Puberty Blocker, Leuprolide, Reduces Sex Differences in Rough-and-Tumble Play and Anxiety-like Behavior in Juvenile Rats.

We examined the effect of the puberty blocker, leuprolide acetate, on sex differences in juvenile rough-and-tumble play behavior and anxiety-like behavior in adolescent male and female rats. We also evaluated leuprolide treatment on gonadal and pituitary hormone levels and activity-regulated cytoskeleton-protein messenger RNA levels within the adolescent amygdala, a region important both for rough-and-tumble play and anxiety-like behavior. Our findings suggest that leuprolide treatment lowered anxiety-like behavior during adolescent development, suggesting that the maturation of gonadotropin-releasing hormone systems may be linked to increased anxiety. These data provide a potential new model to understand the emergence of increased anxiety triggered around puberty. Leuprolide also reduced masculinized levels of rough-and-tumble play behavior, lowered follicle-stimulating hormone, and produced a consistent pattern of reducing or halting sex differences of hormone levels, including testosterone, growth hormone, thyrotropin, and corticosterone levels. Therefore, leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior, allowing for better harmonization of these systems following gender-affirming hormone treatment. These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior.

Brain, behavior, and physiological changes associated with predator stress-An animal model for trauma exposure in adult and neonatal rats.

The use of predators and predator odor as stressors is an important and ecologically relevant model for studying the impact of behavioral responses to threat. Here we summarize neural substrates and behavioral changes in rats resulting from predator exposure. We briefly define the impact predator exposure has on neural targets throughout development (neonatal, juvenile, and adulthood). These findings allow us to conceptualize the impact of predator exposure in the brain, which in turn may have broader implications for human disorders such as PTSD. Importantly, inclusion of sex as a biological variable yields distinct results that may indicate neural substrates impacted by predator exposure differ based on sex.

Polygenic risk scores cannot make their mark on psychiatry without considering epigenetics.

We generally agree with Burt's thesis. However, we note that the author did not discuss epigenetics, the study of how the environment can alter gene structure and function. Given epigenetic mechanisms, the utility of polygenic risk scores (PRS) is limited in studies of development and mental illness. Finally, in this commentary we expand upon the risks of reliance upon PRSs.

Delayed estrogen actions diminish food consumption without changing food approach, motor activity, or hypothalamic activation elicited by corticostriatal µ-opioid signaling.

Mu-opioid receptor (µ-OR) signaling in forebrain sites including nucleus accumbens (Acb) and ventromedial prefrontal cortex (vmPFC) modulates reward-driven feeding and may play a role in the pathophysiology of disordered eating. In preclinical models, intra-Acb or intra-vmPFC µ-OR stimulation causes overeating and vigorous responding for food rewards. These effects have been studied mainly in male animals, despite demonstrated sex differences and estrogen modulation of central reward systems. Hence, the present study investigated sex differences and estrogen modulation of intra-Acb and intra-vmPFC µ-OR-driven feeding behaviors. First, the dose-related effects of intra-Acb and intra-vmPFC infusions of the µ-OR-selective agonist, DAMGO, were compared among intact female, ovariectomized (OVX) female, and intact male rats. The DAMGO feeding dose-effect function was flattened in intact females relative to the robust, dose-dependent effects observed in OVX females and intact males. Thus, in intact females, intra-Acb DAMGO failed to elevate food intake relative to vehicle, while intra-vmPFC DAMGO elevated food intake, but to a smaller degree compared to males and OVX females. Next, to explore the possible role of estrogen in mediating the diminished DAMGO response observed in intact females, OVX rats were given intra-Acb or intra-vmPFC infusions of DAMGO either immediately after a subcutaneous injection of 17-beta-estradiol 3-benzoate (EB; 5 µg/0.1 mL) or 24 h after EB injection. Intra-Acb DAMGO effects were not changed at the immediate post-EB time point. At the delayed post-EB timepoint, significant lordosis was noted and the duration of intra-Acb DAMGO-driven feeding bouts was significantly reduced, with no change in the number of bouts initiated, locomotor hyperactivity, or Fos immunoreactivity in hypothalamic feeding and arousal systems. Similarly, EB failed to alter the motor-activational effects of intra-vmPFC DAMGO while reducing feeding. These findings indicate that delayed, presumably genomically mediated estrogen actions modulate the µ-OR-generated motivational state by reducing consummatory activity while sparing goal-approach and general arousal/activity. The results additionally suggest that EB regulation of consummatory activity occurs outside of forebrain-µ-OR control of hypothalamic systems.

