Oestrogen engages brain MC4R signalling to drive physical activity in female mice.

Oestrogen depletion in rodents and humans leads to inactivity, fat accumulation and diabetes1,2, underscoring the conserved metabolic benefits of oestrogen that inevitably decrease with age. In rodents, the preovulatory surge in 17ß-oestradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we report that a subset of oestrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)3-7 projects to arousal centres in the hippocampus and hindbrain, and enables oestrogen to rebalance energy allocation in female mice. Surges in E2 increase melanocortin-4 receptor (MC4R) signalling in these VMHvl neurons by directly recruiting oestrogen receptor-α (ERα) to the Mc4r gene. Sedentary behaviour and obesity in oestrogen-depleted female mice were reversed after chemogenetic stimulation of VMHvl neurons expressing both MC4R and ERα. Similarly, a long-term increase in physical activity is observed after CRISPR-mediated activation of this node. These data extend the effect of MC4R signalling - the most common cause of monogenic human obesity8 - beyond the regulation of food intake and rationalize reported sex differences in melanocortin signalling, including greater disease severity of MC4R insufficiency in women9. This hormone-dependent node illuminates the power of oestrogen during the reproductive cycle in motivating behaviour and maintaining an active lifestyle in women.

Copolymerization of single-cell nucleic acids into balls of acrylamide gel.

We show the use of 5'-Acrydite oligonucleotides to copolymerize single-cell DNA or RNA into balls of acrylamide gel (BAGs). Combining this step with split-and-pool techniques for creating barcodes yields a method with advantages in cost and scalability, depth of coverage, ease of operation, minimal cross-contamination, and efficient use of samples. We perform DNA copy number profiling on mixtures of cell lines, nuclei from frozen prostate tumors, and biopsy washes. As applied to RNA, the method has high capture efficiency of transcripts and sufficient consistency to clearly distinguish the expression patterns of cell lines and individual nuclei from neurons dissected from the mouse brain. By using varietal tags (UMIs) to achieve sequence error correction, we show extremely low levels of cross-contamination by tracking source-specific SNVs. The method is readily modifiable, and we will discuss its adaptability and diverse applications.

Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis.

Oxytocin is a neuropeptide that can produce anxiolytic effects and promote social approach. However, emerging evidence shows that under some conditions, oxytocin can instead induce anxiety-related behaviors. These diverse effects of oxytocin appear to be mediated by circuit-specific actions. Recent data showed that inhibition of oxytocin receptors (OTRs) in the bed nucleus of the stria terminalis (BNST) was sufficient to increase social approach and decrease social vigilance in female California mice (Peromyscus californicus) exposed to social defeat stress. As a member of the G-protein coupled receptor family, OTRs can induce distinct downstream pathways by coupling to different G-protein isoforms. We show that infusion of carbetocin, a biased OTR-Gq agonist, in the BNST reduced social approach in both female and male California mice. In both females and males, carbetocin also increased social vigilance. To gain insight into cell types that could be mediating this effect, we analyzed previously published single-cell RNAseq data from the BNST and nucleus accumbens (NAc). In the NAc, we and others showed that OTR activation promotes social approach behaviors. In the BNST, Oxtr was expressed in over 40 cell types, that span both posterior and anterior subregions of the BNST. The majority of Oxtr-expressing neurons were GABAergic. In the anterior regions of BNST targeted in our carbetocin experiments, Cyp26b1-expressing neurons had high average Oxtr expression. In the NAc, most Oxtr+ cells were D1 dopamine receptor-expressing neurons and interneurons. These differences in Oxtr cell type distribution may help explain how activation of OTR in BNST versus NAc can have different effects on social approach and social vigilance.

Estrogen Receptor Chromatin Profiling by CUT&RUN.

Gonadal steroid hormones, namely, testosterone, progesterone, and estrogens, influence the physiological state of an organism through the regulation of gene transcription. Steroid hormones activate nuclear hormone receptor (HR), transcription factors (TFs), which bind DNA in a tissue- and cell type-specific manner to influence cellular function. Identifying the genomic binding sites of HRs is essential to understanding mechanisms of hormone signaling across tissues and disease contexts. Traditionally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been used to map the genomic binding of HRs in cancer cell lines and large tissues. However, ChIP-seq lacks the sensitivity to detect TF binding in small numbers of cells, such as genetically defined neuronal subtypes in the brain. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) resolves most of the technical limitations of ChIP-seq, enabling the detection of protein-DNA interactions with as few as 100-1000 cells. In this chapter, we provide a stepwise CUT&RUN protocol for detecting and analyzing the genome-wide binding of estrogen receptor α (ERα) in mouse brain tissue. The steps described here can be used to identify the genomic binding sites of most TFs in the brain.

Area postrema neurons mediate interleukin-6 function in cancer cachexia.

Interleukin-6 (IL-6) has been long considered a key player in cancer cachexia. It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia. However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, is a critical mediator of IL-6 function in cancer cachexia in male mice. We find that circulating IL-6 can rapidly enter the AP and activate neurons in the AP and its associated network. Peripheral tumor, known to increase circulating IL-6, leads to elevated IL-6 in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons and AP network hyperactivity. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an anti-IL-6 antibody attenuates cachexia and the hyperactivity in the AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra, the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing Gfral-expressing AP neurons also attenuates cancer cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer cachexia.

T cell-Mediated Development of Stromal Fibroblasts with an Immune-Enhancing Chemokine Profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAF) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA sequencing of CAFs from mouse pancreatic adenocarcinomas identified a subpopulation of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune-suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

T cell-mediated development of stromal fibroblasts with an immune-enhancing chemokine profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAFs) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA-sequencing of CAFs from mouse pancreatic adenocarcinomas identified a sub-population of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

Area postrema neurons mediate interleukin-6 function in cancer-associated cachexia.

