Deborah M. Hodgson

Endotoxin exposure in early life alters the development of anxiety-like behaviour in the Fischer 344 rat.

Previous research in the rat has demonstrated that neonatal exposure to bacterial endotoxin alters the level of anxiety-like behaviour displayed in adulthood. Currently, however, little is known about the emergence and development of this type of behaviour. Given the ability of neonatal endotoxin exposure to alter neural substrates involved in regulating anxiety, we tested the hypothesis that it may also alter the developmental trajectory of anxiety-like behaviour in the rat. Male Fischer 344 neonatal rats were treated with endotoxin (0.05 mg/kg lipopolysaccharide from Salmonella enteriditis) or vehicle on postnatal days 3 and 5. Age related changes in anxiety-like behaviour were subsequently investigated using the elevated plus maze apparatus at three developmental time points; adolescence (43 days), adulthood (80 days) and senescence (400 days). Neonatal endotoxin exposure was found to significantly increase circulating levels of corticosterone on postnatal days 3 and 5 at 4 h postadministration (P < 0.05). Additionally, endotoxin exposure was found to markedly alter anxiety-like behaviour in adulthood and senescence (P < 0.05). Specifically, adult and senescent endotoxin treated animals displayed significantly more anxiety-like behaviour than vehicle treated controls. Interestingly no significant differences in anxiety-like behaviour were observed between treatment groups during adolescence. These findings highlight the importance of the early life microbial environment in the development of emotional behaviour and suggests that neonatal infection may be an important predictor of susceptibility to anxiety related disorders in adult life.

Neonatal Endotoxin Exposure Influences HPA Responsivity and Impairs Tumor Immunity in Fischer 344 Rats in Adulthood

Recent research in rodents has demonstrated that exposure to bacterial endotoxin during the neonatal period alters the development of the hypothalamic-pituitary-adrenal axis resulting in hypersecretion of corticosterone after stress-exposure in adulthood. Given the known interactions between glucocorticoids and the immune system it was hypothesized that such alterations may impact on immune outcomes. Fischer 344 rats were treated with endotoxin (50 μg/kg Salmonella enteritidis, i.p.) or the vehicle on postpartum d 1, 3, 5, and 7. In adulthood, animals were subjected to chronic stress (6 × 10 h/d restraint stress), and the effect on resistance to tumor colonization (experiment 1) and natural killer cell activity (experiment 2) was assessed. Experiment 3 assessed corticosterone responses to acute stress in adulthood after neonatal endotoxin or saline treatment. Neonatal endotoxin exposure resulted in a 2-fold increase in tumor colonization (p < 0.001) and a significant impairment in the activity of natural killer cells (p < 0.01), cells critically involved in the surveillance and eradication of tumor cells. Neonatal endotoxin exposure also resulted in a significant decrease in gain weight that persisted into adulthood (p < 0.05), and potentiation of corticosterone responses to acute stress in adulthood (p < 0.05). We conclude that neonatal endotoxin exposure produces long-term changes in the hypothalamic-pituitary-adrenal axis, and has significant long-term effects on immune function, specifically in terms of resistance to tumor colonization in adulthood.

Placental Cytokine Expression Covaries with Maternal Asthma Severity and Fetal Sex

In the presence of maternal asthma, we have previously reported reduced placental blood flow, decreased cortisol metabolism, and reductions in fetal growth in response to maternal asthma and asthma exacerbations. We have proposed that these changes in placental function and fetal development may be related to activation of proinflammatory pathways in the placenta in response to maternal asthma. In the present study, we examined the influence of maternal asthma severity, inhaled glucocorticoid treatment, maternal cigarette use, placental macrophage numbers, and fetal sex on placental cytokine mRNA expression from a prospective cohort study of pregnant women with and without asthma. Placental expression of TNF-α, IL-1β, IL-6, IL-8, and IL-5 mRNA were all increased significantly in placentae of female fetuses whose mothers had mild asthma, but no changes were observed in placentae of male fetuses. The proinflammatory cytokines TNF-α, IL-1β, and IL-6 were negatively correlated with female cord blood cortisol, but there were no such correlations in placentae from males. Multivariate analysis indicated the strongest predictor of both cytokine mRNA expression in the placenta and birth weight was fetal cortisol but only in females. Placental cytokine mRNA levels were not significantly altered by inhaled glucocorticoid use, placental macrophage numbers, cigarette use, moderate-severe asthma, or male sex. These data suggest that placental basal cytokine mRNA expression is sex specifically regulated in pregnancies complicated by asthma, and interestingly these changes are more prevalent in mild rather than severe asthma.

Epidural Auditory Event-Related Potentials in the Rat to Frequency and duration Deviants: Evidence of Mismatch Negativity?