DNA topoisomerase Top3ß is impacted by early life stress in the developing female and male rat brain.

DNA topoisomerases are essential for preserving genomic integrity. DNA topoisomerases induce breakage of DNA to facilitate replication and transcription by relaxing DNA and relieving supercoiling. Aberrant expression and deletions of topoisomerases are associated with psychiatric disorders such as schizophrenia and autism. Our study investigated the effects of early life stress (ELS) on three topoisomerases, Top1, Top3α, and Top3ß in the developing rat brain. Newborn rats were exposed to a predator odor stress on postnatal days 1, 2, and 3; brain tissue was collected either 30 min after the last stressor on postnatal day 3 or during the juvenile period. We found that exposure to predator odor resulted in a decrease in Top3ß expression levels in the neonatal male amygdala and in the juvenile prefrontal cortex of males and females. These data suggest that developing males and females respond differently to predator odor-induced stress. As ELS results in lower Top3ß levels, these data suggest that ELS experienced during development may have consequences for genomic structural integrity and increased mental health risk.

Early life stress during the neonatal period alters social play and Line1 during the juvenile stage of development.

Early life stress (ELS) has been shown to have a significant impact on typical brain development and the manifestation of psychological disorders through epigenetic modifications that alter gene expression. Line1, a retrotransposon associated with genetic diversity, has been linked with various psychological disorders that are associated with ELS. Our previous work demonstrated altered Line1 DNA copy number in the neonatal period following stressful experiences; we therefore chose to investigate whether early life stress altered Line1 retrotransposition persists into the juvenile period of development. Our study uses a neonatal predator odor exposure (POE) paradigm to model ELS in rats. We examined Line1 using qPCR to assess Line1 expression levels and DNA copy number in the male and female juvenile amygdala, hippocampus and prefrontal cortex-areas chosen for their association with affective disorders and stress. We report a sex difference in Line1 levels within the juvenile amygdala. We also find that ELS significantly increases Line1 DNA copy number within the juvenile amygdala which correlates with reduced juvenile social play levels, suggesting the possibility that Line1 may influence juvenile social development.

Early life stress increases Line1 within the developing brain in a sex-dependent manner.

Long-interspersing element 1 (Line1)-a retrotransposon that comprises ~17% of the human genome and ~24% of the rat genome -is aberrantly expressed in psychiatric disorders such as schizophrenia, bipolar disorder, and Rett syndrome, suggesting it may play an important role in neurodevelopment. Retrotransposons such as Line1 have the ability to self-replicate via reverse transcription and can subsequently be reinserted throughout the genome, potentially increasing genetic diversity. We sought to understand whether early life stress (ELS), a known risk factor for the development of later psychiatric disorders in humans, would affect Line1 expression and DNA copy number. Our study uses a neonatal predator odor exposure (POE) paradigm to model ELS in rats. We found sex- and region-specific increases in both Line1 Open Reading Frame 1 (ORF1) and ORF2 mRNA following POE-induced stress. Interestingly, ELS increased Line1 DNA copy number within the male hippocampus. These data suggest the possibility that early life stress can mobilize Line1 in a sex- and region-specific manner, resulting in genomic heterogeneity between cells in the brain suggesting that some cells may have a different genetic makeup than others resulting in genomic heterogeneity.

Mu opioid receptors in the medial preoptic area govern social play behavior in adolescent male rats.

