Investigating Resistance to Antidepressants in Animal Models.

Major depressive disorder is one of the most prevalent psychiatric diseases, and up to 30-40% of patients remain symptomatic despite treatment. Novel therapies are sorely needed, and animal models may be used to elucidate fundamental neurobiological processes that contribute to human disease states. We conducted a systematic review of current preclinical approaches to investigating treatment resistance with the goal of describing a path forward for improving our understanding of treatment resistant depression. We conducted a broad literature search to identify studies relevant to the preclinical investigation of treatment resistant depression. We followed PRISMA (Preferred Reporting Items for Systemic Reviews and Meta-Analyses) guidelines and included all relevant studies. We identified 467 studies in our initial search. Of these studies, we included 69 in our systematic review after applying our inclusion/exclusion criteria. We identified 10 broad strategies for investigating treatment resistance in animal models. Stress hormone administration was the most commonly used model, and the most common behavioral test was the forced swim test. We systematically identified and reviewed current approaches for gaining insight into the neurobiology underlying treatment resistant depression using animal models. Each approach has its advantages and disadvantages, but all require careful consideration of their potential limitations regarding therapeutic translation. An enhanced understanding of treatment resistant depression is sorely needed given the burden of disease and lack of effective therapies.

Differential recruitment of brain circuits during fear extinction in non-stressed compared to stress resilient animals.

Dysfunctional fear responses in post-traumatic stress disorder (PTSD) may be partly explained by an inability to effectively extinguish fear responses elicited by trauma-related cues. However, only a subset of individuals exposed to traumatic stress develop PTSD. Therefore, studying fear extinction deficits in animal models of individual differences could help identify neural substrates underlying vulnerability or resilience to the effects of stress. We used a rat model of social defeat in which rats segregate into passively and actively coping rats. In previous work, we showed that passively coping rats exhibit disruptions in social interaction whereas actively coping rats do not display behaviors differently from controls, indicating their resilience. Here, adult male rats exposed to 7 days of social defeat were tested for fear extinction, retention of extinction, and persistence of retention using contextual fear and ethologically-relevant fear tests. Passively coping rats exhibited elevated freezing in response to the previously extinguished context. Analyses of cFos expressing cells across select brain regions showed high correlations within dorsal hippocampal subregions, while passively coping rats had high correlations between the dorsal hippocampus CA1 and the central and basolateral subregions of the amygdala. Importantly, although control and actively coping rats showed similar levels of behavioral extinction, there was little similarity between activated structures, suggesting stress resilience in response to chronic social defeat involves an adaptive differential recruitment of brain circuits to successfully extinguish fear memories.

The importance of REM sleep fragmentation in the effects of stress on sleep: Perspectives from preclinical studies.

Psychological stress poses a risk for sleep disturbances. Importantly, trauma-exposed individuals who develop posttraumatic stress disorder (PTSD) frequently report insomnia and recurrent nightmares. Clinical studies have provided insight into the mechanisms of these sleep disturbances. We review polysomnographic findings in PTSD and identify analogous measures that have been made in animal models of PTSD. There is a rich empirical and theoretical literature on rapid eye movement sleep (REMS) substrates of insomnia and nightmares, with an emphasis on REMS fragmentation. For future investigations of stress-induced sleep changes, we recommend a focus on tonic, phasic and other microarchitectural REMS measures. Power spectral density analysis of the sleep EEG should also be utilized. Animal models with high construct validity can provide insight into gender and time following stressor exposure as moderating variables. Ultimately, preclinical studies with translational potential will lead to improved treatment for stress-related sleep disturbances.

Complete genome resource unravels the close relation of an Indian Xanthomonas oryzae pv. oryzae strain IXOBB0003 with Philippines strain causing bacterial blight of rice.

