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.

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.

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.

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.

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.

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.

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.

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.

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.

Altered corticolimbic connectivity reveals sex-specific adolescent outcomes in a rat model of early life adversity.

Exposure to early-life adversity (ELA) increases the risk for psychopathologies associated with amygdala-prefrontal cortex (PFC) circuits. While sex differences in vulnerability have been identified with a clear need for individualized intervention strategies, the neurobiological substrates of ELA-attributable differences remain unknown due to a paucity of translational investigations taking both development and sex into account. Male and female rats exposed to maternal separation ELA were analyzed with anterograde tracing from basolateral amygdala (BLA) to PFC to identify sex-specific innervation trajectories through juvenility (PD28) and adolescence (PD38;PD48). Resting-state functional connectivity (rsFC) was assessed longitudinally (PD28;PD48) in a separate cohort. All measures were related to anxiety-like behavior. ELA-exposed rats showed precocial maturation of BLA-PFC innervation, with females affected earlier than males. ELA also disrupted maturation of female rsFC, with enduring relationships between rsFC and anxiety-like behavior. This study is the first providing both anatomical and functional evidence for sex- and experience-dependent corticolimbic development.

Having a traumatic childhood increases the risk a person will develop anxiety disorders later in life. Early life adversity affects men and women differently, but scientists do not yet know why. Learning more could help scientists develop better ways to prevent or treat anxiety disorders in men and women who experienced childhood trauma. Anxiety occurs when threat-detecting brain circuits turn on. These circuits begin working in infancy, and during childhood and adolescence, experiences shape the brain to hone the body's responses to perceived threats. Two areas of the brain that are important hubs for anxiety-related brain circuits include the basolateral amygdala (BLA) and the prefrontal cortex (PFC). Now, Honeycutt et al. show that rats that experience early life adversity develop stronger connections between the BLA and PFC, and these changes occur earlier in female rats. In the experiments, one group of rats was repeatedly separated from their mothers and littermates (an early life trauma), while a second group was not. Honeycutt et al. examined the connections between the BLA and PFC in the two groups at three different time periods during their development: the juvenile stage, early adolescence, and late adolescence. The experiments showed stronger connections between the BLA and PFC begin to appear earlier in juvenile traumatized female rats. But these changes did not appear in their male counterparts until adolescence. Lastly, the rats that developed these strengthened BLA-PFC connections also behaved more anxiously later in life. This may mean that the ideal timing for interventions may be different for males and females. More work is needed to see if these results translate to humans and then to find the best times and methods to help people who experienced childhood trauma.

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.

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.

Cross-Generational Transmission of Early Life Stress Effects on HPA Regulators and Bdnf Are Mediated by Sex, Lineage, and Upbringing.

Early life stress (ELS) is a potent developmental disruptor and increases the risk for psychopathology. Various forms of ELS have been studied in both humans and rodents, and have been implicated in altered DNA methylation, gene transcription, stress hormone levels, and behavior. Although recent studies have focused on stress-induced epigenetic changes, the extent to which ELS alters HPA axis function and stress responsivity across generations, whether these effects are sex-specific, and how lineage interacts with upbringing to impact these effects, remain unclear. To address these points, two generations of rodents were utilized, with the first generation subjected to ELS via maternal separation, and the second to a balanced cross-fostering paradigm. We hypothesized that ELS would disrupt normative development in both generations, manifesting as altered methylation and expression of genes associated with stress signaling pathways (Nr3c1, Nr3c2, and Bdnf), blunted corticosterone (CORT), and anxiety-like behaviors. Additionally, we expected deficits in the second generation to be modulated by caretaking environment and for the pattern of results to differ between the sexes. Results suggest that direct exposure to ELS leads to sex-specific effects on gene regulation and HPA functioning in adulthood, with maternal separation leading to increases in Bdnf methylation in both sexes, decreases in Bdnf expression in females, and decreases in Nr3c1 methylation in males, as well as blunted CORT and less anxiety-like behavior in females. These alterations converged with caretaking to impart perturbations upon the subsequent generation. Across sex, ELS lineage led to decreased methylation of Nr3c1, and increased methylation of Bdnf. In fostered animals, upbringing by a previously stressed mother interacted with offspring lineage to impact methylation of Nr3c1 and Bdnf. Upbringing was also implicated in altered anxiety-like behavior in males, and baseline CORT levels in females. Such effects may correspond with observed alterations in maternal behavior across groups. In conclusion, ELS conferred enduring sex-specific alterations, both first-hand and trans-generationally via lineage and upbringing. Importantly, lineage of cross-fostered pups was sufficient to normalize or disturb maternal behavior of foster-dams, an observation requiring further elucidation. These results have implications for multi-generational effects of ELS in humans and may motivate early interventions.

