Prenatal Stress Induces Translational Disruption Associated with Myelination Deficits.

Disruptions to neurodevelopment are known to be linked to behavioral disorders in childhood and into adulthood. The fetal brain is extremely vulnerable to stimuli that alter inhibitory GABAergic pathways and critical myelination processes, programing long-term neurobehavioral disruption. The maturation of the GABAergic system into the major inhibitory pathway in the brain and the development of oligodendrocytes into mature cells capable of producing myelin are integral components of optimal neurodevelopment. The current study aimed to elucidate prenatal stress-induced mechanisms that disrupt these processes and to delineate the role of placental pathways in these adverse outcomes. Pregnant guinea pig dams were exposed to prenatal stress with strobe light exposure for 2 h/day on gestational age (GA) 35, 40, 45, 50, 55, 60, and 65, and groups of fetuses and placentae were collected after the stress exposure on GA40, GA50, GA60, and GA69 (term). Fetal plasma, placental, and brain tissue were collected for allopregnanolone and cortisol quantification with ELISA. Relative mRNA expression of genes of specific pathways of interest was examined with real-time PCR in placental and hippocampal tissue, and myelin basic protein (MBP) was quantified immunohistochemically in the hippocampus and surrounding regions for assessment of mature myelin. Prenatal stress in mid-late gestation resulted in disruptions to the translational machinery responsible for the production of myelin and decreased myelin coverage in the hippocampus and surrounding regions. The male placenta showed an initial protective increase in allopregnanolone concentrations in response to maternal psychosocial stress. The male and female placentae had a sex-dependent increase in neurosteroidogenic enzymes at term following prenatal stress. Independent from exposure to prenatal stress, at gestational day 60 - a critical period for myelin development, the placentae of female fetuses had increased capability of preventing cortisol transfer to the fetus through expression of 11-beta-hydroxysteroid dehydrogenase types 1 and 2. The deficits early in the process of maturation of myelination indicate that the reduced myelination observed at childhood equivalence in previous studies begins in fetal life. This negative programing persists into childhood, potentially due to dysregulation of MBP translation processes. Expression patterns of neurosteroidogenic enzymes in the placenta at term following stress may identify at-risk fetuses that have been exposed to a stressful in utero environment.

Neurosteroid replacement therapy using the allopregnanolone-analogue ganaxolone following preterm birth in male guinea pigs.

BACKGROUND: Children born preterm, especially boys, are at increased risk of developing attention deficit hyperactivity disorder (ADHD) and learning difficulties. We propose that neurosteroid-replacement therapy with ganaxolone (GNX) following preterm birth may mitigate preterm-associated neurodevelopmental impairment. METHODS: Time-mated sows were delivered preterm (d62) or at term (d69). Male preterm pups were randomized to ganaxolone (Prem-GNX; 2.5 mg/kg subcutaneously twice daily until term equivalence), or preterm control (Prem-CON). Surviving male juvenile pups underwent behavioural testing at d25-corrected postnatal age (CPNA). Brain tissue was collected at CPNA28 and mature myelinating oligodendrocytes of the hippocampus and subcortical white matter were quantified by immunostaining of myelin basic protein (MBP). RESULTS: Ganaxolone treatment returned the hyperactive behavioural phenotype of preterm-born juvenile males to a term-born phenotype. Deficits in MBP immunostaining of the preterm hippocampus and subcortical white matter were also ameliorated in animals receiving ganaxolone. However, during the treatment period weight gain was poor, and pups were sedated, ultimately increasing the neonatal mortality rate. CONCLUSION: Ganaxolone improved neurobehavioural outcomes in males suggesting that neonatal treatment may be an option for reducing preterm-associated neurodevelopmental impairment. However, dosing studies are required to reduce the burden of unwanted side effects.

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.

Long-term effects of preterm birth on behavior and neurosteroid sensitivity in the guinea pig.