Xanthomonas oryzae pv. oryzae (Xoo) is a pathogen of concern for rice growers as it limits the production potential of rice varieties worldwide. Due to their high genomic plasticity, the pathogen continues to evolve, nullifying the deployed resistance mechanisms. It is pertinent to monitor the evolving Xoo population for the virulent novel stains, and the affordable sequencing technologies made the task feasible with an in-depth understanding of their pathogenesis arsenals. We present the complete genome of a highly virulent Indian Xoo strain IXOBB0003, predominantly found in northwestern parts of India, by employing next-generation sequencing and single-molecule sequencing in real-time technologies. The final genome assembly comprises 4,962,427 bp and has 63.96% GC content. The pan genome analysis reveals that strain IXOBB0003 houses total of 3655 core genes, 1276 accessory genes and 595 unique genes. Comparative analysis of the predicted gene clusters of coding sequences and protein count of strain IXOBB0003 depicts 3687 of almost 90% gene clusters shared by other Asian strains, 17 unique to IXOBB0003 and 139 CDSs of IXOBB0003 are shared with PXO99A. AnnoTALE-based studies revealed 16 TALEs conferred from the whole genome sequence. Prominent TALEs of our strain are found orthologous to TALEs of the Philippines strain PXO99A. The genomic features of Indian Xoo strain IXOBB0003 and in comparison with other Asian strains would certainly contribute significantly while formulating novel strategies for BB management. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03596-x.

Preclinical and translational models for delirium: Recommendations for future research from the NIDUS delirium network.

Delirium is a common, morbid, and costly syndrome that is closely linked to Alzheimer's disease (AD) and AD-related dementias (ADRD) as a risk factor and outcome. Human studies of delirium have advanced our knowledge of delirium incidence and prevalence, risk factors, biomarkers, outcomes, prevention, and management. However, understanding of delirium neurobiology remains limited. Preclinical and translational models for delirium, while challenging to develop, could advance our knowledge of delirium neurobiology and inform the development of new prevention and treatment approaches. We discuss the use of preclinical and translational animal models in delirium, focusing on (1) a review of current animal models, (2) challenges and strategies for replicating elements of human delirium in animals, and (3) the utility of biofluid, neurophysiology, and neuroimaging translational markers in animals. We conclude with recommendations for the development and validation of preclinical and translational models for delirium, with the goal of advancing awareness in this important field.

Sex-specific alterations in corticotropin-releasing factor regulation of coerulear-cortical network activity.

The locus coeruleus (LC)-norepinephrine system is a stress responsive system that regulates arousal and cognitive functions through extensive projections, including to the prefrontal cortex. LC-cortical circuits are activated by stressors, and this activation is thought to contribute to stress-induced impairments in executive function. Because corticotropin-releasing factor (CRF) is a mediator of stress-induced LC activation, we examined the effects of CRF administered into the LC of male and female rats on network activity of two functionally distinct regions of the PFC, the medial PFC (mPFC) and the orbitofrontal cortex (OFC). Network activity, measured as local field potentials, was recorded in awake animals before and after intra-LC infusion of aCSF or CRF (2 or 20 ng). CRF had qualitatively distinct effects on network activity in males and females with respect to dose, region and timecourse. CRF (20 ng) produced a prominent theta oscillation (7-9 Hz) selectively in female rats shortly after LC infusion and 20 min later. In contrast, in male rats, CRF (2 and 20 ng) decreased the amplitude of power in the 4-6 Hz range in the mPFC 10 min after injection. Lastly, CRF (20 ng) increased mPFC-OFC coherence in females and decreased mPFC-OFC coherence in males. In sum, these results show sex differences in CRF modulation of the LC-norepinephrine system that regulates prefrontal cortical networks, which may underlie sex differences in cognitive and behavioral responses to stress.

The association of polysomnographic sleep on posttraumatic stress disorder symptom clusters in trauma-exposed civilians and veterans.

Study Objectives: Self-reported sleep disturbance has been established as a risk factor and predictor for posttraumatic stress disorder (PTSD); however, less is known about the relationship between objective sleep and PTSD symptom clusters, and the specific role of hyperarousal. The present study examined the relationships between sleep continuity and architecture on PTSD symptom clusters. Methods: Participants underwent two in-laboratory sleep studies to assess sleep continuity and architecture. They also completed the Clinician-Administered PTSD-IV scale and the Structured Clinical Interview for the DSM-IV to assess for PTSD diagnosis and other psychiatric disorders. Results: Sleep continuity (i.e. total sleep time, sleep efficiency percent, wake after sleep onset, sleep latency) was significantly related to PTSD Cluster B (reexperiencing) symptom severity (R 2 = .27, p < .001). Sleep architecture, specifically Stage N1 sleep, was significantly associated with PTSD Cluster B (t = 2.98, p = .004), C (Avoidance; t = 3.11, p = .003), and D (Hyperarosual; t = 3.79, p < .001) symptom severity independently of Stages N2, N3, and REM sleep. REM sleep variables (i.e. REM latency, number of REM periods) significantly predicted Cluster D symptoms (R 2 = .17, p = .002). Conclusions: These data provide evidence for a relationship between objective sleep and PTSD clusters, showing that processes active during Stage N1 sleep may contribute to PTSD symptomatology in civilians and veterans. Further, these data suggest that arousal mechanisms active during REM sleep may also contribute to PTSD hyperarousal symptoms.This paper is part of the War, Trauma, and Sleep Across the Lifespan Collection. This collection is sponsored by the Sleep Research Society.

