation with tri-iodothyronine (T3) or thyroxine (T4) to investigate the consequences of dysthyroid hormone levels on behaviors that denote anxiety. Our data from the open field and the light-dark transition tests suggest that adult onset hypothyroidism in male mice produces a mild anxiogenic effect that is possibly due to unliganded receptor actions. T3 or T4 supplementation reverses this phenotype and euthyroid animals show anxiety that is intermediate between the hypothyroid and thyroid hormone supplemented groups. In addition, T3 but not T4 supplemented animals have lower spine density in the CA1 region of the hippocampus and in the central amygdala suggesting that T3-mediated rescue of the hypothyroid state might be due to lower neuronal excitability in the limbic circuit.

Activation of the GPR30 receptor promotes lordosis in female mice.

BACKGROUND/AIMS: Estrogens are important effectors of reproduction and are critical for upregulating female reproductive behavior or lordosis in females. In addition to the importance of transcriptional regulation of genes by 17ß-estradiol-bound estrogen receptors (ER), extranuclear signal transduction cascades such as protein kinase A (PKA) are also important in regulating female sexual receptivity. GPR30 (G-protein coupled receptor 30), also known as GPER1, a putative membrane ER (mER), is a G protein-coupled receptor that binds 17ß-estradiol with an affinity that is similar to that possessed by the classical nuclear ER and activates both PKA and extracellular-regulated kinase signaling pathways. The high expression of GPR30 in the ventromedial hypothalamus, a region important for lordosis behavior as well as kinase cascades activated by this receptor, led us to hypothesize that GPR30 may regulate lordosis behavior in female rodents. METHOD: In this study, we investigated the ability of G-1, a selective agonist of GPR30, to regulate lordosis in the female mouse by administering this agent prior to progesterone in an estradiol-progesterone priming paradigm prior to testing with stud males. RESULTS: As expected, 17ß-estradiol benzoate (EB), but not sesame oil, increased lordosis behavior in female mice. G-1 also increased lordosis behavior in female mice and decreased the number of rejective responses towards male mice, similar to the effect of EB. The selective GPR30 antagonist G-15 blocked these effects. CONCLUSION: This study demonstrates that activation of the mER GPR30 stimulates social behavior in a rodent model in a manner similar to EB.

Estrogen and testosterone secretion from the mouse brain.

Estrogen and testosterone are typically thought of as gonadal or adrenal derived steroids that cross the blood brain barrier to signal via both rapid nongenomic and slower genomic signalling pathways. Estrogen and testosterone signalling has been shown to drive interlinked behaviours such as social behaviours and cognition by binding to their cognate receptors in hypothalamic and forebrain nuclei. So far, acute brain slices have been used to study short-term actions of 17ß-estradiol, typically using electrophysiological measures. For example, these techniques have been used to investigate, nongenomic signalling by estrogen such as the estrogen modulation of long-term potentiation (LTP) in the hippocampus. Using a modified method that preserves the slice architecture, we show, for the first time, that acute coronal slices from the prefrontal cortex and from the hypothalamus maintained in aCSF over longer periods i.e. 24 h can be steroidogenic, increasing their secretion of testosterone and estrogen. We also show that the hypothalamic nuclei produce more estrogen and testosterone than the prefrontal cortex. Therefore, this extended acute slice system can be used to study the regulation of steroid production and secretion by discrete nuclei in the brain.

Distinct behavioral phenotypes in male mice lacking the thyroid hormone receptor α1 or ß isoforms.

Thyroid hormones influence both neuronal development and anxiety via the thyroid hormone receptors (TRs). The TRs are encoded by two different genes, TRα and TRß. The loss of TRα1 is implicated in increased anxiety in males, possibly via a hippocampal increase in GABAergic activity. We compared both social behaviors and two underlying and related non-social behaviors, state anxiety and responses to acoustic and tactile startle in the gonadally intact TRα1 knockout (α1KO) and TRß (ßKO) male mice to their wild-type counterparts. For the first time, we show an opposing effect of the two TR isoforms, TRα1 and TRß, in the regulation of state anxiety, with α1 knockout animals (α1KO) showing higher levels of anxiety and ßKO males showing less anxiety compared to respective wild-type mice. At odds with the increased anxiety in non-social environments, α1KO males also show lower levels of responsiveness to acoustic and tactile startle stimuli. Consistent with the data that T4 is inhibitory to lordosis in female mice, we show subtly increased sex behavior in α1KO male mice. These behaviors support the idea that TRα1 could be inhibitory to ERα driven transcription that ultimately impacts ERα driven behaviors such as lordosis. The behavioral phenotypes point to novel roles for the TRs, particularly in non-social behaviors such as state anxiety and startle.

