NKCC1 activity modulates formation of functional inhibitory synapses in cultured neocortical neurons.
Intracellular Cl(-) concentration ([Cl(-)](i)) in immature neurons is higher than that expected for a passive distribution, therefore the equilibrium potential for chloride is more positive than the resting membrane potential, and the resulting GABA renders immature neurons depolarization. The higher [Cl(-)](i) in immature neurons is thought to be attributed to the uptake of Cl(-) mediated by NKCC1 (Na(+), K(+)-2Cl(-) cotransporter). Thus, a dysfunction of this transporter could affect synaptic development through a GABA(A) receptor-mediated pathway. To test this possibility, we examined the effects of a Cl(-)-uptake inhibitor on the development of synaptic activities of rat neocortical neurons in culture. Chronic treatment with bumetanide at 10 microM during the culture diminished the amplitude of synaptically-driven rhythmic depolarizing potentials (RDPs) in neurons and also decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but not of spontaneous excitatory postsynaptic currents (sEPSCs). Chronic treatment with bumetanide decreased vesicular GABA transporter (VGAT)-immunopositive particles without affecting paired-pulse ratio of evoked IPSCs (eIPSCs), indicating decrease in the number of functional GABAergic synapses. Acute treatment with bumetanide (10 microM) decreased neuronal [Cl(-)](i), the amplitude of RDPs, and neuronal excitability, while bumetanide had no effect on RDPs and neuronal excitability in the presence of bicuculline. These results suggest that the uptake of Cl(-) by NKCC1 affects the development of inhibitory synapses by promoting a depolarizing GABA-mediated response.
Nakanishi
Keiko
K
Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan. nakanishi@inst-hsc.jp
Yamada
Junko
J
Takayama
Chitoshi
C
Oohira
Atsuhiko
A
Fukuda
Atsuo
A
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology; D050485:GABA Plasma Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology; D007395:Interneurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019579:Neocortex / Q000196:embryology / Q000378:metabolism* / Q000648:ultrastructure; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D009434:Neural Pathways / Q000196:embryology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013569:Synapses / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D018118:Chloride Channels; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; C578877:SLC12A2 protein, human; C578879:Slc12a2 protein, rat; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; D005680:gamma-Aminobutyric Acid