Sex differences in electrophysiological properties and voltage-gated ion channel expression in the paraventricular thalamic nucleus following repeated stress.

BACKGROUND: Habituation to repeated stress refers to a progressive reduction in the stress response following multiple exposures to the same, predictable stressor. We previously demonstrated that the posterior division of the paraventricular thalamic nucleus (pPVT) nucleus regulates habituation to 5 days of repeated restraint stress in male rats. Compared to males, female rats display impaired habituation to 5 days of restraint. To better understand how activity of pPVT neurons is differentially impacted in stressed males and females, we examined the electrophysiological properties of pPVT neurons under baseline conditions or following restraint. METHODS: Adult male and female rats were exposed to no stress (handling only), a single period of 30 min restraint or 5 daily exposures to 30 min restraint. 24 h later, pPVT tissue was prepared for recordings. RESULTS: We report here that spontaneous excitatory post-synaptic current (sEPSC) amplitude was increased in males, but not females, following restraint. Furthermore, resting membrane potential of pPVT neurons was more depolarized in males. This may be partially due to reduced potassium leakage in restrained males as input resistance was increased in male, but not female, rats 24 h following 1 or 5 days of 30-min restraint. Reduced potassium efflux during action potential firing also occurred in males following a single restraint as action potential half-width was increased following a single restraint. Restraint had limited effects on electrophysiological properties in females, although the mRNA for 10 voltage-gated ion channel subunits was altered in the pPVT of female rats. CONCLUSIONS: The results suggest that restraint-induced changes in pPVT activation promote habituation in males. These findings are the first to describe a sexual dimorphism in stress-induced electrophysiological properties and voltage-gated ion channel expression in the pPVT. These results may explain, at least in part, why habituation to 5 days of restraint is disrupted in female rats.

Arc-Mediated Plasticity in the Paraventricular Thalamic Nucleus Promotes Habituation to Stress.

BACKGROUND: Habituation is defined as a progressive decline in response to repeated exposure to a familiar and predictable stimulus and is highly conserved across species. Disrupted habituation is a signature of posttraumatic stress disorder. In rodents, habituation is observed in neural, neuroendocrine, and behavioral responses to repeated exposure to predictable and moderately intense stress or restraint. We previously demonstrated that lesioning the posterior paraventricular thalamic nucleus (pPVT) impairs habituation. However, the underlying molecular mechanisms and specific neural connections among the pPVT and other brain regions that underlie habituation are unknown. METHODS: Behavioral and neuroendocrine habituation was assessed in adult male Sprague Dawley rats using the repeated restraint paradigm. Pan-neuronal and Cre-dependent DREADDs (designer receptors exclusively activated by designer drugs) were used to chemogenetically inhibit the pPVT and the subpopulation of pPVT neurons that project to the medial prefrontal cortex (mPFC), respectively. Activity-regulated cytoskeleton-associated protein (Arc) expression was knocked down in the pPVT using small interfering RNA. Structural plasticity of pPVT neurons was assessed using Golgi staining. Local field potential recordings were used to assess coherent neural activity between the pPVT and mPFC. The attentional set shifting task was used to assess mPFC-dependent behavior. RESULTS: Here, we show that Arc promotes habituation by increasing stress-induced spinogenesis in the pPVT, increasing coherent neural activity with the mPFC, and improving mPFC-mediated cognitive flexibility. CONCLUSIONS: Our results demonstrate that Arc induction in the pPVT regulates habituation and mPFC function. Therapies that improve synaptic plasticity during posttraumatic stress disorder therapy may enhance habituation and the efficacy of posttraumatic stress disorder treatment.

"Smart" drug delivery: A window to future of translational medicine.

