Abstract
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 undergo tightly regulated trafficking processes that are essential for maintenance and physiological modulation of inhibitory strength. The trafficking of GABAARs to and from the membrane is altered during prolonged seizures such as in Status Epilepticus (SE) and has been suggested to contribute to benzodiazepine pharmacoresistance in patients with SE. However, the intracellular signalling mechanisms that cause this modification in GABAAR trafficking remain poorly understood. In this study, we investigate the surface stability of GABAARs during SE utilising the low Mg2+ model in hippocampal rat neurons. Live-cell imaging of super ecliptic pHluorin (SEP)-tagged α2 subunit containing GABAARs during low Mg2+ conditions reveals that the somatic surface receptor pool undergoes down-regulation dependent on N-methyl-d-aspartate receptor (NMDAR) activity. Analysis of the intracellular Ca2+ signal during low Mg2+ using the Ca2+-indicator Fluo4 shows that this reduction of surface GABAARs correlates well with the timeline of intracellular Ca2+ changes. Furthermore, we show that the activation of the phosphatase calcineurin was required for the decrease in surface GABAARs in neurons undergoing epileptiform activity. These results indicate that somatic modulation of GABAAR trafficking during epileptiform activity in vitro is mediated by calcineurin activation which is linked to changes in intracellular Ca2+ concentrations. These mechanisms could account for benzodiazepine pharmacoresistance and the maintenance of recurrent seizure activity, and reveal potential novel targets for the treatment of SE.This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.
Highlights
GABAA Receptors (GABAARs) are ligand-gated chloride permeable ion channels which mediate both phasic and tonic inhibitory neurotransmission in the central nervous system (Jacob et al, 2008; Luscher et al, 2011)
These results indicate that somatic modulation of GABAAR trafficking during epileptiform activity in vitro is mediated by calcineurin activation which is linked to changes in intracellular Ca2þ concentrations
We show that epileptiform activity alters intracellular Ca2þ concentrations, which correlates with the decrease of GABAARs from the surface possibly contributing to pathological signalling during Status Epilepticus (SE)
Summary
GABAA Receptors (GABAARs) are ligand-gated chloride permeable ion channels which mediate both phasic (synaptic) and tonic (extrasynaptic) inhibitory neurotransmission in the central nervous system (Jacob et al, 2008; Luscher et al, 2011) They assemble from five subunits, the composition of which determines the receptors functional and pharmacological properties and the specific location on the neuronal membrane (Luscher et al, 2011; Jacob et al, 2008). We induced prolonged epileptiform bursting activity in vitro by exposing neurons to artificial cerebrospinal fluid (aCSF) lacking Mg2þ (Mangan and Kapur, 2004; Sombati and Delorenzo, 1995) Using this model, we show a decrease in somatic surface GABAARs that is dependent on NMDAR activity and the Ca2þ-dependent phosphatase, calcineurin. We show that epileptiform activity alters intracellular Ca2þ concentrations, which correlates with the decrease of GABAARs from the surface possibly contributing to pathological signalling during SE
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