Early life adversity drives sex-dependent changes in 5-mC DNA methylation of parvalbumin cells in the prefrontal cortex in rats.

Early life adversity (ELA) can result in increased risk for developing affective disorders, such as anxiety or depression, later in life, with women showing increased risk. Interactions between an individual's genes and their environment play key roles in producing, as well as mitigating, later life neuropathology. Our current understanding of the underlying epigenomic drivers of ELA associated anxiety and depression are limited, and this stems in part from the complexity of underlying biochemical processes associated with how early experiences shapes later life behavior. Epigenetic alterations, or experience-driven modifications to DNA, can be leveraged to understand the interplay between genes and the environment. The present study characterized DNA methylation patterning, assessed via evaluation of 5-methylcytosine (5-mC), following ELA in a Sprague Dawley rat model of ELA induced by early caregiver deprivation. This study utilized maternal separation to investigate sex- and age-specific outcomes of ELA on epigenetic patterning in parvalbumin (PV)-containing interneurons in the prefrontal cortex (PFC), a subpopulation of inhibitory neurons which are associated with ELA and affective dysfunction. While global analysis of 5-mC methylation and CpG site specific pyrosequencing of the PV promoter, Pvalb, showed no obvious effects of ELA, when analyses were restricted to assessing 5-mC intensity in colocalized PV cells, there were significant sex and age dependent effects. We found that ELA leads sex-specific changes in PV cell counts, and that cell counts can be predicted by 5-mC intensity, with males and females showing distinct patterns of methylation and PV outcomes. ELA also produced sex-specific effects in corticosterone reactivity, with juvenile females showing a blunted stress hormone response compared to controls. Overall, ELA led to a sex-specific developmental shift in PV profile, which is comparable to profiles that are seen at a later developmental timepoint, and this shift may be mediated in part by epigenomic alterations driven by altered DNA methylation.

Playback of rat 22-kHz ultrasonic vocalizations as a translational assay of negative affective states: An analysis of evoked behavior and brain activity.

The subjective nature of human emotions makes them uniquely challenging to investigate in preclinical models. While behavioral assays in rodents aim to evaluate affect (i.e., anxiety, hypervigilance), they often lack ethological validity. Playback of negatively valenced 22-kHz ultrasonic vocalizations (USVs) in rats shows promise as a translational tool to investigate affective processing. Much like how human facial expressions can communicate internal states, rats emit 22-kHz USVs that similarly convey negative affective states to conspecifics indicating possible threat. 22-kHz USV playback elicits avoidance and hypervigilant behaviors, and recruit brain regions comparable to those seen in human brains evoked by viewing fearful faces. Indeed, 22-kHz playback alters neural activity in brain regions associated with negative valence systems (i.e., amygdala, bed nucleus of the stria terminalis, periaqueductal gray) alongside increases in behaviors typically associated with anxiety. Here, we present evidence from the literature that supports leveraging 22-kHz USV playback in rat preclinical models to obtain clinically relevant and translational findings to identify the neural underpinnings of affective processing and neuropathological dysfunction.

Sex Differences in Affective Dysfunction and Alterations in Parvalbumin in Rodent Models of Early Life Adversity.

