Points of divergence on a bumpy road: early development of brain and immune threat processing systems following postnatal adversity.

Lifelong indices of maladaptive behavior or illness often stem from early physiological aberrations during periods of dynamic development. This is especially true when dysfunction is attributable to early life adversity (ELA), when the environment itself is unsuitable to support development of healthy behavior. Exposure to ELA is strongly associated with atypical sensitivity and responsivity to potential threats-a characteristic that could be adaptive in situations where early adversity prepares individuals for lifelong danger, but which often manifests in difficulties with emotion regulation and social relationships. By synthesizing findings from animal research, this review will consider threat sensitivity through the lenses of associated corticolimbic brain circuitry and immune mechanisms, both of which are immature early in life to maximize adaptation for protection against environmental challenges to an individual's well-being. The forces that drive differential development of corticolimbic circuits include caretaking stimuli, physiological and psychological stressors, and sex, which influences developmental trajectories. These same forces direct developmental processes of the immune system, which bidirectionally communicates with sensory systems and emotion regulation circuits within the brain. Inflammatory signals offer a further force influencing the timing and nature of corticolimbic plasticity, while also regulating sensitivity to future threats from the environment (i.e., injury or pathogens). The early development of these systems programs threat sensitivity through juvenility and adolescence, carving paths for probable function throughout adulthood. To strategize prevention or management of maladaptive threat sensitivity in ELA-exposed populations, it is necessary to fully understand these early points of divergence.

Early life adversity accelerates hypothalamic drive of pubertal timing in female rats with associated enhanced acoustic startle.

Early life adversity in the form of childhood maltreatment in humans or as modeled by maternal separation (MS) in rodents is often associated with an earlier emergence of puberty in females. Earlier pubertal initiation is an example of accelerated biological aging and predicts later risk for anxiety in women, especially in populations exposed to early life trauma. Here we investigated external pubertal markers as well as hypothalamic gene expression of pubertal regulators kisspeptin and gonadotropin-releasing hormone, to determine a biological substrate for MS-induced accelerated puberty. We further investigated a mechanism by which developmental stress might regulate pubertal timing. As kisspeptin and gonadotropin-releasing hormone secretion are typically inhibited by corticotropin releasing hormone at its receptor CRH-R1, we hypothesized that MS induces a downregulation of Crhr1 gene transcription in a cell-specific manner. Finally, we explored the association between pubertal timing and anxiety-like behavior in an acoustic startle paradigm, to drive future preclinical research linking accelerated puberty and anxiety. We replicated previous findings that MS leads to earlier puberty in females but not males, and found expression of kisspeptin and gonadotropin-releasing hormone mRNA to be prematurely increased in MS females. RNAscope confirmed increased expression of these genes, and further revealed that kisspeptin-expressing neurons in females were less likely to express Crhr1 after MS. Early puberty was associated with higher acoustic startle magnitude in females. Taken together, these findings indicate precocial maturation of central pubertal timing mechanisms after MS, as well as a potential role of CRH-R1 in these effects and an association with a translational measure of anxiety.

Infant ultrasonic vocalizations predict adolescent social behavior in rats: Effects of early life adversity.

Early life adversity (ELA) increases risk for psychopathologies that often manifest during adolescence and involve disrupted social functioning. ELA affects development of the prefrontal cortex (PFC), which plays a role in social behavior. PFC oxytocin and vasopressin are important regulators of, first, mother-infant attachment, and, later, social behavior, and are implicated in psychiatric disorders. Here, we tested whether infant social communication is predictive of PFC development and adolescent social behavior. We used the limited bedding (LB) ELA model in rats during postnatal days (P)2-14, and measured isolation-induced ultrasonic vocalizations (USVs) at P10 to characterize differences in an early social response. Rats were tested for dyadic social interaction in adolescence (P34). Adolescent oxytocin receptor (Oxtr) and arginine-vasopressin receptor 1a mRNA were measured in the PFC. Relationships between infant USVs, adolescent behavior, and gene expression were assessed. LB-reared rats exhibited fewer USVs at P10. While social behaviors were not robustly affected by rearing, fewer total and complex-type infant USVs predicted fewer interactions in adolescence. LB increased Oxtr in both sexes but Oxtr was not directly predicted by USVs. Findings support the use of USVs as indicators of differential early life experience in rodents, toward further characterization of early factors associated with vulnerability.

