Neonatal immune challenge alters reproductive development in the female rat

This article is part of a Special Issue "Neuroendocrine-Immune Axis in Health and Disease." Neonatal lipopolysaccharide (LPS) exposure alters neuroendocrine, immune and behavioural responses in adult rats. Recent findings indicate that neonatal LPS treatment may have a more pronounced effect on the mating behaviours of females compared to males. The current study further explored the impact of neonatal inflammation on reproductive development in the female rat. Wistar rats were administered LPS (0.05mg/kg, i.p.) or saline (equivolume) on postnatal days (PNDs) 3 and 5. The immediate effect of treatment was assessed on plasma corticosterone and tyrosine hydroxylase (TH) phosphorylation in the adrenal medulla. Weight gain and vaginal opening were recorded, and oestrous cyclicity was monitored post-puberty and in late adulthood. Blood and ovaries were collected throughout development to assess HPA and HPG hormones and to examine ovarian morphology. Reproductive success in the first (F1) generation and reproductive development in the second (F2) generation were also assessed. Neonatal LPS exposure resulted in increased TH phosphorylation in the neonatal adrenals. LPS treatment increased the corticosterone concentrations of females as juveniles, adolescents and adults, and reduced FSH in adolescence. Increased catch-up growth was evident in LPS-treated females, prompting earlier onset of puberty. Diminished follicular reserve was observed in neonatally LPS-treated females along with the advanced reproductive senescence. While fertility rates were not compromised, higher mortality and morbidity were observed in litters born to LPS-treated mothers. Female offspring of LPS-treated mothers displayed increased corticosterone on PND 14, increased catch-up growth and delayed emergence of the first oestrous cycle. No differences in any of the parameters assessed were observed in F2 males. These data suggest that neonatal immunological challenge has a profound impact on the female reproductive development, via the alteration of metabolic and neuroendocrine factors which regulate sexual maturation. Evidence of altered development in the female, but not male offspring of LPS-treated dams suggests increased susceptibility of females to the deleterious effects of neonatal immunological stress and its possible transferability to a subsequent generation.     

Influence of prenatal stress on the rat hypothalamic-pituitary-adrenal axis activity: the role of the brain glycocorticoid receptors

The effects of prenatal stress on the hypothalamic-pituitary-adrenal (HPA) axis activity and brain glycocorticoid receptors were studied in neonatal male and female offspring, as well as the influence of neonatal glycocorticoid receptors blockade on hormonal stress reactivity of adult rats. The results showed that there were sexual differences in plasma corticosterone level and corticosteroid binding in the cortex and hypothalamus of 5-day old control rats. Prenatal stress increased basal level of corticosterone in female rats, decreased corticosterone binding in hypothalamus and hippocampus of male and female rats, and increased corticosteroid receptor level in the male cortex. Neonatal administration of glycocorticoid receptor antagonist did not change plasma corticosterone level in 5-day old rats, but prolonged hormonal stress response of the HPA axis in adult male rats and increased hormonal stress response in female ones. The character of the IIPA axis activity of male and female rats with neonatal blockade of glycocorticoid receptors correspond to hormonal stress response of prenatal stressed rats. These data suggest that change of brain glycocorticoid receptors function in neonatal period of development might be one of the mechanisms of prenatal stress influence on the HPA axis activity in the adulthood.     

Embryonic GABAB Receptor Blockade Alters Cell Migration,Adult Hypothalamic Structure,and Anxiety- and Depression-Like Behaviors Sex Specifically in Mice

Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA_B receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA_B receptor to a 7-day critical period (E11–E17) during embryonic development. Experiments tested the role of GABA_B receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA_B receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA_B receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA_B receptor antagonist. Embryonic exposure to GABA_B receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA_B receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.     

LPS Exposure Increases Maternal Corticosterone Levels,Causes Placental Injury and Increases IL-1Β Levels in Adult Rat Offspring: Relevance to Autism

Maternal immune activation can induce neuropsychiatric disorders, such as autism and schizophrenia. Previous investigations by our group have shown that prenatal treatment of rats on gestation day 9.5 with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally), which mimics infections by gram-negative bacteria, induced autism-like behavior in male rats, including impaired communication and socialization and induced repetitive/restricted behavior. However, the behavior of female rats was unchanged. Little is known about how LPS-induced changes in the pregnant dam subsequently affect the developing fetus and the fetal immune system. The present study evaluated the hypothalamic-pituitary-adrenal (HPA) axis activity, the placental tissue and the reproductive performance of pregnant Wistar rats exposed to LPS. In the adult offspring, we evaluated the HPA axis and pro-inflammatory cytokine levels with or without a LPS challenge. LPS exposure increased maternal serum corticosterone levels, injured placental tissue and led to higher post-implantation loss, resulting in fewer live fetuses. The HPA axis was not affected in adult offspring. However, prenatal LPS exposure increased IL-1β serum levels, revealing that prenatal LPS exposure modified the immune response to a LPS challenge in adulthood. Increased IL-1β levels have been reported in several autistic patients. Together with our previous studies, our model induced autistic-like behavioral and immune disturbances in childhood and adulthood, indicating that it is a robust rat model of autism.     