BACKGROUND: Ex-preterm children and adolescents are at risk of developing late-onset neurodevelopmental and behavioral disorders. The mechanisms by which this happens are poorly understood and relevant animal models are required. METHODS: Ex-preterm (delivered at 62 d gestation) and term (spontaneously delivered) juvenile guinea pigs underwent behavioral testing at 25 d corrected postnatal age, with tissues collected at 28 d. Neurodevelopmental markers (myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP)) were analyzed in the hippocampus and subcortical white matter by immunohistochemistry. Gamma-aminobutyric acid A (GABAA) receptor subunit mRNA levels were quantified by reverse transcription polymerase chain reaction (RT-PCR), and salivary cortisol measured by enzyme-linked immunosorbent assay. RESULTS: Preterm males travelled greater distances, were mobile for longer, spent more time investigating objects, and approached or interacted with familiar animals more than controls. Myelination and reactive astrocyte coverage was lower in the hippocampus and the subcortical white matter in preterm males. Hippocampal levels of the α5 subunit were also lower in the preterm male brain. Baseline salivary cortisol was higher for preterm males compared to controls. CONCLUSION: We conclude that juvenile ex-preterm male guinea pigs exhibit a hyperactive phenotype and feature impaired neurodevelopment, making this a suitable model for future therapeutic studies.

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.

Potential for a cerebellar role in moderate-late preterm associated behavioural disorders.

Preterm birth is known to cause impaired cerebellar development, and this is associated with the development of neurobehavioral disorders. This review aims to identify the mechanisms through which preterm birth impairs cerebellar development and consequently, increases the risk of developing neurobehavioral disorders. The severity of these disorders is directly related to the degree of prematurity, but it is also evident that even late preterm births are at significantly increased risk of developing serious neurobehavioral disorders. Preterm birth is associated with hypoxic events and increased glutamatergic tone within the neonatal brain which contribute to excitotoxic damage. The cerebellum is a dense glutamatergic region which undergoes relatively late neurodevelopment up to and beyond birth. Evidence indicates that the cerebellum forms reciprocal connections to regions important in behaviour regulation such as the limbic system and frontal cortex. Studies using fMRI (functional magnetic resonance Imaging), BOLD (blood oxygen level dependent) response and morphology studies in humans show the cerebellum is often involved in disorders such as attention deficit hyperactivity disorder (ADHD) and anxiety. The vulnerability of the cerebellum to preterm birth insult and its connections to behaviour associated brain regions implicates it in the development of neurobehavioral disorders. Protection against preterm associated insults on the cerebellum may provide a novel avenue through which ADHD and anxiety can be reduced in children born preterm.

Ongoing effects of preterm birth on the dopaminergic and noradrenergic pathways in the frontal cortex and hippocampus of guinea pigs.

Children born preterm have an increased likelihood of developing neurobehavioral disorders such as attention-deficit hyperactivity disorder (ADHD) and anxiety. These disorders have a sex bias, with males having a higher incidence of ADHD, whereas anxiety disorder tends to be more prevalent in females. Both disorders are underpinned by imbalances to key neurotransmitter systems, with dopamine and noradrenaline in particular having major roles in attention regulation and stress modulation. Preterm birth disturbances to neurodevelopment may affect this neurotransmission in a sexually dimorphic manner. Time-mated guinea pig dams were allocated to deliver by preterm induction of labor (gestational age 62 [GA62]) or spontaneously at term (GA69). The resultant offspring were randomized to endpoints as neonates (24 h after term-equivalence age) or juveniles (corrected postnatal day 40, childhood equivalence). Relative mRNA expressions of key dopamine and noradrenaline pathway genes were examined in the frontal cortex and hippocampus and quantified with real-time PCR. Myelin basic protein and neuronal nuclei immunostaining were performed to characterize the impact of preterm birth. Within the frontal cortex, there were persisting reductions in the expression of dopaminergic pathway components that occurred in preterm males only. Conversely, preterm-born females had increased expression of key noradrenergic receptors and a reduction of the noradrenergic transporter within the hippocampus. This study demonstrated that preterm birth results in major changes in dopaminergic and noradrenergic receptor, transporter, and synthesis enzyme gene expression in a sex- and region-based manner that may contribute to the sex differences in susceptibility to neurobehavioral disorders. These findings highlight the need for the development of sex-based treatments for improving these conditions.