Bundling the haystack to find the needle: Challenges and opportunities in modeling risk and resilience following early life stress.

Various forms of early life adversity (ELA) have been linked with increased risk for negative health outcomes, including neuropsychiatric disorders. Understanding how the complex interplay between types, timing, duration, and severity of ELA, together with individual differences in genetic, socio-cultural, and physiological differences can mediate risk and resilience has proven difficult in population based studies. Use of animal models provides a powerful toolset to isolate key variables underlying risk for altered neural and behavioral maturational trajectories. However, a lack of clarity regarding the unique features of differing forms of adversity, lab differences in the implementation and reporting of methods, and the ability compare across labs and types of ELA has led to some confusion. Here, we highlight the diversity of approaches available, current challenges, and a possible ways forward to increase clarity and drive more meaningful and fruitful implementation and comparison of these approaches.

Effects of early life stress on cocaine conditioning and AMPA receptor composition are sex-specific and driven by TNF.

Exposure to early life adversity can predispose adolescents to the formation of substance abuse disorders. In rodents, early stressors such as repeated maternal separation (MS) impact AMPAR activity in the prefrontal cortex (PFC) and nucleus accumbens (NAc), regions involved in drug-cue association after cocaine-induced conditioned place preference (CPP). Notably, previous reports suggest that the pro-inflammatory cytokine tumor necrosis factor (TNF) regulates AMPAR subunit composition; increased TNF levels are reported to reduce GluA2-positive AMPARs. Since MS can elevate adolescent TNF levels, the stressor may therefore alter AMPAR subunit composition via neuroimmune signaling, thereby affecting cocaine-induced CPP. We tested the specific role of soluble TNF in MS-induced GluA2 loss and cocaine-induced CPP with biologic disruption of TNF signaling. TNF gene and protein expression were elevated in both PFC and NAc of MS males, but not females. GluA2 expression was reduced in both regions in only male MS rats, and systemic treatment with either ibudilast - a phosphodiesterase inhibitor, or XPro1595 - a blood-brain barrier-permeable blocker of soluble TNF - reversed such loss. MS males also formed greater preference for a cocaine-paired environment, the expression of which returned to control levels after XPro1595 administration. These data suggest a sex-specific mechanistic link between TNF signaling and changes in GluA2 expression and drug-cue conditioning, thereby providing further evidence for a role of MS and neuro-immune activity in cortical and striatal AMPAR changes. Moreover, manipulation of the TNF signaling pathway represents a novel approach for influencing response to reinforcing effects of drug use.

Adolescent food restriction in rats alters prefrontal cortex microglia in an experience-dependent manner.

Microglia are resident immune cells of the brain that can regulate neural communication and excitability. Any environmental influence on microglial activity has the potential to alter subsequent neural physiology and behavior. Within the prefrontal cortex, several types of stressors have been shown to increase microglial expression of activation markers such as ionized calcium-binding adapter molecule-1 (Iba-1), which suggests altered microglial activity. Recent reports in rodents suggest that exposure to forms of early-life stress such as maternal separation can alter microglial responsivity to subsequent challenges. Several learning paradigms used in rodents require food restriction to provoke motivational states that facilitate approach behaviors. Here, we tested whether food restriction (increasing from 13 g/day-23 g/day in males and 10 g/day-20 g/day in females, which reduced body weight to 72-84% free-fed weight) in adolescent rats is a sufficient challenge to affect microglial Iba-1 expression, and whether previous exposure to postnatal maternal separation influenced microglial outcomes. We measured prefrontal cortex Iba-1 expression and microglial morphology after 20 days of ad libitum or restricted food availability in males and females with or without exposure to maternal separation. Food-restricted animals displayed higher levels of Iba-1 in the prefrontal cortex, with hyper-ramified microglial morphology in maternally separated males and control females, compared to those that were free-fed. Together, our data provide evidence that food restriction paradigms may have unintended effects in some behavioral protocols.