The capacity of the human brain to detect deviance in the acoustic environment pre-attentively is reflected in a brain event-related potential (ERP), mismatch negativity (MMN). MMN is observed in response to the presentation of rare oddball sounds that deviate from an otherwise regular pattern of frequent background standard sounds. While the primate and cat auditory cortex (AC) exhibit MMN-like activity, it is unclear whether the rodent AC produces a deviant response that reflects deviance detection in a background of regularities evident in recent auditory stimulus history or differential adaptation of neuronal responses due to rarity of the deviant sound. We examined whether MMN-like activity occurs in epidural AC potentials in awake and anesthetized rats to high and low frequency and long and short duration deviant sounds. ERPs to deviants were compared with ERPs to common standards and also with ERPs to deviants when interspersed with many different standards to control for background regularity effects. High frequency (HF) and long duration deviant ERPs in the awake rat showed evidence of deviance detection, consisting of negative displacements of the deviant ERP relative to ERPs to both common standards and deviants with many standards. The HF deviant MMN-like response was also sensitive to the extent of regularity in recent acoustic stimulation. Anesthesia in contrast resulted in positive displacements of deviant ERPs. Our results suggest that epidural MMN-like potentials to HF sounds in awake rats encode deviance in an analogous manner to the human MMN, laying the foundation for animal models of disorders characterized by disrupted MMN generation, such as schizophrenia.

Increased microglial activation in the rat brain following neonatal exposure to a bacterial mimetic

Neonatal lipopolysaccharide (LPS) exposure increases anxiety-like behaviour in adulthood. Our current aim was to examine whether neonatal LPS exposure is associated with changes in microglial activation, and whether these alterations correspond with alterations in behaviour. In adulthood, LPS-treated animals exhibited significantly increased anxiety-like behaviour and hippocampal microglial activation. The efficacy of the LPS challenge was confirmed by increased neonatal plasma corticosterone and tyrosine hydroxylase (TH) phosphorylation in the adrenal medulla. These findings suggest a neuroimmune pathway which may underpin the long-term behavioural and neuroendocrine changes following neonatal infection.

Mismatch Negativity (MMN) in Freely-Moving Rats with Several Experimental Controls

Mismatch negativity (MMN) is a scalp-recorded electrical potential that occurs in humans in response to an auditory stimulus that defies previously established patterns of regularity. MMN amplitude is reduced in people with schizophrenia. In this study, we aimed to develop a robust and replicable rat model of MMN, as a platform for a more thorough understanding of the neurobiology underlying MMN. One of the major concerns for animal models of MMN is whether the rodent brain is capable of producing a human-like MMN, which is not a consequence of neural adaptation to repetitive stimuli. We therefore tested several methods that have been used to control for adaptation and differential exogenous responses to stimuli within the oddball paradigm. Epidural electroencephalographic electrodes were surgically implanted over different cortical locations in adult rats. Encephalographic data were recorded using wireless telemetry while the freely-moving rats were presented with auditory oddball stimuli to assess mismatch responses. Three control sequences were utilized: the flip-flop control was used to control for differential responses to the physical characteristics of standards and deviants; the many standards control was used to control for differential adaptation, as was the cascade control. Both adaptation and adaptation-independent deviance detection were observed for high frequency (pitch), but not low frequency deviants. In addition, the many standards control method was found to be the optimal method for observing both adaptation effects and adaptation-independent mismatch responses in rats. Inconclusive results arose from the cascade control design as it is not yet clear whether rats can encode the complex pattern present in the control sequence. These data contribute to a growing body of evidence supporting the hypothesis that rat brain is indeed capable of exhibiting human-like MMN, and that the rat model is a viable platform for the further investigation of the MMN and its associated neurobiology.

Early life host–bacteria relations and development: Long-term individual differences in neuroimmune function following neonatal endotoxin challenge

Neonatal animals have a proportionately greater risk, relative to the adult animal, of developing a bacterial infection. Research has revealed that such infections can influence biological processes long after the actual infection has been resolved. Indeed, studies examining the long-term alterations induced by early-life infection, simulated using endotoxin, have indicated that some aspects of the systemic inflammatory response in the adult animal are susceptible to modification. Available evidence suggests that altered inflammatory activities observed in the neonatally endotoxin challenged adult may be the result of potentiated hypothalamic pituitary adrenal (HPA) activity, specifically increased corticosterone production. Few studies, however, have examined whether altered corticosterone production is actually associated with changes in systemic inflammation in the neonatally endotoxin challenged adult animal. The aim, therefore, of the current study was to simultaneously examine the relationship between altered inflammatory activities and corticosterone production in the neonatally endotoxin challenged adult rat. Our findings demonstrate no significant differences exist between adults neonatally treated with saline or endotoxin in terms of their production of corticosterone following endotoxin challenge. While not appearing to influence the production of corticosterone neonatal endotoxin challenge did result in a marked attenuation in the adults febrile response following endotoxin challenge. Interestingly, circulating levels of IL-1beta and TNF-alpha were found to be equivalent in neonatal treatment groups following endotoxin administration in adulthood, indicating that the reduction in fever was unlikely to be the result of altered pro-inflammatory cytokine production. Further, no differences were found between neonatal treatment groups in net food consumption, water consumption or weight loss following endotoxin challenge in adulthood. Collectively, these findings demonstrate that neonatal endotoxin challenge does not affect a blanket down regulation of inflammatory processes but rather appears to induce highly specific alterations in the adult male Fischer-344 rat.

