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.

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.

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.

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.