Effects of early adolescent environmental enrichment on cognitive dysfunction, prefrontal cortex development, and inflammatory cytokines after early life stress.

Early postnatal stress such as maternal separation causes cognitive dysfunction later in life, including working memory deficits that are largely mediated by the prefrontal cortex. Maternal separation in male rats also yields a loss of parvalbumin-containing prefrontal cortex interneurons in adolescence, which may occur via inflammatory or oxidative stress mechanisms. Environmental enrichment can prevent several effects of maternal separation; however, effects of enrichment on prefrontal cortex development are not well understood. Here, we report that enrichment prevented cognitive dysfunction in maternally separated males and females, and prevented elevated circulating pro-inflammatory cytokines that was evident in maternally separated males, but not females. However, enrichment did not prevent parvalbumin loss or adolescent measures of oxidative stress. Significant correlations indicated that adolescents with higher oxidative damage and less prefrontal cortex parvalbumin in adolescence committed more errors on the win-shift task; therefore, maternal separation may affect cognitive dysfunction via aberrant interneuron development. © 2015 Wiley Periodicals, Inc. Dev Psychobiol 58: 482-491, 2016.

Impacts of age and environment on postnatal microglial activity: Consequences for cognitive function following early life adversity.

Early life adversity (ELA) increases the likelihood of later-life neuropsychiatric disorders and cognitive dysfunction. Importantly, ELA, neuropsychiatric disorders, and cognitive deficits all involve aberrant immune signaling. Microglia are the primary neuroimmune cells and regulate brain development. Microglia are particularly sensitive to early life insults, which can program their responses to future challenges. ELA in the form of maternal separation (MS) in rats alters later-life microglial morphology and the inflammatory profile of the prefrontal cortex, a region important for cognition. However, the role of microglial responses during MS in the development of later cognition is not known. Therefore, here we aimed to determine whether the presence of microglia during MS mediates long-term impacts on adult working memory. Clodronate liposomes were used to transiently deplete microglia from the brain, while empty liposomes were used as a control. We hypothesized that if microglia mediate the long-term impacts of ELA on working memory in adulthood, then depleting microglia during MS would prevent these deficits. Importantly, microglial function shifts throughout the neonatal period, so an exploratory investigation assessed whether depletion during the early versus late neonatal period had different effects on adult working memory. Surprisingly, empty liposome treatment during the early, but not late, postnatal period induced microglial activity changes that compounded with MS to impair working memory in females. In contrast, microglial depletion later in infancy impaired later life working memory in females, suggesting that microglial function during late infancy plays an important role in the development of cognitive function. Together, these findings suggest that microglia shift their sensitivity to early life insults across development. Our findings also highlight the potential for MS to impact some developmental processes only when compounded with additional neuroimmune challenges in a sex-dependent manner.

Effects of early life adversity and adolescent basolateral amygdala activity on corticolimbic connectivity and anxiety behaviors.

Early postnatal development of corticolimbic circuitry is shaped by the environment and is vulnerable to early life challenges. Prior work has shown that early life adversity (ELA) leads to hyperinnervation of glutamatergic basolateral amygdala (BLA) projections to the prefrontal cortex (PFC) in adolescence. While hyperinnervation is associated with later-life anxiety behaviors, the physiological changes underpinning corticolimbic and behavioral impacts of ELA are not understood. We tested whether postsynaptic BLA-driven PFC activity is enhanced in ELA-exposed animals, using the maternal separation (MS) model of ELA. PFC local-field potential following BLA stimulation was facilitated in MS-exposed adolescents. Since ELA increases activity of the early-developing BLA, while the PFC exhibits protracted development, we further examined impacts of glutamatergic BLA activity during early adolescence on later-life PFC innervation and heightened anxiety. In early adolescence, MS-exposed animals exhibited decreased anxiety-like behavior, and acute adolescent BLA inhibition induced behaviors that resembled those of MS animals. To examine long-lasting impacts of adolescent BLA activity on innervation, BLA-originating axonal boutons in the PFC were quantified in late adolescence after early adolescent BLA inhibition. We further tested whether late adolescent BLA-PFC changes were associated with anxious reactivity expressed as heightened acoustic startle responses. MS rearing increased BLA-PFC innervation and threat reactivity in late adolescence, however early adolescent BLA inhibition was insufficient to prevent MS effects, suggesting that earlier BLA activity or post-synaptic receptor rearrangement in the PFC drives altered innervation. Taken together, these findings highlight both pre- and postsynaptic changes in the adolescent BLA-PFC circuit following ELA.