Prenatal Restraint Stress is Associated with Demethylation of Corticotrophin Releasing Hormone (CRH) Promoter and Enhances CRH Transcriptional Responses to Stress in Adolescent Rats

Maternal stress can disturb normal fetal neurodevelopmental progress, and lead to negative behavioral and neuroendocrine consequences for the offspring. These effects may be related to alterations in the hypothalamic–pituitary–adrenal (HPA) axis. Early life events disrupting the function of the HPA axis may be associated with epigenetic modification. This study investigated the effect of maternal stress on the methylation rate of the corticotrophin-releasing hormone (CRH) promoter and HPA axis response to acute stress in the adolescent offspring of Sprague–Dawley rats. Pregnant dams were randomly assigned to two groups: restraint stress group and normal control group. Adolescent male and female offspring were used from each group. The results showed that prenatal stress is associated with the demethylation of the CRH promoter, and leads to anxiety-like behaviors in adolescent life stages, as well as hyper-responsiveness of the HPA axis. Together, these results imply that prenatal stress alters the normal HPA function, which may be via the epigenetic mechanism.     

Effects of chronic mild stress on behavioral and neurobiological parameters — Role of glucocorticoid

Major depression is thought to originate from maladaptation to adverse events, particularly when impairments occur in mood-related brain regions. Hypothalamus–pituitary–adrenal (HPA) axis is one of the major systems involved in physiological stress response. HPA axis dysfunction and high glucocorticoid concentrations play an important role in the pathogenesis of depression. In addition, astrocytic disability and dysfunction of neurotrophin brain-derived neurotrophin factor (BDNF) greatly influence the development of depression and anxiety disorders. Therefore, we investigated whether depressive-like and anxiety-like behaviors manifest in the absence of glucocorticoid production and circulation in adrenalectomized (ADX) rats after chronic mild stress (CMS) exposure and its potential molecular mechanisms. The results demonstrate that glucocorticoid-controlled rats showed anxiety-like behaviors but not depression-like behaviors after CMS. Molecular and cellular changes included the decreased BDNF in the hippocampus, astrocytic dysfunction with connexin43 (cx43) decreasing and abnormality in gap junction in prefrontal cortex (PFC). Interestingly, we did not find any changes in glucocorticoid receptor (GR) or its chaperone protein FK506 binding protein 51 (FKBP5) expression in the hippocampus or PFC in ADX rats subjected to CMS. In conclusion, the production and circulation of glucocorticoids are one of the contributing factors in the development of depression-like behaviors in response to CMS. In contrast, the effects of CMS on anxiety-like behaviors are independent of the presence of circulating glucocorticoids. Meanwhile, stress decreased GR expression and enhanced FKBP5 expression via higher glucocorticoid exposure. Gap junction dysfunction and changes in BDNF may be associated with anxiety-like behaviors.     

Prenatal exposure to dexamethasone alters hippocampal drive on hypothalamic-pituitary-adrenal axis activity in adult male rats

Glucocorticoids are essential for normal hypothalamic-pituitary-adrenal (HPA) axis activity; however, recent studies warn that exposure to excess endogenous or synthetic glucocorticoid during a specific period of prenatal development adversely affects HPA axis stability. We administered dexamethasone (DEX) to pregnant rats during the last week of gestation and investigated subsequent HPA axis regulation in adult male offspring in unrestrained and restraint-stressed conditions. With the use of real-time PCR and RIA, we examined the expression of regulatory genes in the hippocampus, hypothalamus, and pituitary, including corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptors (GR), mineralcorticoid receptors (MR), and 11-beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1), as well as the main HPA axis hormones, adrenal corticotropic hormone (ACTH) and corticosterone (CORT). Our results demonstrate that the DEX-exposed group exhibited an overall change in the pattern of gene expression and hormone levels in the unrestrained animals. These changes included an upregulation of CRH in the hypothalamus, a downregulation of MR with a concomitant upregulation of 11beta-HSD-1 in the hippocampus, and an increase in circulating levels of both ACTH and CORT relative to unrestrained control animals. Interestingly, both DEX-exposed and control rats exhibited an increase in pituitary GR mRNA levels following a 1-h recovery from restraint stress; however, the increased expression in DEX-exposed rats was significantly less and was associated with a slower return to baseline CORT compared with controls. In addition, circulating levels of ACTH and CORT as well as hypothalamic CRH and hippocampal 11beta-HSD-1 expression levels were significantly higher in the DEX-exposed group compared with controls following restraint stress. Taken together, these data demonstrate that late-gestation DEX exposure in rats is associated with persistent changes in both the modulation of HPA axis activity and the HPA axis-mediated response to stress.     