Localisation of oestrogen receptors in stem cells and in stem cell-derived neurons of the mouse.

Oestrogen receptors (ER) transduce the effects of the endogenous ligand, 17ß-estradiol in cells to regulate a number of important processes such as reproduction, neuroprotection, learning and memory and anxiety. The ERα or ERß are classical intracellular nuclear hormone receptors while some of their variants or novel proteins such as the G-protein coupled receptor (GPCR), GPER1/GPR30 are reported to localise in intracellular as well as plasma membrane locations. Although the brain is an important target for oestrogen with oestrogen receptors expressed differentially in various nuclei, subcellular organisation and crosstalk between these receptors is under-explored. Using an adapted protocol that is rapid, we first generated neurons from mouse embryonic stem cells. Our immunocytochemistry approach shows that the full length ERα (ERα-66) and for the first time, that an ERα variant, ERα-36, as well as GPER1 is present in embryonic stem cells. In addition, these receptors typically decrease their nuclear localisation as neuronal maturation proceeds. Finally, although these ERs are present in many subcellular compartments such as the nucleus and plasma membrane, we show that they are specifically not colocalised with each other, suggesting that they initiate distinct signalling pathways.

Steroid hormone-mediated regulation of sexual and aggressive behaviour by non-genomic signalling.

Sex and aggression are well studied examples of social behaviours that are common to most animals and are mediated by an evolutionary conserved group of interconnected nuclei in the brain called the social behaviour network. Though glucocorticoids and in particular estrogen regulate these social behaviours, their effects in the brain are generally thought to be mediated by genomic signalling, a slow transcriptional regulation mediated by nuclear hormone receptors. In the last decade or so, there has been renewed interest in understanding the physiological significance of rapid, non-genomic signalling mediated by steroids. Though the identity of the membrane hormone receptors that mediate this signalling is not clearly understood and appears to be different in different cell types, such signalling contributes to physiologically relevant behaviours such as sex and aggression. In this short review, we summarise the evidence for this phenomenon in the rodent, by focusing on estrogen and to some extent, glucocorticoid signalling. The use of these signals, in relation to genomic signalling is manifold and ranges from potentiation of transcription to the possible transduction of environmental signals.

mTOR-Dependent Spine Dynamics in Autism.

Autism Spectrum Conditions (ASC) are a group of neurodevelopmental disorders characterized by deficits in social communication and interaction as well as repetitive behaviors and restricted range of interests. ASC are complex genetic disorders with moderate to high heritability, and associated with atypical patterns of neural connectivity. Many of the genes implicated in ASC are involved in dendritic spine pruning and spine development, both of which can be mediated by the mammalian target of rapamycin (mTOR) signaling pathway. Consistent with this idea, human postmortem studies have shown increased spine density in ASC compared to controls suggesting that the balance between autophagy and spinogenesis is altered in ASC. However, murine models of ASC have shown inconsistent results for spine morphology, which may underlie functional connectivity. This review seeks to establish the relevance of changes in dendritic spines in ASC using data gathered from rodent models. Using a literature survey, we identify 20 genes that are linked to dendritic spine pruning or development in rodents that are also strongly implicated in ASC in humans. Furthermore, we show that all 20 genes are linked to the mTOR pathway and propose that the mTOR pathway regulating spine dynamics is a potential mechanism underlying the ASC signaling pathway in ASC. We show here that the direction of change in spine density was mostly correlated to the upstream positive or negative regulation of the mTOR pathway and most rodent models of mutant mTOR regulators show increases in immature spines, based on morphological analyses. We further explore the idea that these mutations in these genes result in aberrant social behavior in rodent models that is due to these altered spine dynamics. This review should therefore pave the way for further research on the specific genes outlined, their effect on spine morphology or density with an emphasis on understanding the functional role of these changes in ASC.

