Microglia phagocytosis mediates the volume and function of the rat sexually dimorphic nucleus of the preoptic area.

The sexually dimorphic nucleus of the preoptic area (SDN-POA) is the oldest and most robust sex difference reported in mammalian brain and is singular for its presence across a wide range of species from rodents to ungulates to man. This small collection of Nissl-dense neurons is reliably larger in volume in males. Despite its notoriety and intense interrogation, both the mechanism establishing the sex difference and the functional role of the SDN have remained elusive. Convergent evidence from rodent studies led to the conclusion that testicular androgens aromatized to estrogens are neuroprotective in males and that higher apoptosis (naturally occurring cell death) in females determines their smaller SDN. In several species, including humans, a smaller SDN correlates with a preference for mating with males. We report here that this volume difference is dependent upon a participatory role of phagocytic microglia which engulf more neurons in the female SDN and assure their destruction. Selectively blocking microglia phagocytosis temporarily spared neurons from apoptotic death and increased SDN volume in females without hormone treatment. Increasing the number of neurons in the SDN in neonatal females resulted in loss of preference for male odors in adulthood, an effect paralleled by dampened excitation of SDN neurons as evidenced by reduced immediate early gene (IEG) expression when exposed to male urine. Thus, the mechanism establishing a sex difference in SDN volume includes an essential role for microglia, and SDN function as a regulator of sexual partner preference is confirmed.

Maternal-fetal transmission of delta-9-tetrahydrocannabinol (THC) and its metabolites following inhalation and injection exposure during pregnancy in rats.

Cannabis use during pregnancy has increased over the past few decades, with recent data indicating that, in youth and young adults especially, up to 22% of people report using cannabis during pregnancy. Animal models provide the ability to study prenatal cannabis exposure (PCE) with control over timing and dosage; however, these studies utilize both injection and inhalation approaches. While it is known that Δ9-tetrahydrocannabinol (THC; primary psychoactive component of cannabis) can cross the placenta, examination of the transmission and concentration of THC and its metabolites from maternal blood into the placenta and fetal brain remains relatively unknown, and the influence of route of administration has never been examined. Pregnant female rats were exposed to either vaporized THC-dominant cannabis extract for pulmonary consumption or subcutaneous injection of THC repeatedly during the gestational period. Maternal blood, placenta, and fetal brains were collected following the final administration of THC for analysis of THC and its metabolites, as well as endocannabinoid concentrations, through mass spectrometry. Both routes of administration resulted in the transmission of THC and its metabolites in placenta and fetal brain. Repeated exposure to inhaled THC vapor resulted in fetal brain THC concentrations that were about 30% of those seen in maternal blood, whereas repeated injections resulted in roughly equivalent concentrations of THC in maternal blood and fetal brain. Neither inhalation nor injection of THC during pregnancy altered fetal brain endocannabinoid concentrations. Our data provide the first characterization of maternal-fetal transmission of THC and its metabolites following both vaporized delivery and injection routes of administration. These data are important to establish the maternal-fetal transmission in preclinical injection and inhalation models of PCE and may provide insight into predicting fetal exposure in human studies.

Idebenone Has Distinct Effects on Mitochondrial Respiration in Cortical Astrocytes Compared to Cortical Neurons Due to Differential NQO1 Activity.