The early life environment markedly influences brain and behavioral development, with adverse experiences associated with increased risk of anxiety and depressive phenotypes, particularly in females. Indeed, early life adversity (ELA) in humans (i.e., caregiver deprivation, maltreatment) and rodents (i.e., maternal separation, resource scarcity) is associated with sex-specific emergence of anxious and depressive behaviors. Although these disorders show clear sex differences in humans, little attention has been paid toward evaluating sex as a biological variable in models of affective dysfunction; however, recent rodent work suggests sex-specific effects. Two widely used rodent models of ELA approximate caregiver deprivation (i.e., maternal separation) and resource scarcity (i.e., limited bedding). While these approaches model aspects of ELA experienced in humans, they span different portions of the pre-weaning developmental period and may therefore differentially contribute to underlying mechanistic risk. This is borne out in the literature, where evidence suggests differences in trajectories of behavior depending on the type of ELA and/or sex; however, the neural underpinning of these differences is not well understood. Because anxiety and depression are thought to involve dysregulation in the balance of excitatory and inhibitory signaling in ELA-vulnerable brain regions (e.g., prefrontal cortex, amygdala, hippocampus), outcomes are likely driven by alterations in local and/or circuit-specific inhibitory activity. The most abundant GABAergic subtypes in the brain, accounting for approximately 40% of inhibitory neurons, contain the calcium-binding protein Parvalbumin (PV). As PV-expressing neurons have perisomatic and proximal dendritic targets on pyramidal neurons, they are well-positioned to regulate excitatory/inhibitory balance. Recent evidence suggests that PV outcomes following ELA are sex, age, and region-specific and may be influenced by the type and timing of ELA. Here, we suggest the possibility of a combined role of PV and sex hormones driving differences in behavioral outcomes associated with affective dysfunction following ELA. This review evaluates the literature across models of ELA to characterize neural (PV) and behavioral (anxiety- and depressive-like) outcomes as a function of sex and age. Additionally, we detail a putative mechanistic role of PV on ELA-related outcomes and discuss evidence suggesting hormone influences on PV expression/function which may help to explain sex differences in ELA outcomes.

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.

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.

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.

Parvalbumin Loss Following Chronic Sub-Anesthetic NMDA Antagonist Treatment is Age-Dependent in the Hippocampus: Implications for Modeling NMDA Hypofunction.

A marked decrease in parvalbumin (PV), a calcium-binding protein specific to a subset of GABAergic neurons, is a consistent finding in postmortem schizophrenic brain tissue. This reduction is selective to PV and is regionally specific, occurring primarily in the prefrontal cortex and hippocampus (HPC) of patients. Rodent models of NMDA receptor hypofunction utilizing NMDA antagonist treatments - e.g. ketamine (KET) - show schizophrenia-like cognitive and behavioral impairments with parallel changes in PV. While decreased PV is considered a hallmark of neuropathology in schizophrenia, previous work elucidating the effects of KET administration on PV are contradictory, with findings suggesting decreased, increased, or no change in PV expression. Upon close examination of the procedures used across studies, there are two primary inconsistencies, including: (1) the age of animals used; and (2) the timeline of post-treatment tissue collection. To better understand whether these key differences impact observed PV changes, the present study investigated the impact of age and time of sacrifice on chronic KET-induced PV changes in the neocortex and HPC. Our findings suggest an effect of age, but not sacrifice timeline, on PV cell count following 14 days of sub-anesthetic KET treatment. We provide evidence that 1-month-old rats exhibit a significant KET-induced HPC PV decrease, while adult rats show a modest increase in HPC PV following chronic KET. Taken together, we propose that PV is a dynamic marker, and that changes in cell counts - and their interpretation - following NDMA antagonist treatment should be considered in the context of age.

RNaseT2 knockout rats exhibit hippocampal neuropathology and deficits in memory.

RNASET2 deficiency in humans is associated with infant cystic leukoencephalopathy, which causes psychomotor impairment, spasticity and epilepsy. A zebrafish mutant model suggests that loss of RNASET2 function leads to neurodegeneration due to the accumulation of non-degraded RNA in the lysosomes. The goal of this study was to characterize the first rodent model of RNASET2 deficiency. The brains of 3- and 12-month-old RNaseT2 knockout rats were studied using multiple magnetic resonance imaging modalities and behavioral tests. While T1- and T2-weighted images of RNaseT2 knockout rats exhibited no evidence of cystic lesions, the prefrontal cortex and hippocampal complex were enlarged in knockout animals. Diffusion-weighted imaging showed altered anisotropy and putative gray matter changes in the hippocampal complex of the RNaseT2 knockout rats. Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed the presence of hippocampal neuroinflammation. Decreased levels of lysosome-associated membrane protein 2 (LAMP2) and elevated acid phosphatase and ß-N-acetylglucosaminidase (NAG) activities indicated that the RNASET2 knockout rats likely had altered lysosomal function and potential defects in autophagy. Object recognition tests confirmed that RNaseT2 knockout rats exhibited memory deficits. However, the Barnes maze, and balance beam and rotarod tests indicated there were no differences in spatial memory or motor impairments, respectively. Overall, patients with RNASET2 deficiency exhibited a more severe neurodegeneration phenotype than was observed in the RNaseT2 knockout rats. However, the vulnerability of the knockout rat hippocampus as evidenced by neuroinflammation, altered lysosomal function and cognitive defects indicates that this is still a useful in vivo model to study RNASET2 function.

