Maternal Separation With Early Weaning Research Articles

Early Life Stress in the Pristane Model of Systemic Lupus Erythematosus increases cardiovascular risk but not kidney disease severity earlier in disease progression

Experiencing Early Life Stress (ELS), such as physical/verbal abuse before the age of 18, has become an increasingly apparent concern in the context of lifelong health. Systemic Lupus Erythematosus (SLE) is one autoimmune condition linked with ELS, predominantly affects women, and has high CVD burden. Exposure to ELS independently increases the risk of CVD and SLE, so we have developed a model to study this gap. The mouse model of ELS, maternal separation with early weaning (MSEW), combined with the pristane model of SLE mimics the accelerated disease (shown by earlier production of autoantibodies, increased vascular stiffness, and vascular dysfunction at 4 months post injection (p.i.) opposed to 7 months p.i.) in ELS. We hypothesized that the accelerated disease and CVD burden in MSEW+SLE mice, as opposed to normally reared (NR)+SLE mice, arose from severe kidney damage and increased blood pressure. MSEW mice were separated from dams for 4 hours/day (postnatal day 2 to 5) and 8 hours/day before weaning at day 17. NR litters were weaned at postnatal day 21. MSEW or NR female mice were randomly administered PBS or pristane through i.p. injection. Urine was collected at 4 months and 7 months p.i. to test for kidney damage markers and telemetry was utilized. MSEW animals develop increased Anti-Smith autoantibodies at 4 months p.i while SLE NR animals had no difference in titer levels compared to NR PBS mice. However, we found no difference in urine NGAL (Neutrophil Gelatinase-Associated Lipocalin) at 4 (p=0.32) or 7 months p.i. (p=0.6) or urine proteinuria 7 months p.i. (p=0.9) between groups. Blood pressure between MSEW+SLE and NR+SLE mice at 4, 5, and 6 months p.i. compared to control mice was not different. At 7 months p.i., SBP in NR+SLE (140.3 ± 10.22, n=4), MSEW+SLE (140.28 ±9.32, n=4) was not statistically greater than the NR/MSEW controls (128.1 ± 3.16, n=5 NR, n=7 MSEW). Ejection fraction in NR+SLE (66.04 ± 3.24, n=3), MSEW+SLE (75.17 ±3.19, n=2) compared to NR/MSEW controls (64.36 ± 2.49, n=5 NR, n=4 MSEW) was slightly changed over time. We see that at 6 months p.i. PWV and adventitial wall thickness is greater in MSEW+SLE (2.89 mm/sec ± 0.11) mice compared to both NR+SLE mice (2.047 mm/sec ± 0.16) and NR control mice (1.49 mm/sec ± 0.12, p=<0.001, F= 26.02), but at seven months PWV is not different between MSEW+SLE (2.94 mm/sec ± 0.13) mice compared to both NR+SLE mice (2.63 mm/sec ± 0.32, p=0.30). The acceleration of autoantibodies and PWV in MSEW+SLE mice, but not kidney or heart function indicates a specific impact on larger vessels and autoimmune cells in SLE. This work furthers the understanding of how ELS accelerates cardiovascular burden in autoimmune diseases and early identifying targets to prevent worsened outcomes in patients exposed to childhood trauma. Crohn’s and Colitis Foundation NIH R25 PROmoTE NIH T32 PRIME F31HL165863-0 NIH P01 HL158500. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early life stress (ELS) promotes B cell expansion and aortic pathology in a mouse model of systemic lupus erythematosus (SLE)