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.

Neuroimmune Impacts of Early-Life Stress on Development and Psychopathology.

Maltreatment and trauma in childhood, termed early-life stress (ELS), has long-term effects on the immune system. ELS impacts immune signaling at the time of exposure but also disrupts the developmental trajectory of certain immunological processes, both in the periphery and in the brain. One consequence of these early alterations is a heightened immune response to stressors later in life. However, chronic and sustained inflammatory response can also lead to excitotoxicity and prevent typical brain development. In this chapter, we discuss current progress toward understanding the contribution of neuroimmune signaling to ELS-attributable dysfunction or maladaptation with a focus on postnatal experiences. To do so we first present an operational definition of ELS. Then, we offer a brief overview of the immune system and neuroimmune development, followed by a section discussing the interaction between immunity, childhood trauma, and mental disorders in humans. We present evidence from animal models about immune alterations after ELS and discuss the ways in which ELS-induced immune changes ultimately affect brain and behavior, as well as the importance of individual differences and future directions in this field. Taken together, we submit that when encountered with ELS, some core brain circuits could develop differently via various mechanisms involving dysfunctional immune reprograming. However, given the remarkable plasticity of both the brain and the immune system, many of the deleterious effects of ELS may be mitigated with interventions that account for sex and target neuroimmune interactions over the lifespan.

22 kHz and 55 kHz ultrasonic vocalizations differentially influence neural and behavioral outcomes: Implications for modeling anxiety via auditory stimuli in the rat.

The communicative role of ultrasonic vocalizations (USVs) in rats is well established, with distinct USVs indicative of different affective states. USVs in the 22 kHz range are typically emitted by adult rats when in anxiety- or fear-provoking situations (e.g. predator odor, social defeat), while 55 kHz range USVs are typically emitted in appetitive situations (e.g., play, anticipation of reward). Previous work indicates that USVs (real-time and playback) can effectively communicate these affective states and influence changes in behavior and neural activity of the receiver. Changes in cFos activation following 22 kHz USVs have been seen in cortical and limbic regions involved in anxiety, including the basolateral amygdala (BLA). However, it is unclear how USV playback influences cFos activity within the bed nucleus of the stria terminalis (BNST), a region also thought to be critical in processing anxiety-related information, and the nucleus accumbens, a region associated with reward. The present work sought to characterize distinct behavioral, physiological, and neural responses in rats presented with aversive (22 kHz) compared to appetitive (55 kHz) USVs or silence. Our findings show that rats exposed to 22 kHz USVs: 1) engage in anxiety-like behaviors in the elevated zero maze, and 2) show distinct patterns of cFos activation within the BLA and BNST that contrast those seen in 55 kHz playback and silence. Specifically, 22 kHz USVs increased cFos density in the anterodorsal nuclei, while 55 kHz playback increased cFos in the oval nucleus of the BNST, without significant changes within the nucleus accumbens. These results provide important groundwork for leveraging ethologically-relevant stimuli in the rat to improve our understanding of anxiety-related responses in both typical and pathological populations.

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 Water Bottle Materials and Filtration on Bisphenol A Content in Laboratory Animal Drinking Water.

Bisphenol A (BPA) is widely used in the polycarbonate plastics and epoxy resins that are found in laboratory animal husbandry materials including cages and water bottles. Concerns about BPA exposure in humans has led to investigations that suggest physiologic health risks including disruptions to the endocrine system and CNS. However, the extent of exposure of laboratory animals to BPA in drinking water is unclear. In the first study, we compared the amount of BPA contamination in water stored in plastic bottles used in research settings with that in glass bottles. The amount of BPA that leached into water was measured across several time points ranging from 24 to 96 h by using a BPA ELISA assay. The results showed that considerable amounts of BPA (approximately 0.15 µg/L) leached from polycarbonate bottles within the first 24 h of storage. In the second study, BPA levels were measured directly from water taken from filtered compared with unfiltered taps. We observed significantly higher BPA levels in water from unfiltered taps (approximately 0.40 µg/L) compared with taps with filtration systems (approximately 0.04 µg/L). Taken together, our findings indicate that the use of different types of water bottles and water sources, combined with the use of different laboratory products (food, caging systems) between laboratories, likely contribute to decreased rigor and reproducibility in research. We suggest that researchers consider reporting the types of water bottles used and that animal care facilities educate staff regarding the importance of flushing nonfiltered water taps when filling animal water bottles.