Evidence for a neuroinflammatory mechanism in delayed effects of early life adversity in rats: relationship to cortical NMDA receptor expression.

Postnatal maternal separation in rats causes a reduction of GABAergic parvalbumin-containing interneurons in the prefrontal cortex that first occurs in adolescence. This parvalbumin loss can be prevented by pre-adolescent treatment with a non-steroidal anti-inflammatory drug that also protects against excitotoxicity. Therefore, the neuropsychiatric disorders associated with early life adversity and interneuron dysfunction may involve neuroinflammatory processes and/or aberrant glutamatergic activity. Here, we aimed to determine whether delayed parvalbumin loss after maternal separation was due to inflammatory activity, and whether central administration of the anti-inflammatory cytokine interleukin (IL)-10 could protect against such loss. We also investigated the effects of maternal separation and IL-10 treatment on cortical NMDA receptor expression. Male rat pups were isolated for 4h/day between postnatal days 2-20. IL-10 was administered intracerebroventricularly through an indwelling cannula between P30 and 38. Adolescent prefrontal cortices were analyzed using Western blotting and immunohistochemistry for parvalbumin and NMDA NR2A subunit expression. We demonstrate that central IL-10 administration during pre-adolescence protects maternally separated animals from parvalbumin loss in adolescence. Linear regression analyses revealed that increased circulating levels of the pro-inflammatory cytokines IL-1ß and IL-6 predicted lowered parvalbumin levels in maternally separated adolescents. Maternal separation also increases cortical expression of the NR2A NMDA receptor subunit in adolescence, which is prevented by IL-10 treatment. These data suggest that inflammatory damage to parvalbumin interneurons may occur via aberrant glutamatergic activity in the prefrontal cortex. Our findings provide a novel interactive mechanism between inflammation and neural dysfunction that helps explain deleterious effects of early life adversity on prefrontal cortex interneurons.

Depressive-like behavior in adolescents after maternal separation: sex differences, controllability, and GABA.

Exposure to adversity during development is an identified risk factor for depression later in life. In humans, early adversity accelerates the onset of depressive symptoms, which manifest during adolescence. Animal studies have used maternal separation as a model of early adversity to produce adult depressive-like behaviors, but have yet to examine these behaviors during adolescence. Moreover, the nature of depressive-like behaviors has not been well characterized in this model. Here, we used the triadic model of learned helplessness to understand controllability, helplessness, and motivational factors following maternal separation in male and female adolescent rats. We found sex-dependent changes in the effects of separation, with males demonstrating loss of controllability in an escapable shock condition, whereas females demonstrated motivational impairment in a no-shock condition. The effect, however, did not endure as adult females were no longer helpless. Reductions in parvalbumin, a GABAergic marker, in the prefrontal cortex of separated subjects relative to age-matched controls were evident and paralleled depressive-like behavior. Understanding the risk factors for depression, the nature of depressive-like behaviors, and their unique sex dependency may ultimately provide insight into improved treatments.

One week of maternal separation induces more frequent, but less predictable, maternal caregiving behaviors.

Rodent models of early life adversity disrupt typical interactions between dams and offspring, impacting pup development over the lifespan. Predictability of caregiver interactions is a critical feature of the environment, and unpredictability is associated with behavioral and cognitive deficits in offspring. In the maternal separation (MS) paradigm, dams are not able to engage with pups while they are separated, and maternal care is impacted even after pups and dams are reunited. Using a 3.5-h daily MS protocol in rats, the present study sought to compare diurnal patterns of maternal behavior, specifically predictability and fragmentation of care, between MS- and control-reared dams. Three observation periods were assessed (1430, 2330, and 0830) between postnatal days 8-9. Frequencies and durations of maternal behaviors were measured, including pup-directed licking/grooming, arched-back and passive nursing, and carrying pups, as well as non-pup-directed self-grooming, rearing, burrowing, nest-building, and eating. The frequency of nest entries was interpreted as a measure of fragmentation, and entropy rate was calculated from transitional probability matrices to measure predictability of maternal behavioral sequences. After dam-pup reunion, MS dams engaged in more bouts of nursing and licking/grooming and more nest entries, and behavioral sequences were less predictable than control dams. MS-induced enhancement of care is a replication of previous research, but unpredictability and fragmented care during MS is a novel finding, as these measures have not been previously reported for MS.

