Maternal stress in pregnancy affects myelination and neurosteroid regulatory pathways in the guinea pig cerebellum.

Prenatal stress predisposes offspring to behavioral pathologies. These may be attributed to effects on cerebellar neurosteroids and GABAergic inhibitory signaling, which can be linked to hyperactivity disorders. The aims were to determine the effect of prenatal stress on markers of cerebellar development, a key enzyme in neurosteroid synthesis and the expression of GABAA receptor (GABAAR) subunits involved in neurosteroid signaling. We used a model of prenatal stress (strobe light exposure, 2 h on gestational day 50, 55, 60 and 65) in guinea pigs, in which we have characterized anxiety and neophobic behavioral outcomes. The cerebellum and plasma were collected from control and prenatally stressed offspring at term (control fetus: n = 9 male, n = 7 female; stressed fetus: n = 7 male, n = 8 female) and postnatal day (PND) 21 (control: n = 8 male, n = 8 female; stressed: n = 9 male, n = 6 female). We found that term female offspring exposed to prenatal stress showed decreased expression of mature oligodendrocytes (∼40% reduction) and these deficits improved to control levels by PND21. Reactive astrocyte expression was lower (∼40% reduction) following prenatal stress. GABAAR subunit (δ and α6) expression and circulating allopregnanolone concentrations were not affected by prenatal stress. Prenatal stress increased expression (∼150-250% increase) of 5α-reductase type-1 mRNA in the cerebellum, which may be a neuroprotective response to promote GABAergic inhibition and aid in repair. These observations indicate that prenatal stress exposure has marked effects on the development of the cerebellum. These findings suggest cerebellar changes after prenatal stress may contribute to adverse behavioral outcomes after exposure to these stresses.

Severity and timing: How prenatal stress exposure affects glial developmental, emotional behavioural and plasma neurosteroid responses in guinea pig offspring.

Prenatal stress has been associated with a variety of developmental changes in offspring, notably those associated with brain development and subsequent risk for neuropathologies later in life. Recently, the importance of the timing and the severity of the stressor during pregnancy has been emphasized with neurosteroids including allopregnanolone implicated in the regulation of stress and also for endogenous neuroprotection in offspring. Prenatal stress was induced using strobe light exposure in pregnant guinea pigs (term 71days) in three defined stress exposure groups (Gestational Age (GA)35-65, GA50-65 and GA60-65). Stress was induced for 2h (9-11am) every 5days via strobe light exposure. A fetal cohort were euthanased at term with fetal brains and plasma collected. Anxiety-like behaviour was evaluated at 18 days of age in a separate cohort of offspring with brains and plasma collected at 21days of age. Markers for mature oligodendrocytes and reactive astrocytes were measured in the CA1 region of the hippocampus and the subcortical white matter. The neurosteroid allopregnanolone was measured by radioimmunoassay in offspring plasma. In the CA1 region of the hippocampus, fetuses from all stress groups showed reduced expression of mature oligodendrocytes and reactive astrocytes. By juvenility, all male stress exposure groups had recovered to levels of unaffected controls with the exception of the GA35-65 stress group. In juvenile females, mature oligodendrocyte marker expression was reduced in all stress groups and reactive astrocyte expression was reduced in the GA35-65 and GA60-65 stress groups by juvenility. Increased reactive astrocyte expression was also apparent in the subcortical white matter in both sexes both at term and at juvenility. Prenatally stressed offspring spent less time exploring in the object exploration test and also entered the inner zone of the open field less than controls at 18days of age. Circulating allopregnanolone concentrations were significantly reduced in GA35-65 and GA 60-65 stress exposed fetuses with those in the GA35-65 stress group remaining reduced by juvenility. This study has shown the effects of differing levels of prenatal stress severity and timing on glial development, emotional behaviour and plasma allopregnanolone concentrations in offspring.

Loss of neurosteroid-mediated protection following stress during fetal life.

Elevated levels of neurosteroids during late gestation protect the fetal brain from hypoxia/ischaemia and promote neurodevelopment. Suppression of allopregnanolone production during pregnancy leads to the onset of seizure-like activity and potentiates hypoxia-induced brain injury. Markers of myelination are reduced and astrocyte activation is increased. The placenta has a key role in maintaining allopregnanolone concentrations in the fetal circulation and brain during gestation and levels decline markedly after both normal and preterm birth. This leads to the preterm neonate developing in a neurosteroid deficient environment between delivery and term equivalence. The expression of 5α-reductases is also lower in the fetus prior to term. These deficiencies in neurosteroid exposure may contribute to the increase in incidence of the adverse patterns of behaviour seen in children that are born preterm. Repeated exposure to glucocorticoid stimulation suppresses 5α-reductase expression and allopregnanolone levels in the fetus and results in reduced myelination. Both fetal growth restriction and prenatal maternal stress lead to increased cortisol concentrations in the maternal and fetal circulation. Prenatal stress results in reduced expression of key GABAA receptor subunits that normally heighten neurosteroid sensitivity. These stressors also result in altered placental allopregnanolone metabolism pathways. These findings suggest that reduced neurosteroid production and action in the perinatal period may contribute to some of the adverse neurodevelopmental and behavioural outcomes that result from these pregnancy compromises. Studies examining perinatal steroid supplementation therapy with non-metabolisable neurosteroid analogues to improve these outcomes are warranted.