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.

Cognitive impairment effects of early life stress in adolescents can be predicted with early biomarkers: Impacts of sex, experience, and cytokines.

Childhood adversity increases vulnerability to psychiatric disorders that emerge in adolescence, in a sex-dependent manner. Early adversity modeled in rodents with maternal separation (MS) affects cognition and medial prefrontal cortex (mPFC) circuitry. Humans and animals exposed to early life adversity also display heightened circulating inflammatory cytokines, however the predictive relationship of these early measures with later behavioral deficits is unknown. Here, male and female rats were exposed to MS or control rearing during the postnatal period (P2-21). Blood samples were taken at distinct developmental time points for analysis of the pro-inflammatory cytokine IL-1ß and the anti-inflammatory cytokines IL-4, and IL-10, followed by win-shift cognitive testing and analysis of mPFC parvalbumin (PVB) immunofluorescent interneurons in adolescence. Regression analyses were conducted to explore the relationship between early cytokines and adolescent behavioral measures. We observed sex- and age-dependent effects of MS on circulating cytokines. MS also yielded adolescent decreases in mPFC PVB and cognitive deficits, which were predicted by early cytokine expression in a sex- and experience-dependent manner. Taken together, the present data reveals that circulating cytokines and PVB levels are predictive of adolescent cognitive deficits, and therefore provide compelling evidence for a putative role of early biomarkers in mediating MS-induced behavioral dysfunction. Importantly, predictive relationships often depended on sex and on MS history, suggesting that early life experiences may yield individualistic mechanisms of vulnerability compared to the general population.

Developmental Age Differentially Mediates the Calcium-Binding Protein Parvalbumin in the Rat: Evidence for a Selective Decrease in Hippocampal Parvalbumin Cell Counts.

Local circuit GABAergic neurons, including parvalbumin (PV)-containing basket cells, likely play a key role in the development, physiology, and pathology of neocortical circuits. Regionally selective and well-defined decreases in PV have been described in human postmortem schizophrenic brain tissue in both the hippocampus and prefrontal cortex. Animal models of schizophreniform dysfunction following acute and/or chronic ketamine treatment have also demonstrated decreases in PV expression. Conflicting reports with respect to PV immunoreactivity following acute and chronic ketamine treatments in rodents question the utility of using PV as a biological marker of pathology-related dysfunction. The current literature lacks sufficient and systematic characterization of normative PV expression in pharmacologically and behaviorally naïve rodent tissue. In order to understand developmental changes in PV and its putative role in neuropathology, we examined the baseline distribution of the number of cells expressing this protein at distinct developmental ages. The present study examined PV cell counts across the septotemporal axis of the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus, as well as within the retrosplenial, somatosensory, and prefrontal cortices, in 1-, 6-, and 12-month-old naïve rats. Our findings suggest that the hippocampal PV+ cell number significantly decreases as a function of age with considerable regional (CA1, CA3, and DG) and septotemporal variation, a finding that was specific to the hippocampus. Additionally, we observed a modest increase in PV cell number within the prefrontal (anterior cingulate) cortex, which is in line with findings indicating a delayed developmental maturation of this region. The present work highlights decreases in PV+ cell counts within the hippocampus across development, and points to the need for a greater understanding of the role of PV and local circuit developmental changes, as well as consideration of their development when modeling developmentally related neuropathological disorders (e.g. schizophrenia, autism).