Age- and sex-specific effects of maternal separation on the acoustic startle reflex in rats: early baseline enhancement in females and blunted response to ambiguous threat.

Early life adversity (ELA) increases the incidence of later-life anxiety disorders. Dysregulated threat processing, including responsivity to ambiguous threats, is an indicator of anxiety disorders and can be influenced by childhood experiences. The acoustic startle response is a defensive reflex displayed by mammals when exposed to sudden intense stimuli reflecting individual variations in vigilance. These measures can be altered by previous experience and experimental modifications, including the introduction of unconditioned aversive stimuli. Rats emit ultrasonic vocalizations (USVs) in the 22 KHz range in negative contexts. As such, 22 KHz USVs are an ethologically relevant social cue of environmental threat shown to induce anxiety-like behavior in recipient rats. Because the timing of symptom manifestation after early life adversity can differ between sexes, the current study sought to identify the age- and sex-specific effects of daily maternal separation (MS) on responsivity to ambiguous threat in rats. In Experiment 1, rat pups underwent MS or control rearing from postnatal day (P) 2-20, then underwent behavioral testing beginning on P24, 34, or 54 to determine whether MS modified the baseline startle response or the modulation of startle by 22 KHz USVs. In Experiment 2, rats were tested in a light-enhanced startle paradigm at P54 after MS or control rearing to determine whether MS influenced light-enhanced startle. Results show an enhancement of the baseline startle magnitude by MS in females at P34. At P54, MS reduced the modulation of the startle response by 22 KHz USVs and prevented light-enhanced startle, indicating an MS-induced deficit in defensive responsivity when exposed to potential threat.

Immune signaling as a node of interaction between systems that sex-specifically develop during puberty and adolescence.

Adolescence is pivotal for neural and behavioral development across species. During this period, maturation occurs in several biological systems, the most well-recognized being activation of the hypothalamic-pituitary-gonadal axis marking pubertal onset. Increasing comparative studies of sex differences have enriched our understanding of systems integration during neurodevelopment. In recent years, immune signaling has emerged as a key node of interaction between a variety of biological signaling processes. Herein, we review the age- and sex-specific changes that occur in neural, hypothalamic-pituitary, and microbiome systems during adolescence. We then describe how immune signaling interacts with these systems, and review recent preclinical evidence indicating that immune signaling may play a central role in integrating changes in their typical and atypical development during adolescence. Finally, we discuss the translational relevance of these preclinical studies to human health and wellness.

Insular cortex corticotropin-releasing factor integrates stress signaling with social affective behavior.

Impairments in identifying and responding to the emotions of others manifest in a variety of psychopathologies. Therefore, elaborating the neurobiological mechanisms that underpin social responses to social emotions, or social affective behavior, is a translationally important goal. The insular cortex is consistently implicated in stress-related social and anxiety disorders, which are associated with diminished ability to make and use inferences about the emotions of others to guide behavior. We investigated how corticotropin-releasing factor (CRF), a neuromodulator evoked upon exposure to stressed conspecifics, influenced the insula. We hypothesized that social affective behavior requires CRF signaling in the insular cortex in order to detect stress in social interactions. In acute slices from male and female rats, CRF depolarized insular pyramidal neurons. In males, but not females, CRF suppressed presynaptic GABAergic inhibition leading to greater excitatory synaptic efficacy in a CRF receptor 1 (CRF1)- and cannabinoid receptor 1 (CB1)-dependent fashion. In males only, insular CRF increased social investigation, and CRF1 and CB1 antagonists interfered with social interactions with stressed conspecifics. To investigate the molecular and cellular basis for the effect of CRF we examined insular CRF1 and CB1 mRNAs and found greater total insula CRF1 mRNA in females but greater CRF1 and CB1 mRNA colocalization in male insular cortex glutamatergic neurons that suggest complex, sex-specific organization of CRF and endocannabinoid systems. Together these results reveal a new mechanism by which stress and affect contribute to social affective behavior.