SLE is an autoimmune disease that primarily affects young women and is associated with high rates of cardiovascular disease (CVD). Exposure to ELS (adversity in childhood such as verbal and physical abuse and/or familial dysfunction) is an independent risk factor for CVD and SLE. ELS heightens the risk of developing SLE and SLE patient-reported disease activity, however the underlying mechanism is unclear. We previously showed in a mouse model of ELS, maternal separation with early weaning (MSEW), that mice with pristane-induced SLE develop autoantibodies and increased aortic stiffness earlier in disease progression compared to normally reared (NR) SLE mice. We hypothesized that disease acceleration in MSEW+SLE mice compared to NR+SLE mice is associated with an expansion of B-cells and aortic structural changes. The pristane-induced model of SLE uses an intraperitoneal injection (i.p.) to induce SLE-like symptoms in female mice. The MSEW protocol separates pups from the dam 4h/day (postnatal, PD 2 to 5) and 8h/day (PD 6 to 16) with weaning at PD17. NR litters are weaned at PD21. At 6-8 weeks of age, female mice were randomly assigned to control (CON) or SLE groups: CON received an i.p. PBS injection, while NR+SLE and MSEW+SLE received an i.p. pristane injection. Spleen and thymus were weighed and collected for B-cell subset analysis with flow cytometry and aorta were dissected for histological assessments. At 4 months-post pristane, spleen weights did not differ between groups, however, we observed that MSEW+SLE mice had significantly larger thymus/body weight (BW) compared to CON and NR+SLE mice (p=0.0004 F=7.5 n=4). At 7 months post-pristane or PBS injection, MSEW+SLE mice and NR+SLE mice had significantly higher splenic weights/BW compared to CON mice (p=0.03 F=4.36, n=6) as expected. Using flow cytometry, we found increased thymic %age of CD19+ B-cells in both NR+SLE and MSEW+SLE compared to CON (p<0.0001 F=57.33, n=8,4,4), whereas we found similar %age of CD19+ B-cells in the aorta or spleen of mice in all groups. MSEW+SLE mice had increased germinal center B-cells compared to CON (CON: 0.77%, NR+SLE: 6.0%, MSEW+SLE 7.4%, p=0.008 F=7.08 n=8,4,4) while NR+SLE mice had increased plasma cells compared to CON (CON: 0.08%, NR+SLE: 1.3%, MSEW+SLE 0.68%, p=0.015 F=5.92 n=8,4,4). At 7 months post-pristane, MSEW+SLE mice had significantly greater aortic wall thickness (um) compared to NR+SLE and NR+CON mice (p=0.008 F=11.99, n=3,3,3), however, aortic collagen content (picrosirius red) was similar. These data suggest a difference in B-cell subset expansion and aortic structural changes may contribute to the MSEW driven acceleration of disease activity. Understanding how ELS accelerates autoimmunity and CVD risk is crucial to preventing worsened outcomes in vulnerable populations of individuals exposed to ELS with SLE. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early life stress induces visual dysfunction and retinal structural alterations in adult mice.

Early life stress (ELS) is defined as a period of severe and/or chronic trauma, as well as environmental/social deprivation or neglect in the prenatal/early postnatal stage. Presently, the impact of ELS on the retina in the adult stage is unknown. The long-term consequences of ELS at retinal level were analyzed in an animal model of maternal separation with early weaning (MSEW), which mimics early life maternal neglect. For this purpose, mice were separated from the dams for 2h at postnatal days (PNDs) 4-6, for 3h at PNDs 7-9, for 4h at PNDs 10-12, for 6h at PNDs 13-16, and weaned at PND17. At the end of each separation period, mothers were subjected to movement restriction for 10min. Control pups were left undisturbed from PND0, and weaned at PND21. Electroretinograms, visual evoked potentials, vision-guided behavioral tests, retinal anterograde transport, and retinal histopathology were examined at PNDs 60-80. MSEW induced long-lasting functional and histological effects at retinal level, including decreased retinal ganglion cell function and alterations in vision-guided behaviors, likely associated to decreased synaptophysin content, retina-superior colliculus communication deficit, increased microglial phagocytic activity, and retinal ganglion cell loss through a corticoid-dependent mechanism. A treatment with mifepristone, injected every 3 days between PNDs 4 and16, prevented functional and structural alterations induced by MSEW. These results suggest that retinal alterations might be included among the childhood adversity-induced threats to life quality, and that an early intervention with mifepristone avoided ELS-induced retinal disturbances.

Abstract P081: Early Life Stress (ELS) Induces Greater Aortic Pulse Wave Velocity (PWV) Through Reactive Oxygen Species (ROS)

ELS, such as verbal and physical abuse or maltreatment, is linked to a greater risk ofcardiovascular disease (CVD) in adulthood. Aortic stiffness, an independent predictor of CVD,has been suggested as a prognostic tool for early detection and intervention in at-riskpopulations. Several human studies have shown that aortic stiffness (assessed by PWV) isincreased in both adults and adolescents with ELS. ELS mouse models, such as maternalseparation with early weaning (MSEW), show aortic endothelial dysfunction with increasedsuperoxide and systemic inflammation, which are both present in humans with ELS exposureand contribute to aortic stiffness. We hypothesized that MSEW mice develop aortic stiffness in aROS-dependent manner at an early age. The MSEW protocol entails separating pups from thedam 4h/day (postnatal, PD 2 to 5) and 8h/day (PD 6 to 16) followed by weaning at PD17.Normally-reared (NR) litters remain with the dam and are weaned at PD21. Female and maleMSEW and NR mice were treated for 2 weeks with the ROS scavenger, TEMPOL (30mg/kg/day; drinking water) from 8 weeks of age. PWV was assessed prior to and after TEMPOLtreatment. PWV was significantly increased in MSEW mice at baseline (NR: 1.95 mm/sec ±0.15, MSEW: 2.60 mm/sec ± 0.17, p=0.009) in both sexes similarly. PWV was not significantlydifferent between MSEW and NR mice after TEMPOL treatment (NR: 1.78 mm/sec ± 0.06,MSEW: 1.97 mm/sec ± 0.12, p<0.05). MSEW mice had significantly more aorticCD11b + Ly6C + CX3CR1 + inflammatory monocytes compared to NR mice (NR: .9% ± 0.31,MSEW 1.5% ± 0.25, p=0.014) at baseline, however TEMPOL did not affect this. TEMPOLtreatment significantly decreased CD11b + F4/80 + MHCII hi inflammatory resident macrophages inMSEW mice compared to NR mice (Median Fluorescent Intensity; NR: 1,235,274 ± 54413,MSEW : 944,446 ±11728, p=0.007). In conclusion, ELS induces increased aortic stiffness in aROS-specific manner at an early age. Inflammatory resident macrophages may contribute to theELS-specific ROS-dependent mechanism. The mechanisms identified in this work arepotentially powerful tools for preventative therapeutics to reduce CVD burden in ELS-populations.

