Minère, Marielle; (2020) Characterising the electrophysiological and behavioural function of neurosteroids at α4-subunit containing GABAA receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

γ-aminobutyric acid type-A (GABAA) receptors are the main mediators of inhibitory transmission within the brain, playing a pivotal role in the excitation-inhibition (E-I) balance. Neurosteroids, potent modulators of all GABAA receptors, provide an important regulatory pathway fine-tuning inhibition. Since the most prevalent extrasynaptic GABAergic receptor incorporates the α4 subunit (containing the neurosteroid binding site) and δ subunit (conferring high neurosteroid sensitivity), we investigated the relevance of neurosteroid modulation at GABAA α4 receptors using electrophysiology and behaviour and a newly generated knock-in mouse model lacking neurosteroid sensitivity at GABAA α4 receptors. Functional properties of recombinantly expressed α4Q246M GABAA receptors were characterised, confirming their insensitivity to neurosteroid potentiation without affecting other functional receptor properties. Quantitative Western Blot (WB) analysis reveals the absence of compensatory GABAA subunit gene expression in α4Q246M mice. Patch clamp electrophysiology experiments indicate a major impairment in the neurosteroid potentiation of tonic inhibition, mediated by extrasynaptic receptors. Furthermore, physiological levels of neurosteroids were unable to control neuronal firing in GABAA α4Q246M receptor hippocampal neurons, indicating the importance of GABAA α4 receptors and neurosteroids in regulating hippocampal cell excitability. Additionally, we found that γ oscillation frequency (a measure of neuronal population activity) in the hippocampus is reduced by neurosteroid application, an effect we failed to observe in α4Q246M slices, implying key role(s) for these receptors and neurosteroids in regulating network activity. Finally, using a kainic acid-induced epilepsy model, the α4Q246M knock-in mouse displays a more severe acute epileptic phenotype and shortened latency for developing spontaneous seizures. Patch clamp recording from these epileptic animals shows that impaired neurosteroid potentiation of tonic inhibition underpins this behavioural phenotype. Overall, this study reveals that neurosteroid modulation of extrasynaptic GABAA α4 receptors is a major regulator of tonic inhibitory tone, neuronal excitability, neural network activity and susceptibility to developing epilepsy.

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