Immunoblot Detection of the Phosphorylation of the Estrogen Receptor α as an Outcome of GPR30 /GPER1 Activation.

Phosphorylation of the serine residues in estrogen receptor (ER) α is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ERα by electrophoretic separation of proteins and subsequent immunoblotting techniques. In particular, phosphorylation of the ERα is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30 or GPER1. Hence, phosphorylation represents a crosstalk event between GPR30 and ERα and may be important in estrogen-regulated physiology.

Microplastics and Their Impact on Reproduction-Can we Learn From the C. elegans Model?

Biologically active environmental pollutants have significant impact on ecosystems, wildlife, and human health. Microplastic (MP) and nanoplastic (NP) particles are pollutants that are present in the terrestrial and aquatic ecosystems at virtually every level of the food chain. Moreover, recently, airborne microplastic particles have been shown to reach and potentially damage respiratory systems. Microplastics and nanoplastics have been shown to cause increased oxidative stress, inflammation, altered metabolism leading to cellular damage, which ultimately affects tissue and organismal homeostasis in numerous animal species and human cells. However, the full impact of these plastic particles on living organisms is not completely understood. The ability of MPs/NPs to carry contaminants, toxic chemicals, pesticides, and bioactive compounds, such as endocrine disrupting chemicals, present an additional risk to animal and human health. This review will discusses the current knowledge on pathways by which microplastic and nanoplastic particles impact reproduction and reproductive behaviors from the level of the whole organism down to plastics-induced cellular defects, while also identifying gaps in current knowledge regarding mechanisms of action. Furthermore, we suggest that the nematode Caenorhabditis elegans provides an advantageous high-throughput model system for determining the effect of plastic particles on animal reproduction, using reproductive behavioral end points and cellular readouts.

Effect of Light-Emitting Diode Phototherapy on Serum Calcium Levels in Neonates With Jaundice.

BACKGROUND: To assess the change in serum total calcium levels during light-emitting diode phototherapy treatment for jaundice in term neonates. METHODS: A prospective observational study was done on 104 term neonates with hyperbilirubinemia in a tertiary care center to investigate the effects of phototherapy using a light-emitting diode device. The total serum bilirubin along with total calcium levels was measured at the start and at the end of phototherapy. Additionally, all the newborns enrolled in the study were evaluated for hypocalcaemia-related symptoms such as jitteriness, irritability/excitability, lethargy, and convulsions. RESULTS: A significant lowering of posttreatment total calcium level compared to that of pretreatment level (p<0.001) was found in our study. Hypocalcemia (serum calcium <8 mg/dL in term neonates) was found in 12.5% of the study subjects. CONCLUSIONS: In the treatment of neonatal jaundice, similar to conventional blue and white light phototherapy, light-emitting diode phototherapy also has hypocalcemia as an adverse effect. We recommend monitoring these babies for hypocalcemia during light-emitting diode phototherapy.

Membrane-initiated actions of estrogens in neuroendocrinology: emerging principles.

Hormonal ligands for the nuclear receptor superfamily have at least two interacting mechanisms of action: 1) classical transcriptional regulation of target genes (genomic mechanisms); and 2) nongenomic actions that are initiated at the cell membrane, which could impact transcription. Although transcriptional mechanisms are increasingly well understood, membrane-initiated actions of these ligands are incompletely understood. Historically, this has led to a considerable divergence of thought in the molecular endocrine field. We have attempted to uncover principles of hormone action that are relevant to membrane-initiated actions of estrogens. There is evidence that the membrane-limited actions of hormones, particularly estrogens, involve the rapid activation of kinases and the release of calcium. Membrane actions of estrogens, which activate these rapid signaling cascades, can also potentiate nuclear transcription. These signaling cascades may occur in parallel or in series but subsequently converge at the level of modification of transcriptionally relevant molecules such as nuclear receptors and/or coactivators. In addition, other hormones or neurotransmitters may also activate cascades to crosstalk with estrogen receptor-mediated transcription. The idea of synergistic coupling between membrane-initiated and genomic actions of hormones fundamentally revises the paradigms of cell signaling in neuroendocrinology.