Effects of fast-acting antidepressant drugs on a postpartum depression mice model

Postpartum depression (PPD) is a severe psychiatric disorder with devastating consequences on child development and mother’s health. Dysregulation of glutamatergic and GABAergic signalling has been described in the corticolimbic system of PPD patients, who also show a downregulation of allopregnanolone levels in serum. Consequently, a synthetic allopregnanolone-based treatment is the current eligible drug to treat PPD patients. Alternatively, ketamine appears to be a promising medication for preventing PPD, nevertheless the differences in efficacy between both treatments remains unknown due to the lack of comparative studies. On this basis, the present study aims to compare the effectiveness of allopregnanolone and ketamine on a PPD-like mouse model. Our results show that postpartum females undergoing a maternal separation with early weaning (MSEW) protocol show increased despair-like behaviour, anhedonia and disrupted maternal care. Such symptoms are accompanied by lower allopregnanolone serum levels, reduction of vesicular transporters of GABA (VGAT) and glutamate (VGLUT1) in the infralimbic cortex (IL), as well as decreased hippocampal cellular proliferation. Furthermore, both drugs prevent despair-like behaviour while only ketamine reverts anhedonia. Both treatments increase hippocampal neurogenesis, while only allopregnanolone raises VGAT and VGLUT1 markers in IL. These findings suggest that ketamine might be even more effective than allopregnanolone, which points out the necessity of including ketamine in clinical studies for PPD patients. Altogether, we propose a new mice model that recapitulates the core symptomatology and molecular alterations shown in PPD patients, which allows us to further investigate both the neurobiology of PPD and the therapeutic potential of antidepressant drugs.

Early Life Stress Induced Hypothalamic–Pituitary–Adrenal (HPA) Axis Dysfunction Promotes Chronicity of Experimental Colitis in Mice

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract with peak onset during adolescence. The etiology of IBD remains poorly understood, but is believed to involve genetic susceptibility, improper immune regulation, and environmental factors. Psychological stress is one environmental trigger that has been associated with an increased risk of inflammatory diseases. Specifically, previous studies have linked early life stress (ELS) with hypothalamic–pituitary–adrenal (HPA) axis dysfunction that ultimately increases susceptibility for adverse health outcomes later in life. The aim of this study was to determine whether ELS in mice affects susceptibility to, and/or severity of colitis induced subsequent to stress exposure. We hypothesized that ELS would contribute to earlier onset disease and exacerbated colitis. We used an established mouse model of ELS, maternal separation with early weaning (MSEW), where newborn mice are separated from their mothers for 4‐8 hours daily from postnatal day (PD) 2 until early weaning on PD 17. Beginning at PD 28, colitis was induced in MSEW mice and their normally‐reared (NR) counterparts by transient blockade of the interleukin‐10 receptor (IL‐10R). Relative to their NR counterparts, MSEW mice exhibited systemic and colon‐specific deficits of the HPA‐axis endpoint molecule corticosterone, prior to induction of colitis. Following disease onset, NR mice were mostly in remission by 15 days after cessation of IL‐10R blockade, whereas MSEW mice displayed sustained histological pathology with an emphasis on epithelial damage, in addition to consistently elevated colonic tumor necrosis factor (Tnf). Furthermore, pharmacologic induction of HPA‐axis dysfunction via early life exposure (PD 1‐14) to the synthetic glucocorticoid, dexamethasone, mirrors the results we observed in the MSEW model of ELS – both prior to and subsequent to colitis induction. Our results indicate that early life exposure to prolonged stress leads to HPA‐axis dysfunction and colonic corticosterone deficits which may subsequently promote persistent colonic inflammation in susceptible hosts.