Chemotherapy is the mainstay of cancer treatment today. Chemotherapeutic drugs are non-selective and can harm both cancer and healthy cells, causing a variety of adverse effects such as lack of specificity, cytotoxicity, short half-life, poor solubility, multidrug resistance, and acquiring cancer stem-like characteristics. There is a paradigm shift in drug delivery systems (DDS) with the advent of smarter ways of targeted cancer treatment. Smart Drug Delivery Systems (SDDSs) are stimuli responsive and can be modified in chemical structure in response to light, pH, redox, magnetic fields, and enzyme degradation can be future of translational medicine. Therefore, SDDSs have the potential to be used as a viable cancer treatment alternative to traditional chemotherapy. This review focuses mostly on stimuli responsive drug delivery, inorganic nanocarriers (Carbon nanotubes, gold nanoparticles, Meso-porous silica nanoparticles, quantum dots etc.), organic nanocarriers (Dendrimers, liposomes, micelles), antibody-drug conjugates (ADC) and small molecule drug conjugates (SMDC) based SDDSs for targeted cancer therapy and strategies of targeted drug delivery systems in cancer cells.

Rethinking stress resilience.

Resilience to stressful life events has received considerable attention in both clinical and preclinical studies. A number of neural substrates have been identified as putatively mediating resilience to stress. However, there remains considerable diversity in how resilience is defined and studied. This article aims to examine how resilience is defined and conceptualized in social psychology, public health, and related fields, to better inform the understanding of stress resilience in the neurobiological context, and to differentiate resilience from other patterns of response to stressful experiences. An understanding of resilience through the lens of clinical and applied sciences is likely to lead to the identification of more robust and reproducible neural substrates, though many challenges remain.

FTY720 (Fingolimod), a modulator of sphingosine-1-phosphate receptors, increases baseline hypothalamic-pituitary adrenal axis activity and alters behaviors relevant to affect and anxiety.

FTY720 (fingolimod) is an analog of sphingosine, a ubiquitous sphingolipid. Phosphorylated FTY720 (FTY720-P) non-selectively binds to sphingosine-1-phosphate receptors (S1PRs) and regulates multiple cellular processes including cell proliferation, inflammation, and vascular remodeling. We recently demonstrated that S1PR3 expression in the medial prefrontal cortex (mPFC) of rats promotes stress resilience and that S1PR3 expression in blood may serve as a biomarker for PTSD. Here we investigate the effects of FTY720 in regulating the stress response. We found that single and repeated intraperitoneal injections of FTY720 increased baseline plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations. FTY720 reduced social anxiety- and despair-like behavior as assessed by increased social interaction time and reduced time spent immobile in the Porsolt forced swim test. In blood, FTY720 administration reduced lymphocyte and reticulocyte counts, but raised erythrocyte counts. FTY720 also reduced mRNA of angiopoietin 1, endothelin 1, plasminogen, TgfB2, Pdgfa, and Mmp2 in the medial prefrontal cortex, suggesting that FTY720 reduced vascular remodeling. The antidepressant-like and anxiolytic-like effects of FTY720 may be attributed to reduced vascular remodeling as increased stress-induced blood vessel density in the brain contributes to behavior associated with vulnerability in rats. Together, these results demonstrate that FTY720 regulates baseline HPA axis activity but reduces social anxiety and despair, providing further evidence that S1PRs are important and novel regulators of stress-related functions.

Advancements in folate receptor targeting for anti-cancer therapy: A small molecule-drug conjugate approach.

Targeted delivery combined with controlled release of drugs has a crucial role in future of personalized medicine. The majority of cancer drugs are intended to interfere with one or more cellular events. Anticancer agents can also be toxic to healthy cells, as healthy cells may also need to proliferate and avoid apoptosis. The focus of this review covers the principles, advantages, drawbacks and summarize criteria that must be met for design of small molecule-drug conjugates (SMDCs) to achieve the desired therapeutic potency with minimal toxicity. SMDCs are composed of a targeting ligand, a releasable bridge, a spacer, and a therapeutic payload. We summarize the criteria for the effective design that influences the selection of tumor specific receptor and optimum elements in the design of SMDCs. We also discuss the criteria for selecting the optimal therapeutic drug payload, spacer and linker. The linker chemistries and cleavage strategies are also discussed. Finally, we review the folate receptor targeting SMDCs that are in preclinical development and in clinical trials.

