Prenatal Hypoxia Induces Cl- Cotransporters KCC2 and NKCC1 Developmental Abnormality and Disturbs the Influence of GABAA and Glycine Receptors on Fictive Breathing in a Newborn Rat.

Céline Caravagna,Sylvie Liabeuf,Laurence Bodineau,Cécile Brocard,Julie Peyronnet,Florence Cayetanot,Jacques-Olivier Coq,Alexis Casciato

doi:10.3389/fphys.2022.786714

Abstract

Prenatal hypoxia is a recognised risk factor for neurodevelopmental disorders associated with both membrane proteins involved in neuron homeostasis, e.g., chloride (Cl–) cotransporters, and alterations in brain neurotransmitter systems, e.g., catecholamines, dopamine, and GABA. Our study aimed to determine whether prenatal hypoxia alters central respiratory drive by disrupting the development of Cl– cotransporters KCC2 and NKCC1. Cl– homeostasis seems critical for the strength and efficiency of inhibition mediated by GABAA and glycine receptors within the respiratory network, and we searched for alterations of GABAergic and glycinergic respiratory influences after prenatal hypoxia. We measured fictive breathing from brainstem in ex vivo preparations during pharmacological blockade of KCC2 and NKCC1 Cl– cotransporters, GABAA, and glycine receptors. We also evaluated the membrane expression of Cl– cotransporters in the brainstem by Western blot and the expression of Cl– cotransporter regulators brain-derived neurotrophic factor (BDNF) and calpain. First, pharmacological experiments showed that prenatal hypoxia altered the regulation of fictive breathing by NKCC1 and KCC2 Cl– cotransporters, GABA/GABAA, and glycin. NKCC1 inhibition decreased fictive breathing at birth in control mice while it decreased at 4 days after birth in pups exposed to prenatal hypoxia. On the other hand, inhibition of KCC2 decreased fictive breathing 4 days after birth in control mice without any change in prenatal hypoxia pups. The GABAergic system appeared to be more effective in prenatal hypoxic pups whereas the glycinergic system increased its effectiveness later. Second, we observed a decrease in the expression of the Cl– cotransporter KCC2, and a decrease with age in NKCC1, as well as an increase in the expression of BDNF and calpain after prenatal hypoxia exposure. Altogether, our data support the idea that prenatal hypoxia alters the functioning of GABAA and glycinergic systems in the respiratory network by disrupting maturation of Cl– homeostasis, thereby contributing to long-term effects by disrupting ventilation.

Highlights

Development of a mammal neural system is influenced by early life experiences
In medullary-spinal cord (MS) preparations, fR increasesd with age in CONT pups (p = 0.0002) without there being any change in prenatal hypoxia (PH) pups, whereas in ponto–medullary–spinal cord (PMS) preparations, fR increased in PH pups (p = 0.0002) without there being any change in CONT groups
Through analysing fictive breathing on ex vivo en bloc preparations under pharmacological applications, we showed that prenatal hypoxia disrupts NKCC1 and KCC2 Cl−dependent regulation of central respiratory drive and influences GABA/GABAA and glycine systems whose cell effects depend on intracellular Cl− concentration

Summary

Introduction

Development of a mammal neural system is influenced by early life experiences. According to the hypothesis of “developmental programming of health and disease” or “fetal origins of disorder later in life,” maternal environment has an impact on foetal development, and even on adult health (Barker et al, 1993; de Boo and Harding, 2006; Li et al, 2012). In immature neurons, limited expression of the K+/Cl− cotransporter (KCC2) that extrudes Cl− from cells and greater expression of the Na+/K+/Cl− cotransporter (NKCC1), which intrudes Cl− into cells lead to high intracellular Cl− concentrations (Blaesse et al, 2009) In such conditions, the activation of GABAA and glycine receptors induces an increase in Cl− outward conductance that depolarises neurons and promotes excitation. The membrane expression of KCC2 increases whereas the NKCC1 expression decreases, leading to a weak intracellular Cl− concentration (Liu and Wong-Riley, 2012) In this condition, the activation of GABAA and/or glycine receptors induces an increase in Cl− inward conductance that hyperpolarises neurons that promote inhibition (Rivera et al, 2004; Vinay and Jean-Xavier, 2008; Stil et al, 2009). As Cl− homeostasis depends on environmental conditions including prenatal stress, we hypothesised that a defect in Cl− cotransporters disrupts GABA modulation of breathing, as observed after a gestational stress (Delhaes et al, 2014; Pozzi et al, 2020)

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