Early-life stress induces emotional and molecular alterations in female mice that are partially reversed by cannabidiol

Gender is considered as a pivotal determinant of mental health. Indeed, several psychiatric disorders such as anxiety and depression are more common and persistent in women than in men. In the past two decades, impaired brain energy metabolism has been highlighted as a risk factor for the development of these psychiatric disorders. However, comprehensive behavioural and neurobiological studies in brain regions relevant to anxiety and depression symptomatology are scarce. In the present study, we summarize findings describing cannabidiol effects on anxiety and depression in maternally separated female mice as a well-established rodent model of early-life stress associated with many mental disorders. Our results indicate that cannabidiol could prevent anxiolytic- and depressive-related behaviour in early-life stressed female mice. Additionally, maternal separation with early weaning (MSEW) caused long-term changes in brain oxidative metabolism in both nucleus accumbens and amygdalar complex measured by cytochrome c oxidase quantitative histochemistry. However, cannabidiol treatment could not revert brain oxidative metabolism impairment. Moreover, we identified hyperphosphorylation of mTOR and ERK 1/2 proteins in the amygdala but not in the striatum, that could also reflect altered brain intracellular signalling related with to bioenergetic impairment. Altogether, our study supports the hypothesis that MSEW induces profound long-lasting molecular changes in mTOR signalling and brain energy metabolism related to depressive-like and anxiety-like behaviours in female mice, which were partially ameliorated by CBD administration.

Prepulse inhibition can predict the motivational effects of cocaine in female mice exposed to maternal separation

The prepulse inhibition (PPI) of the startle response can identify the rodents that are more sensitive to the effects of cocaine. Mice with a lower PPI presented a higher vulnerability to the effects of cocaine and a higher susceptibility to developing a substance use disorder (SUD). Maternal separation with early weaning (MSEW) is a relevant animal model to induce motivational alterations throughout life. Nevertheless, only a few studies on females exist, even though they are more vulnerable to stress- and cocaine-related problems. Hence, the aim of the present study was to evaluate the ability of PPI to identify females with a greater vulnerability to the long-term consequences of early stress on the motivational effects of cocaine. Female mice underwent MSEW and were classified according to their high or low PPI. They were then assessed in the cocaine-induced locomotor sensitization test, the conditioned place preference paradigm or the operant self-administration paradigm. Additionally, they were also evaluated in the passive avoidance task, the tail-suspension and the splash tests. The results revealed that the females with lower PPI presented higher consequences of MSEW on the effects of cocaine and showed an increase in anhedonia-like behaviours. Our findings support that a PPI deficit could represent a biomarker of vulnerability to the effects of cocaine induced by MSEW.

Early life stress induces dysregulation of the heme pathway in adult mice.

Early life stress (ELS) is associated with cardiovascular disease (CVD) risk in adulthood, but the underlying vascular mechanisms are poorly understood. Increased hemoglobin and heme have recently been implicated to mediate endothelial dysfunction in several vascular diseases. Chronic physiological stress is associated with alterations in the heme pathway that have been well‐described in the literature. However, very little is known about the heme pathway with exposure to ELS or chronic psychosocial stress. Utilizing a mouse model of ELS, maternal separation with early weaning (MSEW), we previously reported that MSEW induces endothelial dysfunction via increased superoxide production. We reasoned that heme dysregulation may be one of the culprits induced by MSEW and sustained throughout adulthood; thus, we hypothesized that MSEW induces heme dysfunction. We investigated whether circulating levels of heme, a circulating pro‐oxidant mediator, are increased by MSEW and examined the role of the heme metabolic pathway and heme homeostasis in this process. We found that circulating levels of heme are increased in mice exposed to MSEW and that plasma from MSEW mice stimulated higher superoxide production in cultured mouse aortic endothelial cells (MAECs) compared to plasma from normally reared mice. The heme scavenger hemopexin blunted this enhanced superoxide production. Splenic haptoglobin abundance was significantly lower and hemoglobin levels per red blood cell were significantly higher in MSEW versus control mice. These findings lead us to propose that ELS induces increased circulating heme through dysregulation of the haptoglobin‐hemoglobin system representing a mechanistic link between ELS and CVD risk in adulthood.