Dual isolation of primary neurons and oligodendrocytes from guinea pig frontal cortex.

Primary cell culture is a technique that is widely used in neuroscience research to investigate mechanisms that underlie pathologies at a cellular level. Typically, mouse or rat tissue is used for this process; however, altricial rodent species have markedly different neurodevelopmental trajectories comparatively to humans. The use of guinea pig brain tissue presents a novel aspect to this routinely used cell culture method whilst also allowing for dual isolation of two major cell types from a physiologically relevant animal model for studying perinatal neurodevelopment. Primary neuronal and oligodendrocyte cell cultures were derived from fetal guinea pig's frontal cortex brain tissue collected at a gestational age of 62 days (GA62), which is a key time in the neuronal and oligodendrocyte development. The major advantage of this protocol is the ability to acquire both neuronal and oligodendrocyte cellular cultures from the frontal cortex of one fetal brain. Briefly, neuronal cells were grown in 12-well plates initially in a 24-h serum-rich medium to enhance neuronal survival before switching to a serum-free media formulation. Oligodendrocytes were first grown in cell culture flasks using a serum-rich medium that enabled the growth of oligodendrocyte progenitor cells (OPCs) on an astrocyte bed. Following confluency, the shake method of differential adhesion and separation was utilized via horizontally shaking the OPCs off the astrocyte bed overnight. Therefore, OPCs were plated in 12-well plates and were initially expanded in media supplemented with growth hormones, before switching to maturation media to progress the lineage to a mature phenotype. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on both cell culture types to analyze key population markers, and the results were further validated using immunocytochemistry. Primary neurons displayed the mRNA expression of multiple neuronal markers, including those specific to GABAergic populations. These cells also positively stained for microtubule-associated protein 2 (MAP2; a dendritic marker specific to neurons) and NeuN (a marker of neuronal cell bodies). Primary oligodendrocytes expressed all investigated markers of the oligodendrocyte lineage, with a majority of the cells displaying an immature oligodendrocyte phenotype. This finding was further confirmed with positive oligodendrocyte transcription factor (OLIG2) staining, which serves as a marker for the overall oligodendrocyte population. This study demonstrates a novel method for isolating both neurons and oligodendrocytes from the guinea pig brain tissue. These isolated cells display key markers and gene expression that will allow for functional experiments to occur and may be particularly useful in studying neurodevelopmental conditions with perinatal origins.

Evaluating changes in GABAergic and glutamatergic pathways in early life following prenatal stress and postnatal neurosteroid supplementation.

BACKGROUND: A correct balance of activity of the GABA and glutamate systems is vital for optimal neurodevelopment and general CNS function, and the dysregulation of this balance has been implicated in a number of neurological conditions. Maternal exposure to stressors is known to have long lasting, deleterious impacts on neurobehaviour, and similarly, results in dysregulation of inhibitory and excitatory pathways in the offspring. The current study aimed to examine effects on these pathways in a guinea pig model of prenatal stress and to elucidate whether increased neuroprotective support by postnatal neurosteroid supplementation would ameliorate adverse outcomes. METHODS: Prenatal stress was achieved by exposing pregnant guinea pigs dams to a strobe light for 2hrs/day on gestational age (GA) 50, 55, 60 and 65. Dams were allowed to spontaneously deliver (~GA70) and pups were orally administered either allopregnanolone analogue, ganaxolone (5 mg/kg/day in 45% cyclodextrin), the translocator protein (TSPO) agonist, emapunil (XBD173; 0.3 mg/kg/day in 1% tragacanth gum) or vehicle on postnatal days (PND) 1-7. Hippocampal samples were collected at PND30 to measure relative mRNA expression of components involved in the inhibitory GABAergic pathway and exctitatory glutamatergic pathway by real-time PCR. GABAergic interneurons were quantified by assessing immunohistochemical protein expression of markers parvalbumin, calbindin and calretinin. RESULTS: mRNA expression of GABAergic pathway components at one week of age indicated immature expression profiles of the GABAA receptors as well as decreased GABA synthesis and transport suggesting reduced extrasynaptically-mediated tonic inhibition. Expression profiles of the pathways examined evolved between one week and one month of age but an imbalance in inhibitory/excitatory components persisted. The allopregnanolone analogue ganaxolone offered some protection against excitotoxicity in female hippocampus, however neurosteroid supplementation with ganaxolone or emapunil were unable to fully correct the GABAergic/glutamatergic imbalance observed following prenatal stress. CONCLUSION: Prenatal stress leads to programmed lasting effects on the major inhibitory and excitatory pathways in the guinea pig brain that continue evolving between the equivalent of early and late childhood. Neurosteroid therapies particularly improved outcomes in females. Further studies are required to identify additional therapeutic targets that are able to fully restore imbalances in the excitatory and inhibitory systems, which may act to prevent development of childhood behavioural disorders.