Sex differences in stress-induced sleep deficits.

Sleep disruptions are hallmarks in the pathophysiology of several stress-related disorders, including Major Depressive Disorder (MDD) and Post-Traumatic Stress Disorder (PTSD), both known to disproportionately affect female populations. Although previous studies have attempted to investigate disordered sleep in women, few studies have explored and compared how repeated stress affects sleep in both sexes in either human or animal models. We have previously shown that male rats exhibit behavioral and neuroendocrine habituation to 5 days of repeated restraint, whereas females do not; additional days of stress exposure are required to observe habituation in females. This study examined sex differences in sleep measures prior to, during, and after repeated restraint stress in adult male and female rats. Our data reveal that repeated stress increased time spent awake and decreased slow-wave sleep (SWS) and REM sleep (REMS) in females, and these effects persisted over 2 days of recovery. In contrast, the effects of stress on males were transient. These insomnia-like symptoms were accompanied by a greater number of exaggerated motor responses to waking from REMS in females, a phenotype similar to trauma-related nightmares. In sum, these data demonstrate that repeated stress produces disruptions in sleep that persist days after the stress is terminated in female rats. These disruptions in sleep produced by 5 days of repeated restraint may be due to their lack of habituation.

Passive Coping Strategies During Repeated Social Defeat Are Associated With Long-Lasting Changes in Sleep in Rats.

Exposure to severe stress has immediate and prolonged neuropsychiatric consequences and increases the risk of developing Posttraumatic Stress Disorder (PTSD). Importantly, PTSD develops in only a subset of individuals after exposure to a traumatic event, with the understanding of this selective vulnerability being very limited. Individuals who go on to develop PTSD after a traumatic experience typically demonstrate sleep disturbances including persistent insomnia and recurrent trauma-related nightmares. We previously established a repeated social defeat paradigm in which rats segregate into either passively or actively coping subpopulations, and we found that this distinction correlates with measures of vulnerability or resilience to stress. In this study, we examined differences between these two behavioral phenotypes in sleep changes resulting from repeated social defeat stress. Our data indicate that, compared to control and actively coping rats, passively coping rats have less slow-wave sleep (SWS) for at least 2 weeks after the end of a series of exposures to social defeat. Furthermore, resilient rats show less exaggerated motor activation at awakenings from rapid eye movement (REM) sleep and less fragmentation of REM sleep compared to control and passively coping rats. Together, these data associate a passive coping strategy in response to repeated social defeat stress with persisting sleep disturbances. Conversely, an active coping strategy may be associated with resilience to sleep disturbances. These findings may have both prognostic and therapeutic applications to stress-associated neuropsychiatric disorders, including PTSD.

Interleukin-1α in the ventral hippocampus increases stress vulnerability and inflammation-related processes.

Mechanisms of stress vulnerability remain elusive. Previous research demonstrated that inflammation-related processes in the brain play a role in stress vulnerability. Our previous research showed that inflammatory processes in the ventral hippocampus (vHPC) induced a stress vulnerable phenotype. To further understand neuroinflammatory processes in the vHPC in stressed rats, we determined that protein levels of the pro-inflammatory cytokine interleukin-1-α (IL-1α), but not interleukin-1ß (IL-1ß), were increased in the vHPC of rats vulnerable to the effects of repeated social defeat compared to rats resilient to its effects. Injections of IL-1α into the vHPC increased stress vulnerability as characterized by increases in passive coping during defeat and subsequent decreased social interactions. Conversely, injections of recombinant IL-1 receptor antagonist (IL1-RA) increased latencies to social defeat and decreased anxiety-like behaviors during social interaction, suggesting an reduction in stress vulnerability. Protein analyses revealed increased FosB expression in the vHPC of IL-1α-injected rats, and increased HPA activation following a social encounter. Further analysis of vHPC of IL1-α-injected rats showed increased density of microglia, increased expression of the pro-inflammatory cytokine HMGB1, and increases in a marker for vascular remodeling. Taken together, these data show increasing IL-1α during stress exposure is sufficient to produce a stress vulnerable phenotype potentially by increasing inflammation-related processes in the vHPC.LAY SUMMARYOur previous research demonstrated that inflammation-related processes in the brain play a role in inducing vulnerability to the effects of repeated social stress in rats. Here we demonstrate that a pro-inflammatory cytokine interleukin-1-α (IL-1α) induces inflammatory processes in the vHPC and behavioral vulnerability in stressed rats, whereas blocking IL receptors produces the opposite effects on behavioral vulnerability. Together, these results identify a substrate in the vHPC that produces vulnerability to stress by increasing inflammation-related processes in the vHPC.