Effects of Early Life Stress on the Gut Microbiota of Mice

Exposure to early life stress (ELS) is associated with a greater risk of chronic disease development including depression, cardiovascular disease (CVD), and inflammatory bowel disease (IBD). Changes in the gut microbiota have been linked to IBD and CVD. Rodent models of early life neglect are used to characterize the mechanistic links between ELS exposure and the risk of disease later in life. However, little is understood about ELS exposure and the gut microbiota. We used a mouse model of ELS, maternal separation with early weaning (MSEW) and normally-reared (NR) mice to determine whether the neonate microbiota is altered, and if so, whether the MSEW protocol induces changes in the dam microbiota that are then transmitted to their offspring. MSEW mice (n = 12) were subjected to maternal separation for 4 h per day on postnatal days (PDs) 2–5, and 8 h per day on PDs 6–16, and were subsequently weaned at PD17. NR mice (n = 13) remained undisturbed with dams until weaning on PD21. All mice were maintained on standard chow (NIH-31; NSN 8710-01-005-8438) following weaning. Paired-end Illumina sequencing was conducted on colonic content from PD28 mice and DADA2 was utilized to infer and assign taxonomy to individual amplicon sequence variants (ASVs). The fecal microbiota of dams (n= 5) was also sequenced. Analysis of composition of microbiomes (ANCOM) detected 32 ASVs that had statistically different abundance in MSEW compared to NR mice at PD28. Five of the nine ASVs that increased in MSEW mice were classified as Clostridium senso stricto 1 or Lachnoclostridium, while none the of the 23 ASVs enriched in NR animals belonged to these taxa. Fifty-seven percent (13 of 23) of the ASVs that had greater abundance in NR animals were classified as Lachnospiracea (other than Lachnoclostrium) or Ruminococcaceae. Compared to NR animals, MSEW PD28 mice had reduced phylogenetic diversity (18.7 ± 0.3 compared to 20.3 ± 0.3 in NR; P = 0.003) and Chao1ASV richness (175 ± 8 compared to 198 ± 8 in NR; P = 0.04). Microbial community composition was also altered at PD28 based permutational analysis of variance of Aitchison distance (F(1,24) = 2.29, P = 0.002). The composition, levels of diversity, and abundance of individual ASVs in the microbiota of dams were no different between MSEW and NR cohorts. Thus, the observed microbiota shifts in the young pups are likely driven by endogenous effects of MSEW in the offspring and independent of maternal inheritance. MSEW mice had lower microbial diversity and a reduction in Lachnospiraceae and Ruminococcaceae, which are important fermenters of short-chain fatty acids (SCFAs). Nine different SCFAs were measured in plasma of PD28 mice and MSEW animals had reduced butyrate (P = 0.02) compared to NR controls. Butyrate promote colonic homeostasis as well as regulates metabolic health and CVD risk. Studies are on-going to determine the impact of altered SCFA levels with ELS exposure. This study suggests a direct effect of ELS-exposure on the gut microbiota, which may impact the risk of chronic disease development.

Early life stress in mice promotes chronicity of experimental colitis

Abstract Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract with peak onset during adolescence. The etiology of IBD remains poorly understood, but is believed to involve genetic susceptibility, improper immune regulation, and environmental factors. Psychological stress is one environmental trigger that has been associated with an increased risk of inflammatory diseases. Specifically, previous studies have linked early life stress (ELS) with hypothalamic–pituitary–adrenal (HPA) axis dysfunction leading to adverse health outcomes later in life. Our goal was to determine whether ELS in mice affects susceptibility to, and severity of, colitis – induced subsequent to stress exposure. We used an established mouse model of ELS, maternal separation with early weaning (MSEW), in which newborn mice are separated from their mothers for 4–8 hours daily from day of life (DOL) 2 until early weaning on DOL 17. Then, starting on DOL 28, colitis was induced in MSEW mice and their normally-reared (NR) counterparts by transient blockade of the interleukin-10 receptor (IL-10R). Prior to induction of colitis, MSEW mice showed systemic and colon-specific depletion of corticosterone relative to NR mice. In addition, MSEW mice with active colitis harbored significantly reduced numbers of colonic Foxp3+ T cells compared to colitic NR mice. Finally, whereas NR mice were mostly in remission by 15 days following cessation of IL-10R blockade, MSEW mice showed consistently elevated Tnf in the proximal colon and sustained epithelial damage. Our results suggest that ELS-mediated HPA-axis dysfunction causes hypo-corticosteronism, which may subsequently promote prolonged colonic inflammation in susceptible hosts.

Early life stress in mice alters gut microbiota independent of maternal microbiota inheritance.

Exposure to early life stress (ELS) is associated with a greater risk of chronic disease development including depression and cardiovascular disease. Altered gut microbiota has been linked to both depression and cardiovascular disease in mice and humans. Rodent models of early life neglect are used to characterize the mechanistic links between early life stress (ELS) and the risk of disease later in life. However, little is understood about ELS exposure and the gut microbiota in the young mice and the influence of the maternal inheritance of the gut microbiota. We used a mouse model of ELS, maternal separation with early weaning (MSEW), and normally reared mice to determine whether the neonate microbiota is altered, and if so, are the differences attributable to changes in dam microbiota that are then transmitted to their offspring. Individual amplicon sequence variants (ASVs) displayed differential abundance in the microbiota of MSEW compared with normally reared pups at postnatal day (PD) 28. Additionally, ELS exposure reduced the alpha diversity and altered microbial community composition at PD28. The composition, levels of alpha diversity, and abundance of individual ASVs in the microbiota of dams were similar from MSEW or normally reared cohorts. Thus, the observed shifts in the abundance of individual bacterial ASVs in the neonates and young pups are likely driven by endogenous effects of MSEW in the offspring host and are not due to inherited differences from the dam. This knowledge suggests that exposure to ELS has a direct effect on microbial factors on the risk of chronic disease development.

Early-Life Neglect Alters Emotional and Cognitive Behavior in a Sex-Dependent Manner and Reduces Glutamatergic Neuronal Excitability in the Prefrontal Cortex.