Neonatal endotoxin exposure modifies the acoustic startle response and circulating levels of corticosterone in the adult rat but only following acute stress

A variety of early life stressors have consistently been implicated in the development of psychopathology in adulthood. The current study investigates a rarely considered form of early life stress, bacterial infection, for its ability to induce psychopathology-like symptoms in adult rat. Specifically, neonatal rats were exposed to a simulated bacterial infection. In adulthood the acoustic startle response of these animals was evaluated both prior to and following exposure to restraint stress. Our results indicate that animals neonatally exposed to infection exhibit a significantly exaggerated acoustic startle response but only following exposure to stress. Additionally, we observed that adult animals neonatally exposed to infection, exhibited increased production of circulating corticosterone following stress, indicating potentiated hypothalamic pituitary adrenal axis activity as well as altered novelty seeking behaviour and locomotor activity. These results extend upon existing pre-clinical findings that indicate certain stressful early life events can predispose the adult animal to exhibit abnormal behaviour in adulthood.

Neonatal lipopolysaccharide exposure alters central cytokine responses to stress in adulthood in Wistar rats

“Perinatal programming” is a phenomenon describing how early life environmental conditions can produce long-term physiological alterations that either enhance or inhibit adaptive functioning. Previously, we have demonstrated that neonatal exposure to lipopolysaccharide (LPS) predisposes to anxiety-like behaviour in later life, which was associated with changes to the neuroendocrine response to stress. Given the known interactions between the neuroendocrine and neuroimmune systems, here we investigated whether neonatal exposure to a bacterial mimetic alters neuroimmune responses to acute stress in adulthood. Male and female Wistar rats were administered LPS (0.05 mg/kg, i.p.), or saline vehicle (equivolume) on days 3 and 5 post-partum. One group of rats was euthanised following early life treatment to assess immediate hypothalamic–pituitary–adrenal axis and central cytokine responses to treatment. A second group was assessed in adulthood (85 days) following exposure to either a “stress” (30-min restraint) or “no stress” condition. Blood was collected from all rats at baseline, 30, 60 and 90 min after “stress”, “no stress” treatment to assess peripheral corticosterone responses, and brains were collected 180 min following baseline to assess hippocampal content of interleukin-1β (IL-1β), tumour necrosis factor-α (TNFα) and IL-6 protein. Radioimmunoassay revealed that neonatal LPS treatment resulted in a prolonged corticosterone response to stress in adulthood compared to controls (p < 0.05). Enzyme-linked-immunosorbent assays revealed no group differences in hippocampal IL-6 content. However, brain IL-1β and TNFα protein concentrations were significantly greater in rats neonatally exposed to LPS and then exposed to stress in adulthood when compared to all other groups (p < 0.05). These findings suggest that early life bacterial toxin exposure results in a prolonged neuroendocrine response to acute stress in adulthood, which may be a consequence of increased release of IL-1β and TNFα in the brain.

Effect of Maternal Probiotic Intervention on HPA Axis, Immunity and Gut Microbiota in a Rat Model of Irritable Bowel Syndrome

Objective To examine whether maternal probiotic intervention influences the alterations in the brain-immune-gut axis induced by neonatal maternal separation (MS) and/or restraint stress in adulthood (AS) in Wistar rats. Design Dams had free access to drinking water supplemented with Bifidobacterium animalis subsp lactis BB-12® (3×109 CFU/mL) and Propionibacterium jensenii 702 (8.0×108 CFU/mL) from 10 days before conception until postnatal day (PND) 22 (weaning day), or to control ad lib water. Offspring were subjected to MS from PND 2 to 14 or left undisturbed. From PND 83 to 85, animals underwent 30 min/day AS, or were left undisturbed as controls. On PND 24 and 86, blood samples were collected for corticosterone, ACTH and IgA measurement. Colonic contents were analysed for the composition of microflora and luminal IgA levels. Results Exposure to MS significantly increased ACTH levels and neonatal fecal counts of aerobic and anaerobic bacteria, E. coli, enterococci and clostridia, but reduced plasma IgA levels compared with non-MS animals. Animals exposed to AS exhibited significantly increased ACTH and corticosterone levels, decreased aerobic bacteria and bifidobacteria, and increased Bacteroides and E. coli counts compared to non-AS animals. MS coupled with AS induced significantly decreased anaerobes and clostridia compared with the non-stress adult controls. Maternal probiotic intervention significantly increased neonatal corticosterone levels which persisted until at least week 12 in females only, and also resulted in elevated adult ACTH levels and altered neonatal microflora comparable to that of MS. However, it improved plasma IgA responses, increased enterococci and clostridia in MS adults, increased luminal IgA levels, and restored anaerobes, bifidobacteria and E. coli to normal in adults. Conclusion Maternal probiotic intervention induced activation of neonatal stress pathways and an imbalance in gut microflora. Importantly however, it improved the immune environment of stressed animals and protected, in part, against stress-induced disturbances in adult gut microflora.