Examining Neurosteroid-Analogue Therapy in the Preterm Neonate For Promoting Hippocampal Neurodevelopment.

Background: Preterm birth can lead to brain injury and currently there are no targeted therapies to promote postnatal brain development and protect these vulnerable neonates. We have previously shown that the neurosteroid-analogue ganaxolone promotes white matter development and improves behavioural outcomes in male juvenile guinea pigs born preterm. Adverse side effects in this previous study necessitated this current follow-up dosing study, where a focus was placed upon physical wellbeing during the treatment administration and markers of neurodevelopment at the completion of the treatment period. Methods: Time-mated guinea pigs delivered preterm (d62) by induction of labour or spontaneously at term (d69). Preterm pups were randomized to receive no treatment (Prem-CON) or ganaxolone at one of three doses [0.5 mg/kg ganaxolone (low dose; LOW-GNX), 1.0 mg/kg ganaxolone (mid dose; MID-GNX), or 2.5 mg/kg ganaxolone (high dose; HIGH-GNX) in vehicle (45% ß-cyclodextrin)] daily until term equivalence age. Physical parameters including weight gain, ponderal index, supplemental feeding, and wellbeing (a score based on respiration, activity, and posture) were recorded throughout the preterm period. At term equivalence, brain tissue was collected, and analysis of hippocampal neurodevelopment was undertaken by immunohistochemistry and RT-PCR. Results: Low and mid dose ganaxolone had some impacts on early weight gain, supplemental feeding, and wellbeing, whereas high dose ganaxolone significantly affected all physical parameters for multiple days during the postnatal period when compared to the preterm control neonates. Deficits in the preterm hippocampus were identified using neurodevelopmental markers including mRNA expression of oligodendrocyte lineage cells (CSPG4, MBP), neuronal growth (INA, VEGFA), and the GABAergic/glutamatergic system (SLC32A1, SLC1A2, GRIN1, GRIN2C, DLG4). These deficits were not affected by ganaxolone at the doses used at the equivalent of normal term. Conclusion: This is the first study to investigate the effects of a range of doses of ganaxolone to improve preterm brain development. We found that of the three doses, only the highest dose of ganaxolone (2.5 mg/kg) impaired key indicators of physical health and wellbeing over extended periods of time. Whilst it may be too early to see improvements in markers of neurodevelopment, further long-term study utilising the lower doses are warranted to assess functional outcomes at ages when preterm birth associated behavioural disorders are observed.

Impaired Oligodendrocyte Development Following Preterm Birth: Promoting GABAergic Action to Improve Outcomes.