Early-life neglect in critical developmental periods has been associated with emotional and cognitive consequences. Maternal separation (MS) has been commonly used as a rodent model to identify the developmental effects of child neglect. However, reports have shown considerable variability in behavioral results from MS studies in both mice and rats. Difficulties in developing reliable child neglect models have impeded advances in identifying the effects of early-life stress. Accumulating evidence shows that neuronal intrinsic excitability plays an important role in information processing and storage in the brain. The prefrontal cortex (PFC) integrates information from many cortical and subcortical structures. No studies to date have examined the impact of early-life stress on glutamatergic neuronal excitability in the PFC. This study aimed to develop a reliable child neglect rat model and observe glutamatergic neuronal excitability in the PFC. An MS with early weaning (MSEW) rat model was developed. Rats were separated from the dam for 4 h per day on postnatal days (PNDs) 2–5 and for 8 h per day on PNDs 6–16 and then weaned on PND 17. A battery of behavioral tests was used to assess anxiety-like behavior, coping behavior, working memory, spatial reference memory, and fear memory. The action potentials (APs) of glutamatergic neuronal membranes were recorded. MSEW resulted in anxiety-like behavior, a passive coping strategy and increased fear memory in male rats and decreased locomotor activity in both sexes. MSEW slightly impaired working memory during non-stressful situations in female rats but did not change spatial reference memory or associative learning under stressful circumstances in either sex. MSEW reduced the number of glutamatergic neuron APs in male rats. Our findings showed that MS with early weaning induced anxiety-like behavior in male rats. The reduced glutamatergic neuronal excitability may be associated with the emotional alteration induced by MSEW in male rats. In addition, MSEW induced adaptive modification, which depended on a non-stressful context.

Cocaine-seeking behaviour is differentially expressed in male and female mice exposed to maternal separation and is associated with alterations in AMPA receptors subunits in the medial prefrontal cortex.

According with clinical data, women evolve differently from drug use to drug abuse. Among drugs of abuse, cocaine is the most consumed psychostimulant. Animal studies demonstrated that females show increased motivation to seek cocaine during the self-administration paradigm (SA) than males. Moreover, suffering childhood adversity or major depressive disorder are two factors that could increase the predisposition to suffer cocaine addiction. Maternal separation with early weaning (MSEW) is an animal model that allows examining the impact of early-life stress on cocaine abuse. In this study, we aimed to explore changes in MSEW-induced cocaine-seeking motivation to determine potential associations between despair-like behaviour and cocaine-seeking. We also evaluated possible alterations in the AMPA receptors (AMPArs) composition in the medial prefrontal cortex (mPFC) of these mice. We exposed mice to MSEW and the behavioural tests were performed during adulthood. Moreover, GluA1, GluA2 mRNA and protein expression were evaluated in the mPFC. Results show higher cocaine-seeking in standard nest females, as well as an increase in GluA1 and GluA2 protein expression. Moreover, MSEW induces downregulation of Gria2 and increases the Gria1/Gria2 ratio, only in male mice. In conclusion, female mice show different composition of the AMPA receptor in the mPFC and MSEW alters the glutamatergic system in the mPFC of male mice.

Early-life stress exacerbates the effects of WIN55,212-2 and modulates the cannabinoid receptor type 1 expression

Early-life stress induces an abnormal brain development and increases the risk of psychiatric diseases, including depression, anxiety and substance use disorders. We have developed a reliable model for maternal neglect, named maternal separation with early weaning (MSEW) in CD1 mice. In the present study, we evaluated the long-term effects on anxiety-like behaviours, nociception as well as the Iba1-positive microglial cells in this model in comparison to standard nest (SN) mice. Moreover, we investigated whether MSEW alters the cannabinoid agonist WIN55,212-2 effects regarding reward, spatial and emotional memories, tolerance to different cannabinoid responses, and physical dependence. Adult male offspring of MSEW group showed impaired responses on spatial and emotional memories after a repeated WIN55,212-2 treatment. These behavioural impairments were associated with an increase in basolateral amygdala and hippocampal CB1-expressing fibres and higher number of CB1-containing cells in cerebellum. Additionally, MSEW promotes a higher number of Iba1-positive microglial cells in basolateral amygdala and cerebellum. As for the cannabinoid-induced effects, rearing conditions did not influence the rewarding effects of WIN55,212-2 in the conditioned place preference paradigm. However, MSEW mice showed a delay in the development of tolerance to the cannabinoid effects. Moreover, CB1-positive fibres were reduced in limbic areas in MSEW mice after cannabinoid withdrawal precipitated with the CB1 antagonist SR141617A. These findings support that early-life stress promotes behavioural and molecular changes in the sensitivity to cannabinoids, which are mediated by alterations in CB1 signalling in limbic areas and it induces an increased Iba1-microglial marker which could interfere in emotional memories formation.