Neural systems underlying important behaviors are usually highly conserved across species. The medial preoptic area (MPOA) has been demonstrated to play a crucial role in reward associated with affiliative, nonsexual, social communication in songbirds. However, the role of MPOA in affiliative, rewarding social behaviors (eg, social play behavior) in mammals remains largely unknown. Here we applied our insights from songbirds to rats to determine whether opioids in the MPOA govern social play behavior in rats. Using an immediate early gene (ie, Egr1, early growth response 1) expression approach, we identified increased numbers of Egr1-labeled cells in the MPOA after social play in adolescent male rats. We also demonstrated that cells expressing mu opioid receptors (MORs, gene name Oprm1) in the MPOA displayed increased Egr1 expression when adolescent rats were engaged in social play using double immunofluorescence labeling of MOR and Egr1. Furthermore, using short hairpin RNA-mediated gene silencing we revealed that knockdown of Oprm1 in the MPOA reduced the number of total play bouts and the frequency of pouncing. Last, RNA sequencing differential gene expression analysis identified genes involved in neuronal signaling with altered expression after Oprm1 knockdown, and identified Egr1 as potentially a key modulator for Oprm1 in the regulation of social play behavior. Altogether, these results show that the MPOA is involved in social play behavior in adolescent male rats and support the hypothesis that the MPOA is part of a conserved neural circuit across vertebrates in which opioids act to govern affiliative, intrinsically rewarded social behaviors.

Testosterone-related behavioral and neural mechanisms associated with location preferences: A model for territorial establishment.

Territoriality is an adaptive behavioral trait that is important for animal's fitness and there still remains much to learn about the proximate mechanisms underlying the development of territoriality. We speculate that the formation of a conditioned place preference (CPP), an increased time allocation to the environment where a rewarding experience occurred, contributes to territoriality. Testosterone (T) plays an important role in modulating territorial behaviors and T pulses can induce a CPP. We confirmed previous findings in California mice (Peromyscus californicus) that T pulses can induce a CPP in singly-housed, but not group-housed males. Housing singly may be similar enough to dispersal in nature to initiate similar hormonal and neuroanatomical changes needed for the development of territoriality. We further revealed that T pulses interact with the single housing experience and appear to enhance the motivation to be aggressive towards a stimulus male. On a neural level, being singly housed upregulated levels of androgen receptors in the preoptic area, which positively correlated with the strength of the CPP. We speculate that this change in androgen sensitivity in the preoptic area is characteristic of males that have dispersed, making them more sensitive to T pulses. Also, single housing increased markers of synaptic plasticity in the nucleus accumbens, ventral and dorsal hippocampus, neural changes that may be associated with dispersal, reproduction and territory establishment. These behavioral and neural changes may reflect the life history transition from residing in the natal territory to dispersing and establishing a new territory.

Early life stress alters opioid receptor mRNA levels within the nucleus accumbens in a sex-dependent manner.

Early life stress (ELS) strongly impacts mental health, but little is known about its interaction with biological sex and postnatal development to influence risk and resilience to psychopathologies. A number of psychiatric disorders, such as social anhedonia and drug addiction, involve dysfunctional opioid signaling; moreover, there is evidence for differential central opioid function in males vs. females. The present study examined opioid receptor gene expression in the nucleus accumbens (NAc) and amygdala of male and female rats subjected to a neonatal predator odor exposure (POE) paradigm to model ELS. Brain tissue was collected at two developmental time points: neonatal and juvenile. Results showed that, following the neonatal POE experience, opioid receptor mRNA levels in the NAc were differentially regulated at the neonatal and juvenile time points. POE downregulated neonatal mu- and kappa-opioid receptor mRNA levels in neonatal females, but upregulated mu- and delta-opioid receptor mRNA levels in juvenile females. Intriguingly, POE had no significant effect on NAc opioid receptor mRNA levels in males at either time point, indicating that the impact of POE on opioid system development is sex-dependent. Finally, POE failed to alter amygdalar opioid receptor gene expression in either sex at either time-point. The spatiotemporally- and sex-specific impact of ELS within the developing brain may confer differential risk or resilience for males and females to develop atypical opioid-regulated behaviors associated with conditions such as depression and addiction.

