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.

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.

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.

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.

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.

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.

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.

Activation of the Rostral Intralaminar Thalamus Drives Reinforcement through Striatal Dopamine Release.

Glutamatergic projections of the thalamic rostral intralaminar nuclei of the thalamus (rILN) innervate the dorsal striatum (DS) and are implicated in dopamine (DA)-dependent incubation of drug seeking. However, the mechanism by which rILN signaling modulates reward seeking and striatal DA release is unknown. We find that activation of rILN inputs to the DS drives cholinergic interneuron burst-firing behavior and DA D2 receptor-dependent post-burst pauses in cholinergic interneuron firing. In vivo, optogenetic activation of this pathway drives reinforcement in a DA D1 receptor-dependent manner, and chemogenetic suppression of the rILN reduces dopaminergic nigrostriatal terminal activity as measured by fiber photometry. Altogether, these data provide evidence that the rILN activates striatal cholinergic interneurons to enhance the pursuit of reward through local striatal DA release and introduce an additional level of complexity in our understanding of striatal DA signaling.