Idebenone is a synthetic quinone that on reduction in cells can bypass mitochondrial Complex I defects by donating electrons to Complex III. The drug is used clinically to treat the Complex I disease Leber's hereditary optic neuropathy (LHON), but has been less successful in clinical trials for other neurodegenerative diseases. NAD(P)H:quinone oxidoreductase 1 (NQO1) appears to be the main intracellular enzyme catalyzing idebenone reduction. However, NQO1 is not universally expressed by cells of the brain. Using primary rat cortical cells pooled from both sexes, we tested the hypotheses that the level of endogenous NQO1 activity limits the ability of neurons, but not astrocytes, to use idebenone as an electron donor to support mitochondrial respiration. We then tested the prediction that NQO1 induction by pharmacological activation of the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) enables idebenone to bypass Complex I in cells with poor NQO1 expression. We found that idebenone stimulated respiration by astrocytes but reduced the respiratory capacity of neurons. Importantly, idebenone supported mitochondrial oxygen consumption in the presence of a Complex I inhibitor in astrocytes but not neurons, and this ability was reversed by inhibiting NQO1. Conversely, recombinant NQO1 delivery to neurons prevented respiratory impairment and conferred Complex I bypass activity. Nrf2 activators failed to increase NQO1 in neurons, but carnosic acid induced NQO1 in COS-7 cells that expressed little endogenous enzyme. Carnosic acid-idebenone combination treatment promoted NQO1-dependent Complex I bypass activity in these cells. Thus, combination drug strategies targeting NQO1 may promote the repurposing of idebenone for additional disorders.SIGNIFICANCE STATEMENT Idebenone is used clinically to treat loss of visual acuity in Leber's hereditary optic neuropathy. Clinical trials for several additional diseases have failed. This study demonstrates a fundamental difference in the way idebenone affects mitochondrial respiration in cortical neurons compared with cortical astrocytes. Cortical neurons are unable to use idebenone as a direct mitochondrial electron donor due to NQO1 deficiency. Our results suggest that idebenone behaves as an NQO1-dependent prodrug, raising the possibility that lack of neuronal NQO1 activity has contributed to the limited efficacy of idebenone in neurodegenerative disease treatment. Combination therapy with drugs able to safely induce NQO1 in neurons, as well as other brain cell types, may be able to unlock the neuroprotective therapeutic potential of idebenone or related quinones.

Microglia and sexual differentiation of the developing brain: A focus on extrinsic factors.

Microglia, the innate immune cells of the brain, have recently been removed from the position of mere sentinels and promoted to the role of active sculptors of developing circuits and cells. Alongside their functions in normal brain development, microglia coordinate sexual differentiation of the brain, a set of processes which vary by region and endpoint like that of microglia function itself. In this review, we highlight the ways microglia are both targets and drivers of brain sexual differentiation. We examine the factors that may drive sex differences in microglia, with a special focus on how changing microenvironments in the developing brain dictate microglia phenotypes and discuss how their diverse functions sculpt lasting sex-specific changes in the brain. Finally, we consider how sex-specific early life environments contribute to epigenetic programming and lasting sex differences in microglia identity.

Surprising origins of sex differences in the brain.

This article is part of a Special Issue "SBN 2014". Discerning the biologic origins of neuroanatomical sex differences has been of interest since they were first reported in the late 60's and early 70's. The centrality of gonadal hormone exposure during a developmental critical window cannot be denied but hormones are indirect agents of change, acting to induce gene transcription or modulate membrane bound signaling cascades. Sex differences in the brain include regional volume differences due to differential cell death, neuronal and glial genesis, dendritic branching and synaptic patterning. Early emphasis on mechanism therefore focused on neurotransmitters and neural growth factors, but by and large these endpoints failed to explain the origins of neural sex differences. More recently evidence has accumulated in favor of inflammatory mediators and immune cells as principle regulators of brain sexual differentiation and reveal that the establishment of dimorphic circuits is not cell autonomous but instead requires extensive cell-to-cell communication including cells of non-neuronal origin. Despite the multiplicity of cells involved the nature of the sex differences in the neuroanatomical endpoints suggests canalization, a process that explains the robustness of individuals in the face of intrinsic and extrinsic variability. We propose that some neuroanatomical endpoints are canalized to enhance sex differences in the brain by reducing variability within one sex while also preventing the sexes from diverging too greatly. We further propose mechanisms by which such canalization could occur and discuss what relevance this may have to sex differences in behavior.

Feminization of social play behavior depends on microglia.