Binge Drinking and Intergenerational Implications: Parental Preconception Alcohol Impacts Offspring Development in Rats.

Preconception behaviors and experiences of mothers and fathers can affect future offspring. Recently, our laboratory showed that alcohol-naive offspring of parents who were exposed to repeated binge alcohol during adolescence showed altered DNA methylation patterns in the hypothalamus, a brain region involved in regulation of pubertal development, stress, and behavior. These observations have potentially far-reaching consequences for human health, as more than 4.6 million Americans under the age of 21 years report engaging in the rapid intoxication behavior of binge-pattern alcohol (EtOH) drinking. Therefore, we tested the hypothesis that offspring of binge EtOH‒exposed parents would have altered hypothalamic function manifested phenotypically as improper pubertal development, impaired socialization, and dysregulated stress response. In addition, we tested the hypothesis that parental EtOH exposure would confer adaptive protection from the negative effects of EtOH when offspring were themselves exposed to EtOH. Rats received EtOH via oral gavage once daily for 6 days at both early [postnatal day (PND) 37] and late puberty (PND 67). Animals were paired (EtOH-EtOH, vehicle-vehicle) for mating 24 hours after the last EtOH dose. After weaning, offspring were randomized to vehicle treatment to assess changes in normal development or to EtOH treatment to assess the effect of parental EtOH exposure on offspring response to this treatment. We found that offspring had smaller body weights and displayed fewer play behaviors when parents had been exposed to EtOH before conception. In addition, offspring showed a reduction in pubertal development markers that could indicate that parental preconception EtOH exposure confers maladaptive epigenetic traits in first-generation offspring.

N6-methyladenine is an epigenetic marker of mammalian early life stress.

Recent evidence described 6-methyladenine (6 mA) as a novel epigenetic regulator in a variety of multicellular species, including rodents; however, its capacity to influence gene expression in the mammalian brain remains unknown. We examined if 6 mA is present and regulated by early life stress associated with predator odor exposure (POE) within the developing rat amygdala. Our results provide evidence that 6 mA is present in the mammalian brain, is altered within the Htr2a gene promoter by early life stress and biological sex, and increased 6 mA is associated with gene repression. These data suggest that methylation of adenosine within mammalian DNA may be used as an additional epigenetic biomarker for investigating the development of stress-induced neuropathology.

Sex differences in Gadd45b expression and methylation in the developing rodent amygdala.

Precise spatiotemporal epigenetic regulation of the genome facilitates species-typical development; sexual differentiation of the brain by gonadal hormones and sex chromosomes causes extensive epigenetic reprogramming of many cells in the body, including the brain, and may indirectly predispose males and females to different psychiatric conditions. We and others have demonstrated sex differences in DNA methylation, as well as in the enzymes that form, or 'write', this epigenetic modification. However, while a growing body of evidence suggests that DNA methylation undergoes rapid turnover and is dynamically regulated in vivo, to our knowledge no studies have been done investigating whether sex differences exist in the epigenetic 'erasers' during postnatal development. Here we report sex differences in the expression of growth arrest and DNA damage inducible factor ß (Gadd45b), but not family members α (a) or γ (g), in the neonatal and juvenile rodent amygdala.

Maternal Obesity Affects Inflammatory and Iron Indices in Umbilical Cord Blood.