Maternal separation with early weaning impairs neuron-glia integrity: non-invasive evaluation and substructure demonstration

Astrocytes and oligodendrocytes play essential roles in regulating neural signal transduction along neural circuits in CNS. The perfect coordination of neuron/astrocyte and neuron/oligodendrocyte entities was termed as neuron-glia integrity recently. Here we monitored the status of neuron-glia integrity via non-invasive neuroimaging methods and demonstrated the substructures of it using other approaches in an animal model of maternal separation with early weaning (MSEW), which mimics early life neglect and abuse in humans. Compared to controls, MSEW rats showed higher glutamate level, but lower GABA in prefrontal cortex (PFC) detected by chemical exchange saturation transfer and 1H-MRS methods, lower levels of glial glutamate transporter-1 and ATP-α, but increased levels of glutamate decarboxylase-65 and glutamine synthetase in PFC; reduced fractional anisotropy in various brain regions revealed by diffusion tensor imaging, along with increased levels of N-acetyl-aspartate measured by 1H-MRS; and hypomyelination in PFC as evidenced by relevant cellular and molecular changes.

4578 Early life stress promotes chronicity of experimental colitis

OBJECTIVES/GOALS: The overall goal of this study was to determine the effect of early life stress (ELS) on the intestinal CD4+ T cell immune compartment, at homeostasis and after induction of experimental Inflammatory Bowel Disease (IBD). METHODS/STUDY POPULATION: We used a mouse model of ELS, maternal separation with early weaning (MSEW). We used IL-10 reporter mice to enable analysis of IL-10-producing cells. Mice were examined on postnatal day 28 to determine the impact of ELS on gut regulatory T cells. Plasma levels of corticosterone (rodent stress response hormone) was determined by ELISA. Colitis was induced in MSEW and normal rear (NR) mice via intraperitoneal injection of α-IL-10R every 5 days until day 15. Mice were euthanized on days 20 and 30. Colonic tissue sections were stained for histological analysis. Remaining tissue was further processed for flow cytometric analysis of CD4+ T cells and innate lymphoid cells. RESULTS/ANTICIPATED RESULTS: Plasma corticosterone was elevated in MSEW mice compared to their NR counterparts at 4 weeks of age. We observed that the MSEW stress protocol does not affect the baseline colonic CD4+ T cell or innate lymphoid cell populations. There was a reduction in the intestinal CD4+ T cells and regulatory T cells on day 20 in α-IL-10R MSEW mice compared to NR counterparts. This difference disappeared by day 30. Histological scoring showed no difference in disease severity between α-IL-10R treated MSEW and NR mice on day 20. However, on day 30, when α-IL-10R NR mice are recovering from colitis, MSEW mice showed persistent histological inflammation, mainly attributable to sustained epithelial damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that ELS prolongs intestinal inflammation and impairs epithelial repair. Future studies will focus on elucidating the mechanisms responsible for ELS-dependent impairment of mucosal repair in experimental colitis.

Maternal separation increases cocaine intake through a mechanism involving plasticity in glutamate signalling.

Early-life stress (ELS) is associated with negative consequences, including maladaptive long-lasting brain effects. These alterations seem to increase the likelihood of developing substance use disorders. However, the molecular consequences of ELS are poorly understood. In the present study, we tested the impact of ELS induced by maternal separation with early weaning (MSEW) in CD1 male mice at different phases of cocaine self-administration (SA). We also investigated the subsequent alterations on GluR2, GluR1, cAMP response element-binding (CREB), and CREB-phosphorylation (pCREB) in ventral tegmental area (VTA) and nucleus accumbens (NAc) induced by both MSEW and cocaine SA. Our results show that MSEW animals expressed a higher cocaine intake, an increased vulnerability to the acquisition of cocaine SA, and incapacity to extinguish cocaine SA behaviour. MSEW mice showed decreased GluR2 and increased GluR1 and pCREB in NAc. Also, results displayed reduction of basal levels of GluR1 and CREB and an elevation of GluR1/GluR2 ratio in the VTA. Such results hint at an enhanced glutamatergic function in NAc and increased excitability of VTA DA neurons in maternally separated mice. Altogether, our results suggest that MSEW induces molecular alterations in the brain areas related to reward processing, increasing the vulnerability to depression and cocaine-seeking behaviour.