Preterm birth is associated with poor long-term neurodevelopmental and behavioral outcomes, even in the absence of obvious brain injury at the time of birth. In particular, behavioral disorders characterized by inattention, social difficulties and anxiety are common among children and adolescents who were born moderately to late preterm (32-37 weeks' gestation). Diffuse deficits in white matter microstructure are thought to play a role in these poor outcomes with evidence suggesting that a failure of oligodendrocytes to mature and myelinate axons is responsible. However, there remains a major knowledge gap over the mechanisms by which preterm birth interrupts normal oligodendrocyte development. In utero neurodevelopment occurs in an inhibitory-dominant environment due to the action of placentally derived neurosteroids on the GABAA receptor, thus promoting GABAergic inhibitory activity and maintaining the fetal behavioral state. Following preterm birth, and the subsequent premature exposure to the ex utero environment, this action of neurosteroids on GABAA receptors is greatly reduced. Coinciding with a reduction in GABAergic inhibition, the preterm neonatal brain is also exposed to ex utero environmental insults such as periods of hypoxia and excessive glucocorticoid concentrations. Together, these insults may increase levels of the excitatory neurotransmitter glutamate in the developing brain and result in a shift in the balance of inhibitory: excitatory activity toward excitatory. This review will outline the normal development of oligodendrocytes, how it is disrupted under excitation-dominated conditions and highlight how shifting the balance back toward an inhibitory-dominated environment may improve outcomes.

Effects of prenatal stress on behavioural and neurodevelopmental outcomes are altered by maternal separation in the neonatal period.

BACKGROUND: Chronic psychosocial stress during pregnancy and/or after birth, and the associated elevation in cortisol, is linked with the onset of behavioural disorders in childhood. Previously, prenatal stress has been shown to reduce neurosteroid pathways in the fetus and the levels of the neurosteroid and GABAA receptor agonist, allopregnanolone. In late gestation, elevated levels of GABAergic activity increases inhibitory tone and protects against excessive excitation. These levels of allopregnanolone may also contribute to promoting myelination, thus stress-induced suppression of protective neurosteroid levels may disrupt neurodevelopmental processes and can result in reduced myelination. The objective of this study was to examine whether prenatal and postnatal stress reduces levels of inhibitory pathways to result in behavioural, myelin, and GABAergic/glutamatergic pathway deficits in the hippocampus at a postnatal time point in the guinea pig equivalent to childhood in humans. METHODS: Pregnant guinea pig dams were exposed to prenatal stress (PRE) with strobe light exposure for 2 h/day on gestational age (GA) 50, 55, 60 and 65 (term is ∼GA70), with postnatal stress (POST) caused by maternal separation for 2 h/day from postnatal day (PND) 1-7), or a double-hit of both stressors (PRE + POST). Control dams and offspring groups (CON) were handled at the same time each day without causing stress. Behavioural outcomes were assessed using open field and elevated plus maze testing on PND27. After euthanasia on PND30, plasma samples were collected for steroid quantification of cortisol, allopregnanolone and progesterone by ELISA. Hippocampal samples were collected to assess markers of oligodendrocyte development and mature cells by myelin basic protein (MBP) immunostaining and GABAergic and glutamatergic pathway component gene expression by real time PCR. RESULTS: Male guinea pig offspring exposed to prenatal stress exhibited hyperactive-like behaviour at childhood equivalence, while female offspring displayed anxious-like behaviour, to a lesser extent. In both sexes, MBP immunostaining was significantly decreased in the hippocampal region following prenatal stress, despite normal levels of MBP mRNA, which suggests a disruption to the MBP protein translation pathway. Many components of the GABAergic and glutamatergic pathways were disrupted following prenatal stress, notably GABAA receptor subunits, GABA production and uptake, glutamate ionotropic and metabotropic receptor subunits and glutamate transport. Following prenatal + postnatal stress, many of the behavioural and neurodevelopmental deficits were improved compared to the prenatal stress only group. CONCLUSION: We conclude that prenatal stress disrupts GABAergic and glutamatergic pathways that may contribute to reduced myelination and subsequent behavioural deficits in the offspring. The deficits seen following prenatal stress are ameliorated when paired with subsequent postnatal stress, which highlights the early postnatal period as an important treatment window.

Neurosteroid-based intervention using Ganaxolone and Emapunil for improving stress-induced myelination deficits and neurobehavioural disorders.

