title: Selective inhibition of KCC2 leads to hyperexcitability and epileptiform discharges in hippocampal slices and in vivo.
abstract: 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.
authors.affiliation: Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, Department of Biosciences, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Sudhir
authors.identifier: http://orcid.org/0000-0002-8586-0469
authors.initials: S
authors.lastname: Sivakumaran
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Cardarelli
authors.affiliation: Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Jamie
authors.identifier: http://orcid.org/0000-0002-4085-8605
authors.initials: J
authors.lastname: Maguire
authors.affiliation: Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Matt R
authors.identifier: http://orcid.org/0000-0002-8010-6328
authors.initials: MR
authors.lastname: Kelley
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111.
authors.forename: Liliya
authors.initials: L
authors.lastname: Silayeva
authors.affiliation: Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Danielle H
authors.initials: DH
authors.lastname: Morrow
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111.
authors.forename: Jayanta
authors.initials: J
authors.lastname: Mukherjee
authors.affiliation: Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111.
authors.forename: Yvonne E
authors.initials: YE
authors.lastname: Moore
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, AstraZeneca Neuroscience iMED, Cambridge, Massachusetts 02139, and.
authors.forename: Robert J
authors.identifier: http://orcid.org/0000-0001-9105-7798
authors.initials: RJ
authors.lastname: Mather
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, AstraZeneca Neuroscience iMED, Cambridge, Massachusetts 02139, and.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Duggan
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, AstraZeneca Neuroscience iMED, Cambridge, Massachusetts 02139, and.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Brandon
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, AstraZeneca Neuroscience iMED, Cambridge, Massachusetts 02139, and.
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, AstraZeneca Neuroscience iMED, Cambridge, Massachusetts 02139, and.
authors.forename: Stephen
authors.identifier: http://orcid.org/0000-0001-5320-147X
authors.initials: S
authors.lastname: Zicha
authors.affiliation: Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, Department of Neuroscience, Physiology and Pharmacology, University College, London, WC1E 6BT, United Kingdom Tarek.Deeb@tufts.edu Stephen.Moss@tufts.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Moss
authors.affiliation: AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111, Tarek.Deeb@tufts.edu Stephen.Moss@tufts.edu.
authors.forename: Tarek Z
authors.initials: TZ
authors.lastname: Deeb
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D057809:HEK293 Cells; D006624:Hippocampus / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology*; D027981:Symporters / Q000037:antagonists & inhibitors* / Q000502:physiology*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D000096922:K Cl- Cotransporters
chemical_list: D049994:Sodium Potassium Chloride Symporter Inhibitors; D027981:Symporters; D000096922:K Cl- Cotransporters
keywords: GABAA; KCC2; VU0463271; chloride; seizures; slices