Delayed Mechanical Response to Chemical Kinetics in Self-Oscillating Hydrogels Driven by the Belousov-Zhabotinsky Reaction.

We show experimentally that chemical and mechanical self-oscillations in Belousov-Zhabotinsky hydrogels are inherently asynchronous, that is, there is a detectable delay in swelling-deswelling response after a change in the chemical redox state. This phenomenon is observable in many previous experimental studies and potentially has far-reaching implications for the functionality and response time of the material in future applications; however, so far, it has not been quantified or reported systematically. Here, we provide a comprehensive qualitative and quantitative description of the chemical-to-mechanical delay, and we propose to explain it as a consequence of the slow nonequilibrium swelling-deswelling dynamics of the polymer material. Specifically, standard hydrogel pieces are large enough that transport processes, for example, counterion migration and water diffusion, cannot occur instantaneously throughout the entire gel piece, as opposed to previous theoretical considerations. As a result, the volume response of the polymer to a chemical change may be governed by a characteristic response time, which leads to the emergence of delay in mechanical oscillation. This is supported by our theoretical calculations.

Does GPER1 Play a Role in Sexual Dimorphism?

Estrogens are critical in driving sex-typical social behaviours that are ethologically relevant in mammals. This is due to both production of local estrogens and signaling by these ligands, particularly in an interconnected set of nuclei called the social behavioural network (SBN). The SBN is a sexually dimorphic network studied predominantly in rodents that is thought to underlie the display of social behaviour in mammals. Signalling by the predominant endogenous estrogen, 17ß-estradiol, can be either via the classical genomic or non-classical rapid pathway. In the classical genomic pathway, 17ß-estradiol binds the intracellular estrogen receptors (ER) α and ß which act as ligand-dependent transcription factors to regulate transcription. In the non-genomic pathway, 17ß-estradiol binds a putative plasma membrane ER (mER) such as GPR30/GPER1 to rapidly signal via kinases or calcium flux. Though GPER1's role in sexual dimorphism has been explored to a greater extent in cardiovascular physiology, less is known about its role in the brain. In the last decade, activation of GPER1 has been shown to be important for lordosis and social cognition in females. In this review we will focus on several mechanisms that may contribute to sexually dimorphic behaviors including the colocalization of these estrogen receptors in the SBN, interplay between the signaling pathways activated by these different estrogen receptors, and the role of these receptors in development and the maintenance of the SBN, all of which remain underexplored.

Estrogenic regulation of social behavior and sexually dimorphic brain formation.

It has long been known that the estrogen, 17ß-estradiol (17ß-E), plays a central role for female reproductive physiology and behavior. Numerous studies have established the neurochemical and molecular basis of estrogenic induction of female sexual behavior, i.e., lordosis, in animal models. In addition, 17ß-E also regulates male-type sexual and aggressive behavior. In males, testosterone secreted from the testes is irreversibly aromatized to 17ß-E in the brain. We discuss the contribution of two nuclear receptor isoforms, estrogen receptor (ER)α and ERß to the estrogenic regulation of sexually dimorphic brain formation and sex-typical expression of these social behaviors. Furthermore, 17ß-E is a key player for social behaviors such as social investigation, preference, recognition and memory as well as anxiety-related behaviors in social contexts. Recent studies also demonstrated that not only nuclear receptor-mediated genomic signaling but also membrane receptor-mediated non-genomic actions of 17ß-E may underlie the regulation of these behaviors. Finally, we will discuss how rapidly developing research tools and ideas allow us to investigate estrogenic action by emphasizing behavioral neural networks.

Non-genomic actions of estrogens and their interaction with genomic actions in the brain.