BACKGROUND: Prenatal stress is associated with long-term disturbances in white matter development and behaviour in children, such as attention deficit hyperactivity disorder (ADHD) and anxiety. Oligodendrocyte maturation and myelin formation is a tightly orchestrated process beginning during gestation, and therefore is very vulnerable to the effects of maternal prenatal stresses in mid-late pregnancy. The current study aimed to examine the effects of prenatal stress on components of the oligodendrocyte lineage to identify the key processes that are disrupted and to determine if postnatal therapies directed at ameliorating white matter deficits also improve behavioural outcomes. METHODS: Pregnant guinea pig dams were exposed to control-handling or prenatal stress with strobe light exposure for 2hrs/day on gestational age (GA) 50, 55, 60 and 65, and allowed to spontaneously deliver ~GA70. Pups were administered oral ganaxolone (5 mg/kg/day in 45% cyclodextrin) or the TSPO agonist, emapunil (XBD173; 0.3 mg/kg/day in 1% tragacanth gum) or vehicle, on postnatal days (PND) 1-7. Behavioural outcomes were assessed using open field and elevated plus maze testing on PND7 and PND27. Hippocampal samples were collected at PND30 to assess markers of oligodendrocyte development through assessment of total oligodendrocytes (OLIG2) and mature cells (myelin basic protein; MBP), and total neurons (NeuN) by immunostaining. Real-time PCR was conducted on hippocampal regions to assess markers of the oligodendrocyte lineage, markers of neurogenesis and components of the neurosteroidogenesis pathway. Plasma samples were collected for steroid quantification of cortisol, allopregnanolone, progesterone and testosterone by ELISA. RESULTS: Prenatal stress resulted in hyperactivity in male offspring, and anxiety-like behaviour in female offspring in the guinea pig at an age equivalent to late childhood. Postnatal ganaxolone and emapunil treatment after prenatal stress restored the behavioural phenotype to that of control in females only. The oligodendrocyte maturation lineage, translation of MBP mRNA-to-protein, and neurogenesis were disrupted in prenatally-stressed offspring, resulting in a decreased amount of mature myelin. Emapunil treatment restored mature myelin levels in both sexes, and reversed disruptions to the oligodendrocyte lineage in female offspring, an effect not seen with ganaxolone treatment. CONCLUSION: The marked and persisting behavioural and white matter perturbations observed in a clinically relevant guinea pig model of prenatal stress highlights the need for postnatal interventions that increase myelin repair and improve long-term outcomes. The effectiveness of emapunil treatment in restoring female offspring behaviour, and promoting maturation of myelin indicates that early therapeutic interventions can reverse the damaging effects of major stressful events in pregnancy. Further studies optimising target mechanisms and dosing are warranted.

Perinatal compromise contributes to programming of GABAergic and glutamatergic systems leading to long-term effects on offspring behaviour.

Extensive evidence now shows that adversity during the perinatal period is a significant risk factor for the development of neurodevelopmental disorders long after the causative event. Despite stemming from a variety of causes, perinatal compromise appears to have similar effects on the developing brain, thereby resulting in behavioural disorders of a similar nature. These behavioural disorders occur in a sex-dependent manner, with males affected more by externalising behaviours such as attention deficit hyperactivity disorder (ADHD) and females by internalising behaviours such as anxiety. Regardless of the causative event or the sex of the offspring, these disorders may begin in childhood or adolescence but extend into adulthood. A mechanism by which adverse events in the perinatal period impact later in life behaviour has been shown to be the changing epigenetic landscape. Methylation of the GAD1/GAD67 gene, which encodes the key glutamate-to-GABA-synthesising enzyme glutamate decarboxylase 1, resulting in increased levels of glutamate, is one epigenetic mechanism that may account for a tendency towards excitation in disorders such as ADHD. Exposure of the fetus or the neonate to high levels of cortisol may be the mediator between perinatal compromise and poor behavioural outcomes because evidence suggests that increased glucocorticoid exposure triggers widespread changes in the epigenetic landscape. This review summarises the current evidence and recent literature about the impact of various perinatal insults on the epigenome and the common mechanisms that may explain the similarity of behavioural outcomes occurring following diverse perinatal compromise.