Many sex differences in brain and behavior are established developmentally by the opposing processes of feminization and masculinization, which manifest following differential steroid hormone exposure in early life. The cellular mechanisms underlying masculinization are well-documented, a result of the fact that it is steroid-mediated and can be easily induced in newborn female rodents via exogenous steroid treatment. However, the study of feminization of particular brain regions has largely been relegated to being "not masculinization" given the absence of an identified initiating trigger. As a result, the mechanisms of this key developmental process remain elusive. Here we describe a novel role for microglia, the brain's innate immune cell, in the feminization of the medial amygdala and a complex social behavior, juvenile play. In the developing amygdala, microglia promote proliferation of astrocytes equally in both sexes, with no apparent effect on rates of cell division, but support cell survival selectively in females through the trophic actions of Tumor Necrosis Factor α (TNFα). We demonstrate that disrupting TNFα signaling, either by depleting microglia or inhibiting the associated signaling pathways, prevents the feminization of astrocyte density and increases juvenile play levels to that seen in males. This data, combined with our previous finding that male-like patterns of astrocyte density are sculpted by developmental microglial phagocytosis, reveals that sexual differentiation of the medial amygdala involves opposing tensions between active masculinization and active feminization, both of which require microglia but are achieved via distinct processes.

Astrocyte-Neuron Interactions in Substance Use Disorders.

Engagement of astrocytes within the brain's reward circuitry has been apparent for approximately 30 years, when noncontingent drug administration was observed to lead to cytological markers of reactive astrocytes. Since that time, advanced approaches in rodent behavior and astrocyte monitoring have revealed complex interactions between astrocytes with drug type, animal sex, brain region, and dose and duration of drug administration. A number of studies now collectively reveal that rodent drug self-administration followed by prolonged abstinence results in decreased features of structure and synaptic colocalization of astrocytes. In addition, stimulation of astrocytes in the nucleus accumbens with DREADD receptors or pharmacological compounds opposes drug-seeking behavior. These findings provide a clear path for ongoing investigation into astrocytes as mediators of drug action in the brain and underscore the potential therapeutic utility of astrocytes in the regulation of drug craving and relapse vulnerability.

The transcriptome of playfulness is sex-biased in the juvenile rat medial amygdala: a role for inhibitory neurons.

Social play is a dynamic behavior known to be sexually differentiated; in most species, males play more than females, a sex difference driven in large part by the medial amygdala (MeA). Despite the well-conserved nature of this sex difference and the importance of social play for appropriate maturation of brain and behavior, the full mechanism establishing the sex bias in play is unknown. Here, we explore "the transcriptome of playfulness" in the juvenile rat MeA, assessing differences in gene expression between high- and low-playing animals of both sexes via bulk RNA-sequencing. Using weighted gene co-expression network analysis (WGCNA) to identify gene modules combined with analysis of differentially expressed genes (DEGs), we demonstrate that the transcriptomic profile in the juvenile rat MeA associated with playfulness is largely distinct in males compared to females. Of the 13 play-associated WGCNA networks identified, only two were associated with play in both sexes, and very few DEGs associated with playfulness were shared between males and females. Data from our parallel single-cell RNA-sequencing experiments using amygdala samples from newborn male and female rats suggests that inhibitory neurons drive this sex difference, as the majority of sex-biased DEGs in the neonatal amygdala are enriched within this population. Supporting this notion, we demonstrate that inhibitory neurons comprise the majority of play-active cells in the juvenile MeA, with males having a greater number of play-active cells than females, of which a larger proportion are GABAergic. Through integrative bioinformatic analyses, we further explore the expression, function, and cell-type specificity of key play-associated modules and the regulator "hub genes" predicted to drive them, providing valuable insight into the sex-biased mechanisms underlying this fundamental social behavior.

Viral-mediated inflammation by Poly I:C induces the chemokine CCL5 in NK cells and its receptors CCR1 and CCR5 in microglia in the neonatal rat cerebellum.

