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Intrauterine growth restriction (IUGR) is one of the most common causes of perinatal mortality and morbidity. White matter and neuronal injury are major pathophysiological features of the IUGR neonatal brain. GABAA (γ-aminobutyric acid type A) receptors have been shown to play a role in oligodendrocyte differentiation and proliferation in the neonatal brain and may be a key factor in white matter injury and myelination in IUGR neonates. Whether there are impairments to the GABAergic system and neuronal cytoskeleton in IUGR brain has yet to be elucidated. This study aims to examine GABAA receptor α1 and α3 subunit protein expression and distribution in parietal cortex and hippocampus of the IUGR piglet at four different ages (term = 115 d – days gestational age), 100 d, 104 d, birth (postnatal day 0–P0) and P7 and to examine neuronal and myelination patterns. Significant alterations to GABAA receptor α1 and α3 protein expression levels were observed in the IUGR piglet brain of P7 IUGR piglets with significantly greater α3 expression compared to α1 expression in the hippocampus while there was virtually no difference between the two subunits in the parietal cortex. However a significantly lower α1/α3 ratio was evident in P7 IUGR cortex when compared with P7 NG cortex. Neuronal somatodendrites studied using MAP2 immunohistochemistry showed reduced and disrupted somatodendrites while MBP immunolabelling showed loss of axonal fibres from gestational day 104 d through to P7. These findings provide insights into the effects of IUGR on the development of the GABA system, altered developmental maturation of GABAA receptor subunit expression in the IUGR brain may influence myelination and may partly explain the cognitive disabilities observed in IUGR. Understanding the mechanisms behind grey and white matter injury in the IUGR infant is essential to identifying targets for treatments to improve long-term outcomes for IUGR infants.

GABA is a major neurotransmitter in the mammalian brain. In the mature brain GABA exerts inhibitory actions via the GABAA receptor (GABAAR); however, in the immature brain GABA provides much of the excitatory drive. We examined the expression of 3 predominant GABAA α-subunit proteins in the pig brain at various pre- and postnatal ages. Brain tissue was collected from piglets born via caesarean section at preterm ages 91, 97, 100, and 104 days' gestational age (GA), at term equivalent (114 days' GA, caesarean section) and at term, postnatal day 0 (P0) (spontaneous delivery, term = 115 days). Tissue was obtained from piglets at P4 and P7. Adult tissue from sows was collected postmortem after caesarean section. In all cortical regions and basal ganglia (1) α3 exhibited a significant increase in protein expression at 100 days' GA, (2) α3 expression decreased with age after 100 days' GA, (3) α1 increased with age, with peak expression at P7 in cortices, hippocampus, and thalamus, and (4) α2 protein expression remained relatively constant across the ages examined. The subunit expression of α3 was most abundant at preterm ages, with α1 the predominant subunit expressed postnatally. Immunofluorescent labelling revealed α1 expression on the somatic membranes of pyramidal cells in the cortex and hippocampus, and in the cerebellar Purkinje cells. Positive α3 labelling was apparent on interneurones in the cortex and hippocampus. The switch between dominant α-subunits may coincide with the functional change in GABAergic neurotransmission from excitation to inhibition. Brain growth in the pig closely reflects that in the term human, making the pig a valuable non-primate model for studying development and the effects of insults on the perinatal brain.