Direct and dam-mediated effects of prenatal dexamethasone on emotionality, cognition and HPA axis in adult Wistar rats

Prenatal stress can affect foetal neurodevelopment and result in increased risk of depression in adulthood. It promotes increased maternal hypothalamo–pituitary–adrenal gland (HPA) secretion of glucocorticoid (GC), leading to increased foetal and maternal GC receptor activity. Prenatal GC receptor activity is also increased during prenatal treatment with dexamethasone (DEX), which is commonly prescribed as a prophylactic treatment of preterm delivery associated morbid symptoms. Here, we exposed pregnant Wistar rats to 0.1 mg/kg/d DEX during the last week of pregnancy and performed cross-fostering at birth. In the adult offspring we then studied the effects of prenatal DEX exposure per se and the effects of rearing by a dam exposed to prenatal DEX. Offspring were assessed in the following paradigms testing biobehavioural processes that are altered in depression: progressive ratio schedule of reinforcement (anhedonia), Porsolt forced swim test (behavioural despair), US pre-exposure active avoidance (learned helplessness), Morris water maze (spatial memory) and HPA axis activity (altered HPA function). Responsiveness to a physical stressor in terms of HPA activity was increased in male offspring exposed prenatally to DEX. Despite this increased HPA axis reactivity, we observed no alteration of the assessed behaviours in offspring exposed prenatally to DEX. We observed impairment in spatial memory in offspring reared by DEX exposed dams, independently of prenatal treatment. This study does not support the hypothesis that prenatal DEX exposure leads to depression-like symptoms in rats, despite the observed sex-specific programming effect on HPA axis. It does however emphasise the importance of rearing environment on adult cognitive performances.     

Neonatal immune challenge exacerbates experimental colitis in adult rats: potential role for TNF-alpha

Early life events and childhood infections have been associated with the development and onset of inflammatory bowel disease in adulthood. However, the consequences of neonatal infection in the development and severity of colitis are not established. We investigated the effects of a neonatal (postnatal day 14) or juvenile (postnatal day 28) immune challenge with LPS on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced damage and weight loss, as well as on food intake and body temperature in adult rats. Neonatally (n)LPS-treated rats developed more severe colitis than control animals, reflected in a greater loss of weight and a significantly increased macroscopic tissue damage score. These findings were associated with a hypothermic response after TNBS treatment in nLPS rats, but not in neonatally saline-treated rats receiving TNBS. These differences were not seen after TNBS in rats that had received LPS on postnatal day 28. Plasma corticosterone was measured as an index of adult hypothalamic-pituitary-adrenal (HPA) axis activation as was TNF-alpha, a proinflammatory cytokine associated with inflammatory bowel disease. Four days after TNBS treatment, plasma corticosterone was unaltered in all groups; however, TNF-alpha was significantly increased in adult TNBS-treated rats that had LPS as neonates compared with all other groups. In conclusion, neonatal, but not later, exposure to LPS produces long-term exacerbations in the development of colitis in adults. This change is independent of HPA axis activation 4 days after TNBS treatment but is associated with increased circulating TNF-alpha, suggestive of an exaggerated immune response in adults exposed to neonatal infection.     