The contribution of orexins to sex differences in the stress response.

Women are twice as likely as men to suffer from stress-related psychiatric disorders, such as post-traumatic stress disorder (PTSD) and Major Depressive Disorder (MDD), however, the biological basis of these sex differences is not fully understood. Interestingly, orexins are known to be dysregulated in these disorders. This review first discusses the important role of orexins regulating the response to stress. Next, we review the evidence for sex differences in the orexin system, in which the majority of both preclinical and clinical studies have reported higher orexin system expression in females. Finally, we discuss the functional consequences of these sex differences in orexin expression. Most importantly, the preclinical literature reveals that higher orexin system activity in females contributes to exaggerated neuroendocrine and behavioral responses to stress. In sum, the available data suggests that orexins may be important in the etiology of stress-related psychiatric disorders that present differently in men and women. Thus, targeting orexins could potentially ameliorate many phenotypes of stress-related illness in a sex-specific way.

The gut microbiome regulates the increases in depressive-type behaviors and in inflammatory processes in the ventral hippocampus of stress vulnerable rats.

Chronic exposure to stress is associated with increased incidence of depression, generalized anxiety, and PTSD. However, stress induces vulnerability to such disorders only in a sub-population of individuals, as others remain resilient. Inflammation has emerged as a putative mechanism for promoting stress vulnerability. Using a rodent model of social defeat, we have previously shown that rats with short-defeat latencies (SL/vulnerable rats) show increased anxiety- and depression-like behaviors, and these behaviors are mediated by inflammation in the ventral hippocampus. The other half of socially defeated rats show long-latencies to defeat (LL/resilient) and are similar to controls. Because gut microbiota are important activators of inflammatory substances, we assessed the role of the gut microbiome in mediating vulnerability to repeated social defeat stress. We analyzed the fecal microbiome of control, SL/vulnerable, and LL/resilient rats using shotgun metagenome sequencing and observed increased expression of immune-modulating microbiota, such as Clostridia, in SL/vulnerable rats. We then tested the importance of gut microbiota to the SL/vulnerable phenotype. In otherwise naive rats treated with microbiota from SL/vulnerable rats, there was higher microglial density and IL-1ß expression in the vHPC, and higher depression-like behaviors relative to rats that received microbiota from LL/resilient rats, non-stressed control rats, or vehicle-treated rats. However, anxiety-like behavior during social interaction was not altered by transplant of the microbiome of SL/vulnerable rats into non-stressed rats. Taken together, the results suggest the gut microbiome contributes to the depression-like behavior and inflammatory processes in the vHPC of stress vulnerable individuals.

The effects of early life adversity on growth, maturation, and steroid hormones in male and female rats.

Early life adversity is a risk factor for psychiatric disorders, yet the mechanisms by which adversity increases this risk are still being delineated. Here, we used a limited bedding and nesting (LBN) manipulation in rats that models a low resource environment to examine effects on growth, developmental milestones, and endocrine endpoints. In LBN, dams and pups, from pups' postnatal days 2-9, are exposed to an environment where dams lack proper materials to build a nest. This manipulation is compared to control housing conditions, where rat dams have access to ample nesting materials and enrichment throughout pups' development. We found that the LBN condition altered maternal care, increasing pup-directed behaviors while reducing self-care. This, perhaps compensatory, increase in nursing and attention to pups did not mitigate against changes in metabolism, as LBN reduced weight gain in both sexes and this effect persisted into adulthood. Although adult stress hormone levels in both sexes and vaginal opening and estrous cycle length in females were not disrupted, there was other evidence of endocrine dysregulation. Compared to controls, LBN rats of both sexes had shortened anogenital distances, indicating reduced androgen exposure. LBN males also had higher plasma estradiol levels in adulthood. This combination of results suggests that LBN causes a demasculinizing effect in males that could contribute to lasting changes in the brain and behavior. Importantly, alterations in metabolic and endocrine systems due to early life adversity could be one mechanism by which stress early in life increases risk for later disease.