Reduced Neurosteroid Exposure Following Preterm Birth and Its' Contribution to Neurological Impairment: A Novel Avenue for Preventative Therapies.

Children born preterm are at an increased risk of developing cognitive problems and neuro-behavioral disorders such as attention deficit hyperactivity disorder (ADHD) and anxiety. Whilst neonates born at all gestational ages, even at term, can experience poor cognitive outcomes due to birth-complications such as birth asphyxia, it is becoming widely known that children born preterm in particular are at significant risk for learning difficulties with an increased utilization of special education resources, when compared to their healthy term-born peers. Additionally, those born preterm have evidence of altered cerebral myelination with reductions in white matter volumes of the frontal cortex, hippocampus and cerebellum evident on magnetic resonance imaging (MRI). This disruption to myelination may underlie some of the pathophysiology of preterm-associated brain injury. Compared to a fetus of the same post-conceptional age, the preterm newborn loses access to in utero factors that support and promote healthy brain development. Furthermore, the preterm ex utero environment is hostile to the developing brain with a myriad of environmental, biochemical and excitotoxic stressors. Allopregnanolone is a key neuroprotective fetal neurosteroid which has promyelinating effects in the developing brain. Preterm birth leads to an abrupt loss of the protective effects of allopregnanolone, with a dramatic drop in allopregnanolone concentrations in the preterm neonatal brain compared to the fetal brain. This occurs in conjunction with reduced myelination of the hippocampus, subcortical white matter and cerebellum; thus, damage to neurons, astrocytes and especially oligodendrocytes of the developing nervous system can occur in the vulnerable developmental window prior to term as a consequence reduced allopregnanolone. In an effort to prevent preterm-associated brain injury a number of therapies have been considered, but to date, other than antenatal magnesium sulfate and corticosteroid therapy, none have become part of standard clinical care for vulnerable infants. Therefore, there remains an urgent need for improved therapeutic options to prevent brain injury in preterm neonates. The actions of the placentally derived neurosteroid allopregnanolone on GABAA receptor signaling has a major role in late gestation neurodevelopment. The early loss of this intrauterine neurotrophic support following preterm birth may be pivotal to development of neurodevelopmental morbidity. Thus, restoring the in utero neurosteroid environment for preterm neonates may represent a new and clinically feasible treatment option for promoting better trajectories of myelination and brain development, and therefore reducing neurodevelopmental disorders in children born preterm.

Administration of Progesterone Throughout Pregnancy Increases Maternal Steroids Without Adverse Effect on Mature Oligodendrocyte Immunostaining in the Guinea Pig.

Progesterone is administered to pregnant women at risk of premature labor, despite systematic reviews showing conflicting outcomes regarding its use, highlighting doubt over the effectiveness of the therapy. Progesterone can be rapidly metabolized into a number of steroids, but to date, there has been a lack of investigation into the fetal steroid profiles following administration and whether this impacts fetal neurodevelopment. The objective of this study was to determine the effect of progesterone treatment on allopregnanolone and cortisol levels in the fetus and on a marker of myelination in the fetal brain. We used a guinea pig model where pregnant dams were administered vehicle (ß-cyclodextrin) or progesterone orally throughout pregnancy (GA29-61). Maternal and fetal fluids and tissues were collected at both preterm (GA61) and term (GA68) ages. Maternal and fetal progesterone and cortisol were analyzed by enzyme immunoassay and allopregnanolone by radioimmunoassay. Measurement of myelination of fetal brains (hippocampus, cingulum, and subcortical white matter) at preterm and term ages was performed by immunohistochemistry staining for myelin basic protein. We found that dams receiving progesterone had significantly elevated progesterone and cortisol concentrations, but there was no effect on allopregnanolone. Interestingly, the increased cortisol concentrations were not reflected in the fetuses, and there was no effect of progesterone treatment on myelination. Therefore, we conclude that in our guinea pig model, maternal administration of progesterone has no effect on cortisol levels or markers of mature oligodendrocytes in the fetus and suggest this is potentially due to the protective cortisol barrier in the placenta.