Prenatal Stress Alters Hippocampal Neuroglia and Increases Anxiety in Childhood.

Prenatal stress has been associated with detrimental outcomes of pregnancy, including altered brain development leading to behavioural pathologies. The neurosteroid allopregnanolone has been implicated in mediating some of these adverse outcomes following prenatal stress due to its potent inhibitory and anxiolytic effects on the brain. The aims of the current study were to characterise key markers for brain development as well as behavioural parameters, adrenocortical responses to handling and possible neurosteroid influences towards outcomes in guinea pig offspring in childhood. Pregnant guinea pig dams were exposed to strobe light for 2 h (9-11 a.m.) on gestational days 50, 55, 60, and 65 and were left to deliver spontaneously at term and care for their litter. Behavioural testing (open-field test, object exploration test) of the offspring was performed at postnatal day 18 (with salivary cortisol and DHEA measured), and brains were collected at post-mortem on day 21. Markers of brain development myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) were assessed via immunohistochemistry, and the neurosteroid allopregnanolone and its rate-limiting enzymes 5α-reductase types 1 and 2 (5αR1/2) were measured in neonatal brains by radioimmunoassay, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot, respectively. Brain-derived neurotrophic factor protein was measured as a marker of synaptic plasticity, and GABAA receptor subunit expression was also assessed using RT-PCR. Neonates born from mothers stressed during late pregnancy showed a reduction in both MBP (p < 0.01) and GFAP (p < 0.05) expression in the CA1 region of the hippocampus at 21 days of age. Pups of prenatally stressed pregnancies also showed higher levels of anxiety and neophobic behaviours at the equivalent of childhood (p < 0.05). There were no significant changes observed in allopregnanolone levels, 5αR1/2 expression, or GABAA receptor subunit expression in prenatally stressed neonates compared to controls. This study shows alterations in markers of myelination and reactive astrocytes in the hippocampus of offspring exposed to prenatal stress. These changes are also observed in offspring that show increased anxiety behaviours at the equivalent of childhood, which indicates ongoing structural and functional postnatal changes after prenatal stress exposure.

Effects of prenatal stress on fetal neurodevelopment and responses to maternal neurosteroid treatment in Guinea pigs.

BACKGROUND: Maternal psychosocial stress during pregnancy is associated with adverse neonatal outcomes. These outcomes result from changes in fetal brain development and lead to disrupted cognitive, behavioural and emotional development. The neurosteroid allopregnanolone has been shown to reduce neural excitability and aid in protecting the fetal brain from excitotoxic insults. The objectives of this study were to assess the effect of prenatal maternal stress on fetal brain development with and without maternal allopregnanolone treatment. METHODS: Pregnant guinea pigs were subjected to stress induced by exposure to a strobe light at 50, 55, 60 and 65 days gestation. Salivary cortisol levels were measured before and after each exposure. Fetal brains were assessed for markers of brain development using immunohistochemistry and plasma allopregnanolone was measured by radioimmunoassay. RESULTS: Female, but not male prenatal stress-exposed fetuses demonstrated higher brain-to-liver ratios (BLR). Male fetuses showed significantly reduced expression of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and both males and females showed reduced expression of microtubule-associated protein 2 (MAP2). These markers were not affected by maternal allopregnanolone treatment. However, maternal allopregnanolone treatment resulted in an increase in fetal plasma allopregnanolone concentrations in control pregnancies but concentrations were not raised after prenatal stress exposure. CONCLUSIONS: These findings indicate that the effects of prenatal stress on fetal brain development are sexually dimorphic with more pronounced negative effects seen on male neurodevelopment. Allopregnanolone treatment was not effective in raising fetal plasma concentrations after prenatal stress suggesting a stress-induced dysregulation of neurosteroid pathways during gestation. Interestingly, this study directly implicates prenatal stress in the disruption of fetal neurosteroid levels, such that it may mediate some of the deleterious effects on fetal neurodevelopment by facilitating a deficit in normal endogenous neuroprotective mechanisms.