Sivakumaran Sudhir, Cardarelli Ross A, Maguire Jamie, Kelley Matt R, Silayeva Liliya, Morrow Danielle H, Mukherjee Jayanta, Moore Yvonne E, Mather Robert J, Duggan Mark E, Brandon Nicholas J, Dunlop John, Zicha Stephen, Moss Stephen J, Deeb Tarek Z
Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, Department of Biosciences, University of Helsinki, 00014 Helsinki, Finland.
AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111.
J Neurosci. 2015 May 27;35(21):8291-6. doi: 10.1523/JNEUROSCI.5205-14.2015.
GABA(A) 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 E(GABA) values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg(2+) 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.
GABA(A)受体形成Cl(-)可渗透通道，介导大脑中大部分快速突触抑制。K(+)/Cl(-)共转运体KCC2是神经元建立低细胞内Cl(-)水平的主要机制，这被认为能够实现GABA能对神经元活动的抑制控制。然而，广泛使用的KCC2抑制剂速尿在切片和体内具有抗癫痫疗效，但具有非选择性，这导致了关于KCC2如何影响GABA能对神经元同步性控制的相互矛盾的方案。在这里，我们使用选择性KCC2抑制剂VU0463271 [N-环丙基-N-(4-甲基-2-噻唑基)-2-[(6-苯基-3-哒嗪基)硫代]乙酰胺]来研究KCC2功能的影响。应用VU0463271导致E(GABA)值发生可逆的去极化偏移，并增加培养的海马神经元的放电。在低镁(2+)条件下，将VU0463271应用于小鼠海马切片会诱导不间断的反复癫痫样放电。最后，将VU0463271单独微量注入小鼠背侧海马体中会迅速引起癫痫样放电。我们的研究结果表明，KCC2功能在体外和体内都是一个关键的抑制因子。