The principal function of the γ-aminobutyric acid (GABA) system in the adult brain is inhibition; however, in the neonatal brain, GABA provides much of the excitatory drive. As the brain develops, transmembrane chloride gradients change and the inhibitory role of GABA is initiated and continues throughout juvenile and adult life. Previous studies have shown that GABAA receptor subunit expression is developmentally regulated, and it is thought that the change in GABA function from excitation to inhibition corresponds to the changeover in expression of ‘immature’ to ‘mature’ subunit isoforms. We examined the protein expression pattern and distribution of GABA type A (GABAA) receptor α1-, α3- and β2-subunits in the parietal cortex and hippocampus of the developing piglet brain. Four perinatal ages were studied; 14 days preterm (P–14), 10 days preterm (P–10), day of birth (P0) and at postnatal day 7 (P7). Animals were obtained by either caesarean section or spontaneous birth. Protein expression levels and subunit localization were analysed by Western blotting and immunohistochemistry, respectively. In the cortex and hippocampus, GABAA receptor α1-subunit showed greatest expression at P7 when compared to all other age groups (p < 0.05). In contrast, α3 expression in the cortex was elevated in preterm brain, peaking at P0, followed by a significant reduction by P7 (p < 0.05); a similar trend was observed in the hippocampus. GABAA receptor β2-subunit protein expression appeared relatively constant across all time points studied in both the cortex and hippocampus. Immunolabelling of the α1-, α3- and β2-subunits was observed throughout all cortical layers at every age. GABAA receptor α3-subunit appeared to show specific localization to layers V and VI whilst labelling for the β2-subunit was observed in layer IV. In the hippocampus of all animals, the α1- and β2-subunits were shown to immunolabel various cells and processes in the dentate gyrus (DG), CA1 and CA3; the α3-subunit was barely observed except at the stratum moleculare of the DG. We report for the first time the ontogenesis of GABAA receptor subunits α1, α3 and β2 in the perinatal pig brain.

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.

Background Large animal models are an essential tool in the development of rationally-based new clinical therapies for preterm infants. We provide a description of the newborn pig as a model of the preterm neonate in terms of growth parameters, physiology and the requirement for intensive care over a range of gestational ages. Methods Twenty-nine litters of piglets (n = 298) were delivered by caesarean section at six timepoints during gestation from 91d to 113d (term = 115d). Two groups, at 91 and 97d gestation, also received maternal glucocorticoid treatment. At four of these timepoints, piglets (n = 79) were ventilated, sedated and monitored using standard neonatal intensive care techniques for up to 8 h in various experimental protocols. Results Body weight increased from mean 697 g (SD 193) at 91d gestation to 1331 g (SD 368) at 113d gestation. Piglets delivered at 97d gestation were able to be resuscitated and kept alive for at least 8 h on respiratory support after surfactant administration. Maternal glucocorticoid treatment 48 h and 24 h hours prior to delivery reduced the requirement for ventilator support and improved cardiovascular stability. Conclusion The pig provides a relevant model for the study of human preterm physiology and for investigation of novel therapies to improve outcomes.

Background and Aims: Antiepileptic drugs (AEDs) do not effectively control seizures in newborn brain and there is growing concern that use of standard AEDs such as phenobarbital may exacerbate seizures and brain injury. Recent data suggests this may be due to the changing role of the GABAA receptor (an AED target) during normal neurodevelopment, a process tightly coupled to developmental expression of the chloride co-transporter proteins NKCC1 and KCC2. The timing of the 'switch' in GABAA receptor function from excitation to inhibition is unclear and in human may not be completed until 3-4 months of age. The aim of this project was to establish the developmental profile of NKCC1, KCC2 and the GABAA receptor µ1 and µ3 subunits in piglet brain. Methods: Preterm and term piglets of several different gestational ages around birth were obtained by caesarean section or spontaneous delivery. Brain tissue was collected from several brain regions and expression analysed by western blot. Results: In parietal cortex the switch between NKCC1 and KCC2 and, GABAA receptor µ1 and µ3 expression was evident by P3. The switch in GABAA receptor µ1 and µ3 expression had not yet occurred in the frontal, temporal or occipital cortex. NKCC1, KCC2 analysis is ongoing. Conclusions: Efficacy of AEDs in neonatal brain may depend on regional and temporal maturation of inhibition. Furthermore changes in GABAA receptor expression as we have demonstrated following HI and seizures will influence receptor pharmacology. There is a critical need to develop effective treatment strategies specific to the neonatal brain.