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Author(s): Ross A Cardarelli [1, 10]; Karen Jones [2, 10]; Lucie I Pisella [3, 4, 10]; Heike J Wobst [1, 5]; Lisa J McWilliams [6]; Paul M Sharpe [2]; Matthew [...]

GABAA receptors form Cl- permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K+/Cl- cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl- levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in EGABA values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg2+ conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factor ex vivo and in vivo. [ABSTRACT FROM AUTHOR]

Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABAAR) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an a and γ subunit of GABAARs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), β3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABAAR surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABAARs occurred within 24 h of flurazepam treatment, whereas GABAARs incorporating α1 subunits showed little alteration. The GABAAR surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABAAR surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABAAR endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABAARs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABAARs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABAAR trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment. [ABSTRACT FROM AUTHOR]