Eckel, R;
(2015)
The Trafficking of GABAARs During Prolonged Seizure Activity.
Doctoral thesis , UCL (University College London).
Abstract
Status Epilepticus (SE), or the maximum expression of epilepsy, manifests as recurrent self-sustaining seizures resulting in significant morbidity and mortality. Fast inhibitory signalling in the mammalian brain is mediated by gamma-aminobutyric acid type A receptors (GABAARs), which are targets for anti-epileptic therapy such as benzodiazepines. GABAARs are subject to tightly regulated trafficking processes, essential for maintenance and physiological modulation of inhibitory strength. These processes can be altered during prolonged seizures such as SE and it is unclear whether these alterations contribute to the progressive development of benzodiazepine pharmacoresistance in patients with SE. Further, the intracellular signalling mechanisms that cause this modification in GABAAR trafficking remain poorly understood. The work presented in this thesis is centered around the activity-dependent trafficking of GABAARs and aims to investigate molecular mechanisms causing altered trafficking of GABAARs during SE. The zero Mg2+ model was utilised to induce SE in hippocampal rat neurons. This was combined with live-cell imaging of super ecliptic pHluorin (SEP)-tagged α2 subunit-containing GABAARs and revealed an acute N-methyl-d-aspartate receptor (NMDAR) dependent down-regulation of somatic surface GABAARs. Analysis of intracellular Ca2+ traces during zero Mg2+ treatment shows that GABAAR down-regulation correlates well with the timeline of intracellular Ca2+ changes. Furthermore, data from single particle tracking (SPT) of endogenous GABAARs using Quantum dots (QDs) shows that diffusion dynamics of synaptic GABAARs are altered after prolonged exposure to zero Mg2+. Both of the zero Mg2+ induced alterations, the decrease in GABAARs from the soma and the increase in diffusion dynamics of dendritic GABAARs, suggest altered surface behavior of GABAARs during SE. Finally, these changes were sensitive to inhibition of the Ca2+ dependent phosphatase calcineurin, indicating a Ca2+-dependent intracellular signalling mechanism. Further, a subunit-dependent modification of surface GABAARs was detected using the electrical stimulation model to induce SE in vivo.
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