Objectives: To study the effect of viral inflammation induced by Polyinosinic:polycytidylic acid (PIC) on the cerebellum during a critical period of development in rats. Methods: Neonatal rat pups were treated with PIC on postnatal days (PN) 8 and 10 after which we quantified RNA using Nanostring, qRT-PCR and RNAscope and analyzed immune cells through flow cytometry and immunohistochemistry on PN11. Using the same paradigm, we also analyzed play juvenile behavior, anxiety-like behavior, motor balance using the balance beam and the rotarod assays as well as fine motor behavior using the sunflower seed opening test. Results: We determined that male and female pups treated with PIC reacted with a significant increase in CCL5, a chemotactic cytokine that attracts T-cells, eosinophils and basophils to the site of inflammation, at PN11. PIC treatment also increased the expression of two receptors for CCL5, CCR1 and CCR5 in the cerebellar vermis in both males and females at PN11. In-situ hybridization (RNAscope®) for specific transcripts revealed that microglia express both CCL5 receptors under inflammatory and non-inflammatory conditions in both males and females. PIC treatment also increased the total number of CCL5+ cells in the developing cerebellum which were determined to be both natural killer cells and T-cells. There were modest but significant impacts of PIC treatment on large and fine motor skills and juvenile play behavior. Conclusions: Our findings suggest an important role for CCL5 and other immune cells in mediating inflammation in the developing cerebellum that potentially impact the maturation of cerebellar neurons during a critical period of development.

Neurochemical Characterization of Dopaminoceptive Cells in Song Control Nuclei of Canaries and Their Activation During Song Production: A Multiplex Fluorescent In Situ Hybridization Study.

Highly sensitive in situ hybridization procedures (RNAScope) were used to quantify the expression of three dopamine receptors (Drd1, Drd2, and Drd3) in two song control nuclei (HVC and the Area X of the basal ganglia) that are known to receive dopaminergic inputs and in the periaqueductal gray (PAG) of male and female canaries. Both sexes were treated with testosterone to ensure they would sing actively. We also determined the excitatory versus inhibitory phenotype of the cells expressing these receptors as well as their activation following a period of song production. The three receptor types were identified in each brain area, with the exception of Drd3 in Area X. The density of cells expressing each receptor varied as a function of receptor type and brain area. Surprisingly few sex differences were detected; they do not seem to explain the sex differences in testosterone-induced song. Overall, the density of Drd-positive cells was much lower in PAG than in the two song control nuclei. In HVC, the majority of cells expressing the three receptor subtypes were VGlut2-positive, whereas colocalization with Vglut2 occurred in few cells in Area X and in an intermediate proportion of cells in PAG. The number of inhibitory cells expressing dopamine receptors was limited. Most dopaminoceptive cells in Area X did not express either excitatory or inhibitory markers. Finally, cellular activation during singing behavior, as measured by the expression of Egr1, was observed in cells expressing each of the three dopamine receptor subtypes, except Drd3 in the PAG.

Abstinence from cocaine self-administration promotes microglia pruning of astrocytes which drives cocaine-seeking behavior.

Rodent drug self-administration leads to compromised ability of astrocytes to maintain glutamate homeostasis within the brain's reward circuitry, as well as reductions in surface area, volume, and synaptic colocalization of astrocyte membranes. However, the mechanisms driving astrocyte responses to cocaine are unknown. Here, we report that long-access cocaine self-administration followed by prolonged home cage abstinence results in decreased branching complexity of nucleus accumbens astrocytes, characterized by the loss of peripheral processes. Using a combination of confocal fluorescence microcopy and immuno-gold electron microscopy, we show that alterations in astrocyte structural features are driven by microglia phagocytosis, as labeled astrocyte membranes are found within microglia phagolysosomes. Inhibition of complement C3-mediated phagocytosis using the neutrophil inhibitory peptide (NIF) rescued astrocyte structure and decreased cocaine seeking behavior following cocaine self-administration and abstinence. Collectively, these results provide evidence for microglia pruning of accumbens astrocytes across cocaine abstinence which mediates cocaine craving.

Social play experience in juvenile rats is indispensable for appropriate socio-sexual behavior in adulthood in males but not females.