Birth and Neonatal Transition in the Guinea Pig: Experimental Approaches to Prevent Preterm Birth and Protect the Premature Fetus.

The guinea pig (Cavia porcellus) displays many features of gestational physiology that makes it the most translationally relevant rodent species. Progesterone production undergoes a luteal to placental shift as in human pregnancy with levels rising during gestation and with labor and delivery occurring without a precipitous decline in maternal progesterone levels. In contrast to other laboratory rodents, labor in guinea pigs is triggered by a functional progesterone withdrawal, which involves the loss of uterine sensitivity to progesterone like in women. In both species the amnion membrane is a major source of labor-inducing prostaglandins, which promote functional progesterone withdrawal by modifying myometrial progesterone receptor expression. These similar features appear to result from convergent evolution rather than closer evolutionally relationship to primates compared to other rodents. Nevertheless, the similarities in the production, metabolism and actions of progesterone and prostaglandins allow information gained in pregnant guinea pigs to be extended to pregnant women with confidence. This includes exploring the effects of pregnancy complications including growth restriction and the mechanisms by which stressful conditions increase the incidence of preterm labor. The relatively long gestation of the guinea pig and the maturity of the pups at birth particularly in brain development means that a greater proportion of brain development happens in utero. This allows adverse intrauterine conditions to make a sustained impact on the developing brain like in compromised human pregnancies. In addition, the brain is exposed to a protective neurosteroid environment in utero, which has been suggested to promote development in the guinea pig and the human. Moreover, in utero stresses that have been shown to adversely affect long term neurobehavioral outcomes in clinical studies, can be modeled successfully in guinea pigs. Overall, these parallels to the human have led to increasing interest in the guinea pig for translational studies of treatments and therapies that potentially improve outcomes following adverse events in pregnancy and after preterm birth.

Disruptions to the cerebellar GABAergic system in juvenile guinea pigs following preterm birth.

BACKGROUND: Children that are born preterm are at an increased risk of developing cognitive problems and behavioural disorders, such as attention deficit hyperactivity disorder (ADHD). There is increasing interest in the role of the cerebellum in these processes and the potential involvement of GABAergic pathways in neurodevelopmental disorders. We propose that preterm birth, and the associated loss of the trophic intrauterine environment, alters the development of the cerebellum, contributing to ongoing neurobehavioral disorders. METHODS: Guinea pigs were delivered preterm (GA62) or spontaneously at term (GA69), and tissues collected at corrected postnatal day (PND) 28. Neurodevelopmental and GABAergic markers myelin basic protein (MBP), neuronal nuclei (NeuN), calbindin (Purkinje cells), and GAD67 (GABA synthesis enzyme) were analysed in cerebellar lobules IX and X by immunohistochemistry. Protein expression of GAD67 and GAT1 (GABA transporter enzyme) were quantified by western blot, whilst neurosteroid-sensitive GABAA receptor subunits were measured by RT-PCR. RESULTS: MBP immunostaining was increased in lobule IX of preterm males, and reduced in lobule X of preterm females when compared to their term counterparts. GAD67 staining was decreased in lobule IX and X of the preterm males, but only in lobule X of the preterm females compared to term cohorts for each sex. Internal granule cell layer width of lobule X was decreased in preterm cohorts of both sexes compared to terms. There were no differences between gestational age groups for NeuN staining, GAD67 and GAT1 protein expression as measured by western blotting, or GABAA receptor subunits as measured by RT-PCR between preterm and term for either sex. CONCLUSIONS: The present findings suggest that components of the cerebellar GABAergic system of the ex-preterm cerebellum are disrupted. The higher expression of myelin in the preterm males may be due to a deficit in axonal pruning, whereas females have a deficit in myelination at 28 corrected days of age. Together these ongoing alterations may contribute to the neurodevelopmental and behavioural disorders observed in those born preterm.

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.