Ligands for the nuclear receptor superfamily have at least two mechanisms of action: (a) classical transcriptional regulation of target genes (genomic mechanisms); and (b) non-genomic actions, which are initiated at the cell membrane, which could also impact transcription. Though transcriptional mechanisms are increasingly well understood, membrane-initiated actions of these ligands are incompletely understood. This has led to considerable debate over the physiological relevance of membrane-initiated actions of hormones versus genomic actions of hormones, with genomic actions predominating in the endocrine field. There is good evidence that the membrane-limited actions of hormones, particularly estrogens, involve the rapid activation of kinases and the release of calcium and that these are linked to physiologically relevant scenarios in the brain. We show evidence in this review, that membrane actions of estrogens, which activate these rapid signaling cascades, can also potentiate nuclear transcription in both the central nervous system and in non-neuronal cell lines. We present a theoretical scenario which can be used to understand this phenomenon. These signaling cascades may occur in parallel or in series but subsequently, converge at the modification of transcriptionally relevant molecules such as nuclear receptors and/or coactivators. In addition, other non-cognate hormones or neurotransmitters may also activate cascades to crosstalk with estrogen receptor-mediated transcription, though the relevance of this is less clear. The idea that coupling between membrane-initiated and genomic actions of hormones is a novel idea in neuroendocrinology and provides us with a unified view of hormone action in the central nervous system.

Membrane-initiated nuclear trafficking of the glucocorticoid receptor in hypothalamic neurons.

Glucocorticoid binding to the intracellular glucocorticoid receptor (GR) stimulates the translocation of the GR from the cytosol to the nucleus, which leads to the transactivation or transrepression of gene transcription. However, multiple lines of evidence suggest that glucocorticoid signaling can also be initiated from the plasma membrane. Here, we provide evidence for membrane-initiated glucocorticoid signaling by a membrane-impermeant dexamethasone-bovine serum albumin (Dex-BSA) conjugate, which induced GR nuclear trafficking in hypothalamic neurons in vitro and in vivo. The GR nuclear translocation induced by a membrane-impermeant glucocorticoid suggests trafficking of an unliganded GR. The membrane-initiated GR trafficking was not blocked by inhibiting ERK MAPK, p38 MAPK, PKA, Akt, Src kinase, or calcium signaling, but was inhibited by Akt activation. Short-term exposure of hypothalamic neurons to dexamethasone (Dex) activated the glucocorticoid response element (GRE), suggesting transcriptional transactivation, whereas exposure to the Dex-BSA conjugate failed to activate the GRE, suggesting differential transcriptional activity of the liganded compared to the unliganded GR. Microarray analysis revealed divergent transcriptional regulation by Dex-BSA compared to Dex. Together, our data suggest that signaling from a putative membrane glucocorticoid receptor induces the trafficking of unliganded GR to the nucleus, which elicits a pattern of gene transcription that differs from that of the liganded receptor. The differential transcriptional signaling by liganded and unliganded receptors may contribute to the broad range of genetic regulation by glucocorticoids, and may help explain some of the different off-target actions of glucocorticoid drugs.

GPER1/GPR30 in the brain: Crosstalk with classical estrogen receptors and implications for behavior.

The GPER1/GPR30 is a membrane estrogen receptor (mER) that binds 17ß-estradiol (17ß-E) with high affinity and is thought to play a role in cancer progression and cardiovascular health. Though widespread in the central nervous system, less is known about this receptor's function in the brain. GPER1 has been shown to activate kinase cascades and calcium flux within cells rapidly, thus fitting in with the idea of being a mER that mediates non-genomic signaling by estrogens. Signaling from GPER1 has been shown to improve spatial memory, possibly via release of neurotransmitters and generation of new spines on neurons in the hippocampus. In addition, GPER1 activation contributes to behaviors that denote anxiety and to social behaviors such as social memory and lordosis behavior in mice. In the male hippocampus, GPER1 activation has also been shown to phosphorylate the classical intracellular estrogen receptor (ER)α, suggesting that crosstalk with ERα is important in the display of these behaviors, many of which are absent in ERα-null mice. In this review, we present a number of categories of such crosstalk, using examples from literature. The function of GPER1 as an ERα collaborator or as a mER in different tissues is relevant to understanding both normal physiology and abnormal pathology, mediated by estrogen signaling.

Detection of the Phosphorylation of the Estrogen Receptor α as an Outcome of GPR30 Activation.

Phosphorylation of the serine residues in estrogen receptor (ER) α is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ERα by electrophoretic separation of proteins and subsequent immuno-blotting techniques. In particular, phosphorylation of the ERα is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30. Hence, phosphorylation represents a cross talk event between GPR30 and ERα and may be important in estrogen-regulated physiology.