Interleukin-6 (IL-6) has been long considered a key player in cancer-associated cachexia 1-15 . It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia 16-20 . However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, mediate the function of IL-6 in cancer-associated cachexia in mice. We found that circulating IL-6 can rapidly enter the AP and activate AP neurons. Peripheral tumor, known to increase circulating IL-6 1-5,15,18,21-23 , leads to elevated IL-6 and neuronal hyperactivity in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an IL-6 antibody prevents cachexia, reduces the hyperactivity in an AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra , the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing of Gfral-expressing AP neurons also ameliorates the cancer-associated cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer-associated cachexia.

Epigenetic Mechanisms of Brain Sexual Differentiation.

Across vertebrate species, gonadal hormones coordinate physiology with behavior to facilitate social interactions essential for reproduction and survival. In adulthood, these hormones activate neural circuits that regulate behaviors presenting differently in females and males, such as parenting and territorial aggression. Yet long before sex-typical behaviors emerge at puberty, transient hormone production during sensitive periods of neurodevelopment establish the circuits upon which adult hormones act. How transitory waves of early-life hormone signaling exert lasting effects on the brain remains a central question. Here we discuss how perinatal estradiol signaling organizes cellular and molecular sex differences in the rodent brain. We review classic anatomic studies revealing sex differences in cell number, volume, and neuronal projections, and consider how single-cell sequencing methods enable distinction between sex-biased cell-type abundance and gene expression. Finally, we highlight the recent discovery of a gene regulatory program activated by estrogen receptor α (ERα) following the perinatal hormone surge. A subset of this program displays sustained sex-biased gene expression and chromatin accessibility throughout the postnatal sensitive period, demonstrating a bona fide epigenetic mechanism. We propose that ERα-expressing neurons throughout the social behavior network use similar gene regulatory programs to coordinate brain sexual differentiation.

Signatures of sex: Sex differences in gene expression in the vertebrate brain.

Women and men differ in disease prevalence, symptoms, and progression rates for many psychiatric and neurological disorders. As more preclinical studies include both sexes in experimental design, an increasing number of sex differences in physiology and behavior have been reported. In the brain, sex-typical behaviors are thought to result from sex-specific patterns of neural activity in response to the same sensory stimulus or context. These differential firing patterns likely arise as a consequence of underlying anatomic or molecular sex differences. Accordingly, gene expression in the brains of females and males has been extensively investigated, with the goal of identifying biological pathways that specify or modulate sex differences in brain function. However, there is surprisingly little consensus on sex-biased genes across studies and only a handful of robust candidates have been pursued in the follow-up experiments. Furthermore, it is not known how or when sex-biased gene expression originates, as few studies have been performed in the developing brain. Here we integrate molecular genetic and neural circuit perspectives to provide a conceptual framework of how sex differences in gene expression can arise in the brain. We detail mechanisms of gene regulation by steroid hormones, highlight landmark studies in rodents and humans, identify emerging themes, and offer recommendations for future research. This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Sex Determination.

Sex Differences in the Epigenome: A Cause or Consequence of Sexual Differentiation of the Brain?

Females and males display differences in neural activity patterns, behavioral responses, and incidence of psychiatric and neurological diseases. Sex differences in the brain appear throughout the animal kingdom and are largely a consequence of the physiological requirements necessary for the distinct roles of the two sexes in reproduction. As with the rest of the body, gonadal steroid hormones act to specify and regulate many of these differences. It is thought that transient hormonal signaling during brain development gives rise to persistent sex differences in gene expression via an epigenetic mechanism, leading to divergent neurodevelopmental trajectories that may underlie sex differences in disease susceptibility. However, few genes with a persistent sex difference in expression have been identified, and only a handful of studies have employed genome-wide approaches to assess sex differences in epigenomic modifications. To date, there are no confirmed examples of gene regulatory elements that direct sex differences in gene expression in the brain. Here, we review foundational studies in this field, describe transcriptional mechanisms that could act downstream of hormone receptors in the brain, and suggest future approaches for identification and validation of sex-typical gene programs. We propose that sexual differentiation of the brain involves self-perpetuating transcriptional states that canalize sex-specific development.

OPRM1 A118G and serum ß-endorphin interact with sex and digit ratio (2D:4D) to influence risk and course of alcohol dependence.

Activation of mesolimbic mu-opioid receptors by their endogenous ligand, ß-endorphin, can mediate the rewarding effects of alcohol. However, there is conflicting evidence on the relationship between the mu-opioid receptor (OPRM1) A118G single nucleotide polymorphism (SNP) and alcohol dependence risk. Preclinical evidence suggests that sex and sex hormone-dependent prenatal brain organization may interact with the opioid system to influence alcohol drinking behavior. We genotyped 200 alcohol-dependent patients and 240 healthy individuals for the OPRM1 A118G SNP and measured serum ß-endorphin level at recruitment and after acute withdrawal. We then determined the association between these factors and alcohol dependence risk and 24-month outcome in the context of both sex and second-to-fourth digit lengths ratio (2D:4D) - a biomarker of prenatal sex hormone levels. The OPRM1 A118G AA genotype associated with elevated risk of alcohol-related hospital readmission, more readmissions, and fewer days until first readmission in male patients only. After normalizing patient 2D:4D against control 2D:4D, we found that normalized 2D:4D ratios were lower in male 118G patients than male AA patients, suggesting prenatal androgens interact with OPRM1 to influence alcohol dependence risk. In addition, ß-endorphin levels after acute withdrawal correlated negatively with withdrawal severity in females but not in males, which may indicate ß-endorphin protects against withdrawal-induced stress in a sex-specific manner.