Social play is a dynamic and rewarding behavior abundantly expressed by most mammals during the juvenile period. While its exact function is debated, various rodent studies on the effects of juvenile social isolation suggest that participating in play is essential to appropriate behavior and reproductive success in adulthood. However, the vast majority of these studies were conducted in one sex only, a critical concern given the fact that there are known sex differences in play's expression: across nearly all species that play, males play more frequently and intensely than females, and there are qualitative sex differences in play patterns. Further limiting our understanding of the importance of play is the use of total isolation to prevent interactions with other juveniles. Here, we employed a novel cage design to specifically prevent play in rats while allowing for other forms of social interaction. We find that play deprivation during the juvenile period results in enduring sex-specific effects on later-life behavior, primarily in males. Males prevented from playing as juveniles exhibited decreased sexual behavior, hypersociability, and increased aggressiveness in adulthood, with no effects on these measures in females. Importantly, play deprivation had no effect on anxiety-like behavior, object memory, sex preference, or social recognition in either sex, showing the specificity of the identified impairments, though there were overall sex differences in many of these measures. Additionally, acute play deprivation impaired performance on a test of prosocial behavior in both sexes, indicating a difference in the motivation and/or ability to acquire this empathy-driven task. Together, these findings provide novel insight into the importance and function of juvenile social play and how this differs in males and females.

The mouse claustrum synaptically connects cortical network motifs.

Spatially distant areas of the cerebral cortex coordinate their activity into networks that are integral to cognitive processing. A common structural motif of cortical networks is co-activation of frontal and posterior cortical regions. The neural circuit mechanisms underlying such widespread inter-areal cortical coordination are unclear. Using a discovery based functional magnetic resonance imaging (fMRI) approach in mouse, we observe frontal and posterior cortical regions that demonstrate significant functional connectivity with the subcortical nucleus, the claustrum. Examining whether the claustrum synaptically supports such frontoposterior cortical network architecture, we observe cortico-claustro-cortical circuits reflecting the fMRI data: significant trans-claustral synaptic connectivity from frontal cortices to posteriorly lying sensory and sensory association cortices contralaterally. These data reveal discrete cortical pathways through the claustrum that are positioned to support cortical network motifs central to cognitive control functions and add to the canon of major extended cortico-subcortico-cortical systems in the mammalian brain.

Fatty acid amide hydrolase inhibition and N-arachidonoylethanolamine modulation by isoflavonoids: A novel target for upcoming antidepressants.

Modulation of the endocannabinoid system (ECS) is a novel putative target for therapeutic intervention in depressive disorders. Altering concentrations of one of the principal endocannabinoids, N-arachidonoylethanolamine, also known as anandamide (AEA) can affect depressive-like behaviors through several mechanisms including anti-inflammatory, hormonal, and neural circuit alterations. Recently, isoflavonoids, a class of plant-derived compounds, have been of therapeutic interest given their ability to modulate the metabolism of the endogenous ligands of the ECS. To determine the therapeutic potential of isoflavonoids, we screened several candidate compounds (Genistein, Biochanin-A, and 7-hydroxyflavone) in silico to determine their binding properties with fatty acid amide hydrolase (FAAH), the primary degrative enzyme for AEA. We further validated the ability of these compounds to inhibit FAAH and determined their effects on depressive-like and locomotor behaviors in the forced swim test (FST) and open field test in male and female mice. We found that while genistein was the most potent FAAH inhibitor, 7-hydroxyflavone was most effective at reducing immobility time in the forced swim test. Finally, we measured blood corticosterone and prefrontal cortex AEA concentrations following the forced swim test and found that all tested compounds decreased corticosterone and increased AEA, demonstrating that isoflavonoids are promising therapeutic targets as FAAH inhibitors.

Phagocytic microglia in development: Are they what they eat?

Microglia, the innate immune cells of the brain, are indispensable for proper brain development. As professional phagocytes, microglia engulf other cells within distinct developmental niches to sculpt the architecture of the brain. Here, I highlight the age-, brain region-, and substrate-dependent diversity of developmental phagocytosis, and pose the idea that phagocytosis may, in turn, drive changes in microglia phenotype. Ultimately, phagocytosis might be just as important for shaping microglia function as it is for shaping the brain.

Generation of an Iba1-EGFP Transgenic Rat for the Study of Microglia in an Outbred Rodent Strain.

