Elsevier

Neuropharmacology

Volume 45, Issue 4, September 2003, Pages 461-472
Neuropharmacology

Steroid-sensitive GABAA receptors in the fetal sheep brain

https://doi.org/10.1016/S0028-3908(03)00196-5Get rights and content

Abstract

Neuroactive steroids such as allopregnanolone (3α-hydroxy-5α-pregnan-20-one) influence central nervous system (CNS) excitability by increasing GABA (gamma aminobutyric acid) inhibitory activity. Allopregnanolone concentrations are higher in the fetal compared to the adult ovine brain, suggesting that this neurosteroid may have a role in regulating fetal CNS activity during gestation. We examined the localisation of allopregnanolone-sensitive GABAA receptors in the fetal brain to determine if their sensitivity to allopregnanolone changed during late gestation. The binding of [35S] tert-butylbicyclophosothionate (TBPS) was used to identify the GABA-chloride ion receptor complex in fetal sheep brains at 90–95, 115–120 and 140–145 days gestational age (GA; term ~147 days), by autoradiography. Allopregnanolone (200 nM) reduced [35S]TBPS binding by 70–100% throughout the brain at all fetal ages examined. The levels of [35S]TBPS binding increased with advancing gestation in all regions examined except some areas of the medulla. Functionally related nuclei and brain areas associated with regulating somato/viscerosensory functions displayed high levels of [35S]TBPS binding from mid-gestation. These results indicate that allopregnanolone may interact with GABAA receptors to inhibit fetal CNS activity from mid-gestation. This inhibition may contribute to maintaining the sleep-like behaviour and low incidence of arousal-type activity typical of fetal life, and may be neuroprotective by limiting excitatory neurotransmission.

Introduction

Neuroactive steroids, including allopregnanolone, are potent GABAA receptor ligands that enhance GABA-activated chloride ion conductance at low concentrations (Gee et al., 1987, Majewska et al., 1986). Allopregnanolone influences CNS excitability and behaviour in adult animals, and may have neuroprotective actions in the brain (Baulieu, 1998, Compagnone and Mellon, 2000). The placenta secretes large amounts of progesterone and progesterone metabolites during pregnancy, many of which are neuroactive (Majewska, 1992), and have an influence on maternal behaviour during late gestation (Lephart et al., 1996). In addition, we recently showed that fetal behaviour is changed markedly by alterations of progesterone synthesis and metabolism in pregnant sheep. For example, administration of progesterone to pregnant sheep in late gestation suppresses the CNS activities associated with fetal arousal, whereas inhibition of progesterone synthesis increases the incidence of fetal arousal (Nicol et al., 1997). Inhibition of the catabolism of progesterone to allopregnanolone by 5α-reductase increased CNS excitability and the incidence of fetal arousal (Nicol et al., 2001). These observations suggest that 5α-reduced metabolites of progesterone may have a role in suppressing fetal CNS activity. This suppression may create resistance to excitotoxicity and have a neuroprotective action during late gestation.

Studies in prenatal rats have identified differences in the expression of 5α-reductase between brain regions (Poletti et al., 1998). This finding suggests that neuroactive steroid concentrations may be greater in regions of high expression and these steroids may have a more marked role in neural modulation at these sites. We have found that 5α-reductase type-II is highly expressed in the fetal motor cortex, throughout the hypothalamus, hippocampus and brainstem from at least 115 days GA (Petratos et al., 2000, Nguyen et al., 2003). Expression was seen in neurones and some glial cells and was higher than levels observed in postnatal lambs. Our previous study using [35S]TBPS binding in homogenates of fetal and adult cortex showed that, in the presence of physiological concentrations of GABA, allopregnanolone reduced [35S]TBPS binding to GABAA receptors. Importantly, the fetal cortex was more sensitive to allopregnanolone than preparations of the adult sheep cortex (Crossley et al., 2000).

The aim of the present study was to extend these findings by using autoradiography to demonstrate the localisation of GABAA receptors, and then to examine their sensitivity to allopregnanolone in the fetal ovine brain from approximately mid to late gestation.

Section snippets

Brain tissue collection

Timed-pregnant Merino-Border Leicester cross ewes were used for these studies. The use of these animals and all procedures was approved by the Standing Committee on Ethics in Animal Experimentation of Monash University. The ewes were euthanised by an overdose of sodium pentobarbitone (Lethobarb 160 mg/kg i.v.; Virbac Pty Ltd, NSW, Australia), which was administered by rapid intravenous injection. The fetuses were immediately delivered by caesarean section and then decapitated. Brains were

Results

In the preliminary study, allopregnanolone was shown to inhibit [35S]TBPS binding in a concentration-dependent manner in the presence of GABA (5 μM) in cortical sections of fetal sheep brain (140–145 days GA). The concentration of allopregnanolone (in the presence of GABA) required to inhibit 50% of [35S]TBPS binding was approximately 200–500 nM (Fig. 1A), whereas 5 μM GABA alone reduced binding by 26%. Therefore, an allopregnanolone concentration of 200 nM was employed in the autoradiographic

Discussion

This study is the first to show localisation of neurosteroid modulation of [35S]TBPS binding to GABAA receptors in the developing sheep brain, a species with a long gestation and in which considerable brain maturation occurs in utero. The principle findings of the study were that binding to the GABAA receptor increased with advancing GA and that, in the presence of a low concentration of GABA, allopregnanolone caused displacement of [35S]TBPS binding. Although the exact location of the steroid

Acknowledgements

This work was supported by National Health and Medical Research Council of Australia grants to D.W. Walker and J.J. Hirst. D.W. Walker, A.J. Lawrence and P.M. Beart are Fellows of the National Health and National Research Council of Australia.

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