Mouse models of altered CRH-binding protein expression

CRH is the key physiological mediator of the endocrine, autonomic, and behavioral responses to stress. The recent characterization of urocortin, a new mammalian CRH-like ligand, adds to the complexity of the CRH system. Both CRH and urocortin mediate their endocrine and/or synaptic effects via two classes of CRH receptors. Similarly, both CRH and urocortin bind to the CRH-binding protein (CRH-BP). This secreted binding protein is smaller than the CRH receptors, but binds CRH and urocortin with an affinity equal to or greater than that of the receptors, and blocks CRH-mediated ACTH release in vitro. Several regions of CRH-BP expression colocalize with sites of CRH synthesis or release, suggesting that this binding protein may have a profound impact on the biological activity of CRH (or urocortin). While in vitro and in vivo studies have characterized the biochemical properties and regulation of the CRH-BP, animal models of altered CRH-BP expression can provide additional information on the in vivo role of this important modulatory protein. This review focuses on three mouse models of CRH-BP overexpression or deficiency. These animal models show numerous physiological changes in the HPA axis and in energy balance, with additional alterations in anxiogenic behavior. These changes are consistent with the hypothesis that CRH-BP plays an important in vivo modulatory role by regulating levels of "free" CRH and other CRH-like peptides in the pituitary and central nervous system.     

Dissecting the genetic effect of the CRH system on anxiety and stress-related behaviour

Corticotropin-releasing hormone (CRH) plays a central role in the adaptation of the body to stress. CRH integrates the endocrine, autonomic and behavioural responses to stress acting as a secretagogue within the line of the hypothalamic pituitary adrenocortical (HPA) system and as a neurotransmitter modulating synaptic transmission in the central nervous system. Accumulating evidence suggests that the neuroendocrine and behavioural symptoms observed in patients suffering from major depression are at least in part linked to a hyperactivity of the CRH system. Genetic modifications of the CRH system by conventional and conditional gene targeting strategies in the mouse allowed us to study the endogenous mechanisms underlying HPA system regulation and CRH-related neuronal circuitries involved in pathways mediating anxiety and stress-related behaviour.     

An animal model of eating disorders associated with stressful experience in early life

Experience of childhood abuse is prevalent among patients with eating disorders, and dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is implicated in its pathophysiology. Neonatal maternal separation is considered as an animal model of stressful experience early in life. Many of studies have demonstrated its impact both on the activity of HPA axis and the development of psycho-emotional disorders later in life. In this paper, a series of our researches on developing an animal model of eating disorders is reviewed. An animal model of neonatal maternal separation was used; Sprague-Dawley pups were separated from dam daily for 180 min during the first 2 weeks of life (MS) or undisturbed. Anxiety-/depression-like behaviors were observed in MS rats at the age of two months with decreased serotonergic activity in the hippocampus and the raphe. Post-weaning social isolation promoted food intake and weight gain of adolescent MS pups, with impacts on anxiety-like behaviors. Sustained hyperphagia was observed in the MS pups subjected to a fasting/refeeding cycle repeatedly during adolescence, with increased plasma corticosterone levels. Anhedonia, major symptom of depression, to palatable food was observed in adolescent MS pups with blunted response of the mesolimbic dopaminergic activity to stress. Results suggest that neonatal maternal separation lead to the development of eating disorders when it is challenged with social or metabolic stressors later in life, in which dysfunctions in the HPA axis and the brain monoaminergic systems may play important roles.     

Effect of LPS treatment on tyrosine hydroxylase expression and Parkinson-like behaviors

Puberty is a critical period of development during which the brain undergoes reorganizing and remodeling. Exposure to stress during this period is thought to interfere with normal brain development and increase susceptibility to mental illnesses. In female mice, pubertal exposure to lipopolysaccharide (LPS), a bacterial endotoxin, has been shown to alter sexual, anxiety-like, and depression-like behaviors and cognition in an enduring manner. However, the mechanisms underlying these effects remain unknown. The present study examined age and sex difference in tyrosine hydroxylase (TH) expression and dopamine-dependent and Parkinson-like behaviors following LPS treatment. The results show that LPS treatment during adulthood causes an enduring increase in TH expression in many of the brain regions examined. In contrast, there is no change in TH expression following LPS treatment during puberty. However, pubertal LPS treatment induces enduring behavioral deficits in tests of Parkinson-like behaviors, more so in male than in female mice. These results suggest that the low levels of TH following exposure to pubertal immune challenge may predispose mice to Parkinson-like behavior. These findings add to our understanding of stress and immune responses during puberty and their impact on mental health later in life.     