Early Life Stress in Mice Alters Microbial Composition

Early‐life stress (ELS) increases the risk of developing cardiovascular disease (CVD) and other chronic inflammatory diseases including inflammatory bowel disease (IBD). Early‐life perturbations in the gut microbiota that enhance a pro‐inflammatory state or reduce microbial metabolites that are important for regulating metabolic homeostasis may predispose children to developing IBD or CVD later in life. Previous investigations of ELS and the gut microbiota have not focused on early stages of gut microbial colonization and community development. We used a mouse model of ELS, maternal separation with early weaning (MSEW), and hypothesized that MSEW would lead to changes in the gut microbiota indicative of a pro‐inflammatory community composition. MSEW mice were subjected to maternal separation for 4 h per day on postnatal days (PDs) 2–5, and 8 h per day on PDs 6–16, and were subsequently weaned at PD 17. Normally reared (NR) mice remained undisturbed with dams until weaning on PD 21. All mice were maintained on standard chow (NIH‐31; NSN 8710‐01‐005‐8438) following weaning. We examined colonic microbiota composition in the MSEW and NR groups via Illumina sequencing of the 16s rRNA gene at PD 10 (n = 9 NR; 14 MSEW) during the separation protocol, and PD 28 (n = 13 NR; 12 MSEW) subsequent to weaning. Fecal microbiota of dams (n = 6) were also sequenced to evaluate potential maternal contribution of microbiota to their pups. Cardiac norepinephrine (NE) levels confirmed a stress phenotype; male MSEW mice had significantly greater NE at PD 28 (P = 0.01, 3.54 ± 0.38 NE/protein (ng/mg), n = 5) compared to NR male mice (2.06 ± 0.38 NE/protein (ng/mg), n = 4). Differences in microbial composition and diversity were not evident between MSEW and NR mice at PD 10 but were significant at PD 28. There were no changes in the microbial composition of dams when they were assigned to MSEW or NR (P = 0.55), providing evidence that microbiota differences detected at PD 28 were not inherited from their mothers. The microbiota of MSEW mice at PD 28 displayed reduced phylogenetic diversity (P = 0.03), a reflection of microbial functional trait diversity. Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify microbial taxa with differential abundances between MSEW and NR mice at PD 28. MSEW mice were enriched in the bacterial families Lactobacillaceae (P = 0.011) and Peptostreptococcaceae (P = 0.017). Microbial communities of NR mice were enriched in the short‐chain fatty acid (SCFA) fermenting families Ruminococcaceae (P = 0.011) and Bacteroidaceae (P = 0.004). Microbial‐produced SCFAs promote colonic homeostasis as well as regulate metabolic health and CVD risk. Further studies are on‐going to determine SCFAs in MSEW and NR mice. In summary, MSEW mice had lower relative abundances of SCFA fermenting taxa and were enriched in taxa, Peptostreptococcacea and Lactobacillaceae, that promote inflammation and degradation of the colonic mucus barrier in mice.Support or Funding InformationFunding SourcesThis work was supported by National Institute of Health Institutional Research and Academic Career Development Award (to KMK), T32 DK007545 and PhRMA Translational Medicine and Therapeutics Postdoctoral Fellowship (to KCM), and a Research Initiatives Award from the Crohn’s and Colitis Foundation (to CLM and JSP).

Early Life Stress Mediates Enhanced Aortic Vasorelaxation in Sv129 Mice

Early life stress (ELS) is an under‐appreciated risk factor for cardiovascular disease (CVD). In humans, adverse childhood experiences (ACEs) or ELS is recognized as inducing behavioral or emotional stress. We reported that young adults exposed to ACEs display enhanced vascular stiffness, increased peripheral resistance, and elevated plasma ET‐1. We hypothesized that vascular endothelial‐derived ET‐1 is associated with vascular dysfunction after exposure to ELS. We hypothesized that endothelial‐derived ET‐1 is linked to vascular dysfunction in ELS. To test this hypothesis, we utilized vascular endothelial‐specific ET‐1 knockout (VEETKO) and flox control mice exposed to normal rearing (NR) or maternal separation with early weaning (MSEW). Briefly, MSEW involves maternal separation for 4h/day (postnatal (PD) 2 to 5), 8h/day (PD6 to 16), and weaned at PD17. NR litters were weaned at PD21. Male and female adult NR and MSEW, flox control (n=3–5) and VEETKO (n=5) mice were used to study acetylcholine (Ach)‐ and sodium nitroprusside (SNP)‐mediated aortic vasorelaxation. Interestingly, aorta from MSEW flox mice showed significantly greater maximal Ach‐mediated vasorelaxation compared to NR flox mice (Flox‐control 74.7% SEM 4.15, Flox‐MSEW 93.7% SEM 1.63, VEETKO‐control 78.0% SEM 6.81, VEETKO‐MSEW 86.9% SEM 2.05; p=0.0023). EC50 responses to Ach were not significantly different (Flox‐NR −6.22 SEM 0.330, Flox‐MSEW −6.88 SEM 0.124, VEETKO‐NR −6.70 SEM 0.144, VEETKO‐MSEW −6.88 SEM 0.268; p=0.066). All responses to SNP were similar. The VEETKO and flox control mice are on a Sv129 background. We previously reported that C57Bl/6J mice exposed to MSEW displayed endothelial dysfunction when compared to NR. Thus, these data suggest that exposure to MSEW has a different mechanistic response to the MSEW protocol with different strains of mice. Future studies will include behavioral studies to determine whether the MSEW protocol in Sv129 mice is distinct from the pro‐anxiety response reported in C57Bl/6J mice.Support or Funding InformationFunding K01‐DK‐105038 KAH, RO1 HL136936 JSP and DMPPROmoTE R25 DK 112731