OBJECTIVE: To determine the impact of maternal obesity and gestational weight gain across pregnancy on fetal indices of inflammation and iron status. STUDY DESIGN: Eighty-five healthy term newborns delivered via elective cesarean were categorized by 2 maternal body mass index (BMI) thresholds; above or below 30 kg/m(2) or above or below 35 kg/m(2). Umbilical cord plasma levels of C-reactive protein, interleukin (IL)-6, tumor necrosis factor (TNF)-α, ferritin, and hepcidin were assayed. Cytokines released by phytohemagglutinin-stimulated umbilical cord mononuclear cells (MNCs) were assayed. RESULTS: Maternal class II obesity, defined as BMI of 35 kg/m(2) and above, predicted higher C-reactive protein and TNF-α in umbilical cord plasma (P < .05 for both), and also proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) from stimulated MNC (P < .05 for all). The rise in plasma TNF-α and MNC TNF-α was not linear but occurred when the threshold of BMI 35 kg/m(2) was reached (P < .005, P < .06). Poorer umbilical cord iron indices were associated with maternal obesity. When ferritin was low, IL-6 was higher (P < .04), but this relationship was present primarily when maternal BMI exceeded 35 kg/m(2) (P < .03). Ferritin was correlated with hepcidin (P < .0001), but hepcidin was unrelated to either maternal BMI or inflammatory indices. CONCLUSIONS: Class II obesity and above during pregnancy is associated with fetal inflammation in a threshold fashion. Although maternal BMI negatively impacted fetal iron status, hepcidin, related to obesity in adults, was related to iron status and not obesity in fetuses. Pediatricians should be aware of these relationships.

Gadd45b is an epigenetic regulator of juvenile social behavior and alters local pro-inflammatory cytokine production in the rodent amygdala.

Precise regulation of the epigenome during perinatal development is critical to the formation of species-typical behavior later in life. Recent data suggests that Gadd45b facilitates active DNA demethylation by recruiting proteins involved in base excision repair (BER), which will catalyze substitution of 5-methyl-cytosine (5mC) for an unmodified cytosine. While a role for Gadd45b has been implicated in both hippocampal and amygdalar learning tasks, to the best of our knowledge, no study has been done investigating the involvement of Gadd45b in neurodevelopmental programming of social behavior. To address this, we used a targeted siRNA delivery approach to transiently knock down Gadd45b expression in the neonatal rat amygdala. We chose to examine social behavior in the juvenile period, as social deficits associated with neurodevelopmental disorders tend to emerge in humans at an equivalent age. We find that neonatal Gadd45b knock-down results in altered juvenile social behavior and reduced expression of several genes implicated in psychiatric disorders, including methyl-CpG-binding protein 2 (MeCP2), Reelin, and brain derived neurotrophic factor (BDNF). We furthermore report a novel role for Gadd45b in the programmed expression of α2-adrenoceptor (Adra2a). Consistent with Gadd45b's role in the periphery, we also observed changes in the expression of pro-inflammatory cytokines interleukin-6 (Il-6) and interleukin-1beta (Il-1beta) in the amygdala, which could potentially mediate or exacerbate effects of Gadd45b knockdown on the organization of social behavior. These data suggest a prominent role for Gadd45b in the epigenetic programming of complex juvenile social interactions, and may provide insight into the etiology of juvenile behavioral disorders such as ADHD, autism, and/or schizophrenia.

MeCP2 regulates GFAP expression within the developing brain.

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurodevelopmental disorder that primarily affects females. Individuals with RTT have increased glial fibrillary acidic protein (GFAP) expression in the brain. GFAP is an intermediate filament protein that is expressed predominately within astrocytes in the CNS. MeCP2 binds to methylated regions of the GFAP promoter region and suppresses GFAP expression in vitro. Therefore, we wanted to determine if transiently reducing MeCP2 expression would increase GFAP expression in the developing rat brain. Male and female rats received infusions of either MeCP2 or control siRNA targeting the amygdala during the first 3 days of postnatal life. Brains were collected after 6h or 2 weeks following the last infusion. MeCP2 siRNA increased GFAP mRNA and protein within the female, but not the male, amygdala on postnatal day (PN) 2. Two weeks following the infusion, levels returned to normal. MeCP2 siRNA targeting the hypothalamus also increases GFAP mRNA within the female hypothalamus on PN2, suggesting that the regulation is not brain region-specific. It appears that MeCP2 does not regulate all astrocyte markers in the developing female brain, but specifically regulates GFAP expression, as levels of S100ß and vimentin were not altered in the female amygdala at either time point. These data contribute to the idea that the role of MeCP2 differs in the developing male versus female brain. Further elucidating the regulation and function of GFAP can contribute to our understanding of MeCP2 function and perhaps RTT etiology.