Trajectories of Mother-Infant Communication: An Experiential Measure of the Impacts of Early Life Adversity.

Caretaking stability in the early life environment supports neurobehavioral development, while instability and neglect constitute adverse environments that can alter maturational processes. Research in humans suggests that different types of early life adversity (ELA) can have differential effects on caretaker relationships and later cognitive and social development; however, identifying mechanistic underpinnings will require animal models with translational validity. Two common rodent models, maternal separation (MS) and limited bedding (LB), influence the mother-infant relationship during a critical window of development. We hypothesized that these paradigms may affect the development of communication strategies on the part of the pup. Ultrasonic vocalizations (USVs) are a care-eliciting mechanism and ethologically relevant response to stressors in the rat pup. USV emission rates and acoustic parameters change throughout early development, presenting the opportunity to define developmental milestones in USVs that would reflect neurobehavioral aberrations if disrupted. This study investigated the effects of MS or LB on the dam-pup relationship by quantifying pup USVs, maternal behavior, and the relationship between the two. First, we used a generalized additive model approach to establish typical developmental trajectories of USV acoustic properties and determine windows of change in MS or LB rearing. Additionally, we quantified maternal behaviors and the predictability of maternal care sequences using an entropy rate calculation. MS and LB each shifted the developmental trajectories of USV acoustic parameters and call types in a sex-specific manner. MS more often impacted male USVs, while LB impacted female USVs. MS dams spent more time passive nursing, and LB dams spent more time on the nest. The predictability of maternal care was associated with the rate of USV emissions exclusively in females. Taken together, findings demonstrate sex- and model-specific effects of rearing environments on a novel developmental trajectory involving the mother-infant relationship, facilitating the translation of animal ELA paradigms to assess later-life consequences.

A two-hit adversity model in developing rats reveals sex-specific impacts on prefrontal cortex structure and behavior.

Adversity early in life substantially impacts prefrontal cortex (PFC) development and vulnerability to later-life psychopathology. Importantly, repeated adverse experiences throughout childhood increase the risk for PFC-mediated behavioral deficits more commonly in women. Evidence from animal models points to effects of adversity on later-life neural and behavioral dysfunction; however, few studies have investigated the neurobiological underpinnings of sex-specific, long-term consequences of multiple developmental stressors. We modeled early life adversity in rats via maternal separation (postnatal day (P)2-20) and juvenile social isolation (P21-35). In adulthood, anxiety-like behavior was assessed in the elevated zero maze and the presence and structural integrity of PFC perineuronal nets (PNNs) enwrapping parvalbumin (PV)-expressing interneurons was quantified. PNNs are extracellular matrix structures formed during critical periods in postnatal development that play a key role in the plasticity of PV cells. We observed a female-specific effect of adversity on hyperactivity and risk-assessment behavior. Moreover, females - but not males - exposed to multiple hits of adversity demonstrated a reduction in PFC PV cells in adulthood. We also observed a sex-specific, potentiated reduction in PV + PNN structural integrity. These findings suggest a sex-specific impact of repeated adversity on neurostructural development and implicate PNNs as a contributor to associated behavioral dysfunction.

Region-specific Effects of Maternal Separation on Perineuronal Net and Parvalbumin-expressing Interneuron Formation in Male and Female Rats.