Pleiotropic effects of corticotropin releasing hormone on normal human skin keratinocytes

The hypothalamic-pituitary-adrenal (HPA) axis is the major stress response system. Several components of the HPA axis, such as corticotropin-releasing hormone (CRH) and POMC peptides and their receptors are also present in the skin. In earlier studies, we showed that CRH inhibits cellular proliferation of immortalized human keratinocytes. We now examine further the functional activity of the HPA axis in the skin, by characterizing the actions of CRH on normal foreskin keratinocytes. The CRH receptor was detected as CRH-R1 antigen at 47 kDa in the cultured keratinocytes by Western blotting, and immunohistochemistry demonstrated its presence in the epidermal and follicular keratinocytes. CRH is also biologically active in cultured keratinocytes, where it inhibits proliferation and enhances the interferon-gamma-stimulated expression of the hCAM and ICAM-1 adhesion molecules and of the HLA-DR antigen. These effects were concentration-dependent, with maximal activity at CRH 10(-7) M. Thus, in the keratinocyte, the most important cellular component of the epidermis, CRH appears to induce a shift in energy metabolism away from proliferation activity, and toward the enhancement of immunoactivity. Therefore, similar to its central actions, cutaneous CRH may also he involved in the stress response, but at a highly localized level.     

Gestational Hypoxia Induces Sex-Differential Methylation of Crhr1 Linked to Anxiety-like Behavior

Stress during gestation increases vulnerability to disease and changes behavior in offspring. We previously reported that hypoxia and restraint during pregnancy sensitized the hypothalamic–pituitary–adrenal (HPA) axis and induced anxiety-like behavior in the adult offspring. Here, we report that gestational intermittent hypoxia (GIH) elicited a sex-dependent anxiety-like behavior in male P90 offspring and activation of corticotropin-releasing hormone (CRH) and CRH type-1 receptor (CRHR1) mRNA in the hypothalamic paraventricular nucleus (PVN) and in male E19 hypothalamus. These linked to demethylation at several specific sites of CpG island of Crhr1 promoter in P90 PVN and E19 embryo hypothalamus in GIH groups. Crhr1 DNA demethylation is more crucial in CpG island 1 than island 2 for activation of CRHR1 mRNA. DNMT3b is required for the Crhr1 DNA methylation than DNMT1 and DNMT3a in increased CRHR1 mRNA. We first address a novel hypothesis that GIH-induced male-sex-dependent demethylation at CpG sites of Crhr1 DNA in promoter triggers elevation of CRHR1 mRNA in PVN and anxiety-like behavior in adult offspring.     

Binge-pattern alcohol exposure during puberty induces long-term changes in HPA axis reactivity

Adolescence is a dynamic and important period of brain development however, little is known about the long-term neurobiological consequences of alcohol consumption during puberty. Our previous studies showed that binge-pattern ethanol (EtOH) treatment during pubertal development negatively dysregulated the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis, as manifested by alterations in corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP), and corticosterone (CORT) during this time period. Thus, the primary goal of this study was to determine whether these observed changes in important central regulators of the stress response were permanent or transient. In this study, juvenile male Wistar rats were treated with a binge-pattern EtOH treatment paradigm or saline alone for 8 days. The animals were left undisturbed until adulthood when they received a second round of treatments consisting of saline alone, a single dose of EtOH, or a second binge-pattern treatment paradigm. The results showed that pubertal binge-pattern EtOH exposure induced striking long-lasting alterations of many HPA axis parameters. Overall, our data provide strong evidence that binge-pattern EtOH exposure during pubertal maturation has long-term detrimental effects for the healthy development of the HPA axis.     

The Sustained Phase of Tyrosine Hydroxylase Activation In vivo

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthetic pathway for catecholamine synthesis. Stress triggers an increase in TH activity, resulting in increased release of catecholamines from both neurons and the adrenal medulla. In response to stress three phases of TH activation have been identified (acute, sustained and chronic) and each phase has a unique mechanism. The acute and chronic phases have been studied in vivo in a number of animal models, but to date the sustained phase has only been characterised in vitro. We aimed to investigate the effects of dual exposure to lipopolysaccharide (LPS) in neonatal rats on TH protein, TH phosphorylation at serine residues 19, 31 and 40 and TH activity in the adrenal gland over the sustained phase. Wistar rats were administered LPS (0.05?mg/kg, intraperitoneal injection) or an equivolume of non-pyrogenic saline on days 3 and 5 postpartum. Adrenal glands were collected at 4, 24 and 48?h after the drug exposure on day 5. Neonatal LPS treatment resulted in increases in TH phosphorylation of Ser40 at 4 and 24?h, TH phosphorylation of Ser31 at 24?h, TH activity at 4 and 24?h and TH protein at 48?h. We therefore have provided evidence for the first time that TH phosphorylation at Ser31 and Ser40 occurs for up to 24?h in vivo and leads to TH activation independent of TH protein synthesis, suggesting that the sustained phase of TH activation occurs in vivo.