Abstract
Immature neurons dominantly express the Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) rather than the K+-Cl- cotransporter isoform 2 (KCC2). The intracellular chloride ion concentration ([Cl-]i) is higher in immature neurons than in mature neurons; therefore, γ-aminobutyric acid type A (GABAA) receptor activation in immature neurons does not cause chloride ion influx and subsequent hyperpolarization. In our previous work, we found that midazolam, benzodiazepine receptor agonist, causes less sedation in neonatal rats compared to adult rats and that NKCC1 blockade by bumetanide enhances the midazolam-induced sedation in neonatal, but not in adult, rats. These results suggest that GABA receptor activation requires the predominance of KCC2 over NKCC1 to exert sedative effects. In this study, we focused on CLP290, a novel KCC2-selective activator, and found that midazolam administration at 20 mg/kg after oral CLP290 intake significantly prolonged the righting reflex latency even in neonatal rats at postnatal day 7. By contrast, CLP290 alone did not exert sedative effects. Immunohistochemistry showed that midazolam combined with CLP290 decreased the number of phosphorylated cAMP response element-binding protein-positive cells in the cerebral cortex, suggesting that CLP290 reverted the inhibitory effect of midazolam. Moreover, the sedative effect of combined CLP290 and midazolam treatment was inhibited by the administration of the KCC2-selective inhibitor VU0463271, suggesting indirectly that the sedation-promoting effect of CLP290 was mediated by KCC2 activation. To our knowledge, this study is the first report showing the sedation-promoting effect of CLP290 in neonates and providing behavioral and histological evidence that CLP290 reverted the sedative effect of GABAergic drugs through the activation of KCC2. Our data suggest that the clinical application of CLP290 may provide a breakthrough in terms of midazolam-resistant sedation.
Highlights
Introduction γAminobutyric acid (GABA) is an inhibitory neurotransmitter that binds to the GABA receptor
To examine whether K+-Cl- cotransporter isoform 2 (KCC2) activation promotes the sedative effect of midazolam in neonates and adults, CLP290 was administered, a KCC2 activator already used in several publications [10,13,15,16,17,22]
To better understand the difference in the sedation-promoting effect of CLP290 and that of bumetanide previously shown by Koyama et al, we considered midazolam at 20 mg/kg was suitable for the subsequent assay in neonates [12,19]
Summary
Introduction γAminobutyric acid (GABA) is an inhibitory neurotransmitter that binds to the GABA receptor. Immature neurons maintain relatively higher intracellular chloride ion levels ([Cl-]i) compared to those in mature neurons and, GABAA receptor activation does not induce chloride ion influx [4,5]. Instead, it may induce chloride ion efflux, leading to neuronal depolarization [1,6]. The switch in the functional role of the GABAA receptor from excitatory to inhibitory GABA signaling occurs between PND8 and 14, which correlates with the switch in dominant expression from NKCC1 to KCC2 [6,7,8]
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