Neuroscience has been transformed by the ability to genetically modify inbred mice, including the ability to express fluorescent markers specific to cell types or activation states. This approach has been put to particularly good effect in the study of the innate immune cells of the brain, microglia. These specialized macrophages are exceedingly small and complex, but also highly motile and mobile. To date, there have been no tools similar to those in mice available for studying these fundamental cells in the rat brain, and we seek to fill that gap with the generation of the genetically modified Sprague Dawley rat line: SD-Tg(Iba1-EGFP)Mmmc Using CRISPR-Cas/9 technology, we knocked in EGFP to the promoter of the gene Iba1 With four male and three female founders confirmed by quantitative PCR analysis to have appropriate and specific insertion, we established a breeding colony with at least three generations of backcrosses to obtain stable and reliable Iba1-EGFP expression. The specificity of EGFP expression to microglia was established by flow cytometry for CD45low/CD11b+ cells and by immunohistochemistry. Microglial EGFP expression was detected in neonates and persisted into adulthood. Blood macrophages and monocytes were found to express low levels of EGFP, as expected. Last, we show that EGFP expression is suitable for live imaging of microglia processes in acute brain slices and via intravital two-photon microscopy.

Assessing Rough-and-tumble Play Behavior in Juvenile Rats.

Play is a complex social behavior that is highly conserved across mammals. In most species, males engage in more frequent and vigorous play as juveniles than females, which reflects subtle yet impactful sex differences in brain circuitry and development. In this protocol, we describe a behavioral testing paradigm to assess social play in male and female juvenile rats. We highlight the behavior scoring criteria for distinguishing rough-and-tumble play from other play-related social behaviors. By analyzing both sexes, play behavior can be leveraged as a powerful tool to understand the sex-specific development and expression of social behavior.

Developmental origins of sex differences in the neural circuitry of play.

Social play consists of reciprocal physical interactions between conspecifics with many features conserved across species, including the propensity for males to engage in play more frequently and with higher physical intensity. Animal models, such as the laboratory rat, reveal that the underlying neural circuitry of play is subject to sexual differentiation during a critical period early in life. In this review, we discuss the developmental processes that produce distinct neural nodes which modulate both shared and sex-specific aspects of play with a focus on the medial amygdala, lateral septum, and prefrontal cortex. While the cellular mechanisms determining sex differences in play are beginning to be uncovered, the ultimate advantages of play continue to be debated.

Microglial Phagocytosis of Newborn Cells Is Induced by Endocannabinoids and Sculpts Sex Differences in Juvenile Rat Social Play.

Brain sex differences are established developmentally and generate enduring changes in circuitry and behavior. Steroid-mediated masculinization of the rat amygdala during perinatal development produces higher levels of juvenile rough-and-tumble play by males. This sex difference in social play is highly conserved across mammals, yet the mechanisms by which it is established are unknown. Here, we report that androgen-induced increases in endocannabinoid tone promote microglia phagocytosis during a critical period of amygdala development. Phagocytic microglia engulf more viable newborn cells in males; in females, less phagocytosis allows more astrocytes to survive to the juvenile age. Blocking complement-dependent phagocytosis in males increases astrocyte survival and prevents masculinization of play. Moreover, increased astrocyte density in the juvenile amygdala reduces neuronal excitation during play. These findings highlight novel mechanisms of brain development whereby endocannabinoids induce microglia phagocytosis to regulate newborn astrocyte number and shape the sexual differentiation of social circuitry and behavior. VIDEO ABSTRACT.

Microglia: Driving critical periods and sexual differentiation of the brain.

The proverbial role of microglia during brain development is shifting from passive members of the brain's immune system to active participants that are able to dictate enduring outcomes. Despite these advances, little attention has been paid to one of the most critical components of early brain development-sexual differentiation. Mounting evidence suggests that the normal developmental functions microglia perform-cell number regulation and synaptic connectivity-may be involved in the sex-specific patterning of the brain during these early sensitive periods, and may have lasting sex-dependent and sex-independent effects on behavior. In this review, we outline the known functions of microglia during developmental sensitive periods, and highlight the role they play in the establishment of sex differences in brain and behavior. We also propose a framework for how researchers can incorporate microglia in their study of sex differences and vice versa. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 580-592, 2018.