Early life experiences play a vital role in contributing to healthy brain development. Adverse experiences have a lasting impact on the prefrontal cortex (PFC) and basolateral amygdala (BLA), brain regions associated with emotion regulation. Early life adversity via maternal separation (MS) has sex-specific effects on expression of parvalbumin (PV), which is expressed in fast-spiking GABAergic interneurons that are preferentially enwrapped by perineuronal nets (PNNs). Importantly, PNN formation coincides with the closure of developmental critical periods and regulates PV-expressing interneuron activity. Since aberrant PNN organization has been reported following adverse experiences in adolescent and adult rats, we investigated the impact of adversity early in life in the form of MS on the developing brain. Rat pups were separated from their dams for 4 h per day from postnatal day (P) 2-20. Tissue sections from juvenile (P20), adolescent (P40), and early adult (P70) animals containing the PFC and BLA were fluorescently stained to visualize Wisteria floribunda agglutinin+ PNNs and PV-expressing interneurons, and density and intensity was quantified. Our results confirm past reports that PFC PNNs form gradually throughout development; however, PNN density plateaus in adolescence, while intensity continues to increase into adulthood. Importantly, MS delays PNN formation in the prelimbic PFC and results in sex-specific aberrations in PNN structural integrity that do not appear until adulthood. The present findings reveal sex-, age-, and region-specific effects of early life adversity on PNN and PV maturation, implicating neuroplastic alterations following early life adversity that may be associated with sex differences in psychopathology and resilience.

Sex differences in prefrontal cortex microglia morphology: Impact of a two-hit model of adversity throughout development.

Neuroimmune mechanisms play critical roles in brain development and can be impacted by early life adversity. Microglia are the resident immune cells in the brain, with both sex-specific and region-specific developmental profiles. Since early life adversity is associated with several neuropsychiatric disorders with developmental pathogeneses, here we investigated the degree to which maternal separation (MS) impacted microglia over development. Microglia are dynamic cells that alter their morphology in accordance with their functions and in response to stressors. While males and females reportedly display different microglial morphology in several brain regions over development and following immune and psychological challenges, little is known about such differences in the prefrontal cortex (PFC), which regulates several early life adversity-attributable disorders. Additionally, little is known about the potential for early life adversity to prime microglia for later immune challenges. In the current study, male and female rats were exposed to MS followed by lipopolysaccharide administration in juvenility or adolescence. The prelimbic and infralimbic PFC were then separately analyzed for microglial density and morphology. Typically developing males expressed smaller soma and less arborization than females in juvenility, but larger soma than females in adolescence. MS led to fewer microglia in the infralimbic PFC of adolescent males. Both MS and lipopolysaccharide administration affected morphological characteristics in juvenile males and females, with MS exposure leading to a greater increase in soma size following lipopolysaccharide. Interestingly, effects of MS and lipopolysaccharide were not observed in adolescence, while notable sex differences in PFC microglial morphology were apparent. Taken together, these findings provide insight into how PFC microglia may differentially respond to challenges over development in males and females.

Region-specific effects of maternal separation on oxidative stress accumulation in parvalbumin neurons of male and female rats.

Early life adversity in humans is linked to cognitive deficits and increased risk of mental illnesses, including depression, bipolar disorder, and schizophrenia, with evidence for different vulnerabilities in men versus women. Modeling early life adversity in rodents shows similar neuropsychological deficits that may partially be driven by sex-dependent dysfunction in parvalbumin (PV) interneurons in the prefrontal cortex (PFC), hippocampus (HPC), and basolateral amygdala (BLA). Research demonstrates that PV interneurons are particularly susceptible to oxidative stress; therefore, accumulation of oxidative damage may drive PV dysfunction following early life adversity. The goal of this study was to quantify oxidative stress accumulation in PV neurons in rats exposed to maternal separation (MS). Pups were separated from their dam and littermates for 4 h per day from postnatal day (P)2 to 20. Serial sections from the PFC, HPC, and BLA of juvenile (P20) rats of both sexes were immunohistochemically stained with antibodies against PV and 8-oxo-dG, a marker for oxidative DNA damage. PV cell counts, colocalization with 8-oxo-dG, and intensity of each signal were measured in each region to determine the effects of MS and establish whether MS-induced oxidative damage varies between sexes. A significant increase in colocalization of PV and 8-oxo-dG was found in the PFC and HPC, indicating increased oxidative stress in that cell population following MS. Region-specific sex differences were also revealed in the PFC, BLA, and HPC. These data identify oxidative stress during juvenility as a potential mechanism mediating PV dysfunction in individuals with a history of early life adversity.