Neonatal RU-486 (mifepristone) exposure increases androgen receptor immunoreactivity and sexual behavior in male rats.

Progesterone and progestin receptors (PRs) are known to play a role in the development of brain physiology and behavior in many different species. The distribution and regulation of PRs within the developing brain suggest that they likely contribute to the organization of the brain and behavior in a sex-specific manner. We examined the role of PR signaling during development on the organization of adult sexual behavior and androgen receptor (AR) expression in the brain. We administered the PR antagonist, RU-486, subcutaneously to male and female rats on postnatal days 1-7 (0=day of birth) and examined adult sexual behavior and AR-immunoreactivity (AR-ir) in the adult brain. A typical sex difference in lordosis quotient (LQ) was observed and neonatal RU-486 treatment did not alter this behavior. In contrast, neonatal RU-486 treatment increased adult male sexual behavior and AR-ir in several brain areas in males. These data indicate that a transient disruption in PR signaling during development can have lasting consequences on the male brain and may increase male sexual behavior in part by increasing AR expression, and therefore androgen sensitivity, in adulthood.

Permanent and plastic epigenesis in neuroendocrine systems.

The emerging area of neuroepigenetics has been linked to numerous mental health illnesses. Importantly, a large portion of what we know about early gene×environment interactions comes from examining epigenetic modifications of neuroendocrine systems. This review will highlight how neuroepigenetic mechanisms during brain development program lasting differences in neuroendocrine systems and how other neuroepigenetic processes remain plastic, even within the adult brain. As epigenetic mechanisms can either be stable or plastic, elucidating the mechanisms involved in reversing these processes could aid in understanding how to reverse pathological epigenetic programming.

Maternal touch moderates sex differences in juvenile social play behavior.

Additional somatosensory contact of preterm human infants improves a variety of developmental assessment scores, but less is known about its lasting consequences. In rodents, maternal contact may influence the programming of juvenile social play behavior. Therefore, we used a paradigm where we can control the levels of somatosensory contact associated with maternal care. We find that additional somatosensory contact of offspring can have lasting consequences on juvenile social play behavior in a sex-dependent manner. Specifically, additional somatosensory stimuli reduced male social play behavior, but did not change female play behavior. We then examined if this additional infant contact altered some neurobiological substrates associated with play within the juvenile amygdala. Control males had lower levels of 5HT2a receptor mRNA levels contrasted to females; however, similar to its sex-dependent effect on juvenile social play, males that received additional somatosensory contact had higher serotonin 5HT2a receptor mRNA levels than control males. No difference was found in females. As serotonin signaling typically opposes juvenile play behavior, these data suggest that maternal touch can program lasting differences in juvenile social play and 5HT2a receptors mRNA levels within the juvenile amygdala.

Gender differences in neurodevelopment and epigenetics.

The concept that the brain differs in make-up between males and females is not new. For example, it is well established that anatomists in the nineteenth century found sex differences in human brain weight. The importance of sex differences in the organization of the brain cannot be overstated as they may directly affect cognitive functions, such as verbal skills and visuospatial tasks in a sex-dependent fashion. Moreover, the incidence of neurological and psychiatric diseases is also highly dependent on sex. These clinical observations reiterate the importance that gender must be taken into account as a relevant possible contributing factor in order to understand the pathogenesis of neurological and psychiatric disorders. Gender-dependent differentiation of the brain has been detected at every level of organization--morphological, neurochemical, and functional--and has been shown to be primarily controlled by sex differences in gonadal steroid hormone levels during perinatal development. In this review, we discuss howthe gonadal steroid hormone testosterone and its metabolites affect downstream signaling cascades, including gonadal steroid receptor activation, and epigenetic events in order to differentiate the brain in a gender-dependent fashion.