Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury.
After axotomy, application of muscimol, a GABA(A) receptor agonist, induced an increase in intracellular Ca(2+) ([Ca(2+)](i)) in dorsal motor neurons of the vagus (DMV neurons). Elevation of [Ca(2+)](i) by muscimol was blocked by bicuculline, tetrodotoxin, and Ni(2+). In axotomized DMV neurons measured with gramicidin perforated-patch recordings, reversal potentials of the GABA(A) receptor-mediated response, presumably equal to the equilibrium potential of Cl(-), were more depolarized than that in intact neurons. Thus, GABA(A) receptor-mediated excitation is suggested to be attributable to Cl(-) efflux out of the cell because of increased intracellular Cl(-) concentration ([Cl(-)](i)) in axotomized neurons. Regulation of [Cl(-)](i) in both control and injured neurons was disturbed by furosemide and bumetanide and by manipulating cation balance across the membrane, suggesting that functional alteration of furosemide-sensitive cation-Cl(-) cotransporters is responsible for the increase of [Cl(-)](i) after axotomy. In situ hybridization revealed that neuron-specific K(+)-Cl(-) cotransporter (KCC2) mRNA was significantly reduced in the DMV after axotomy compared with that in control neurons. Similar expression of Na(+), K(+)-Cl(-) cotransporter mRNA was observed between axotomized and control DMV neurons. Thus, axotomy led to disruption of [Cl(-)](i) regulation attributable to a decrease of KCC2 expression, elevation of intracellular Cl(-), and an excitatory response to GABA. A switch of GABA action from inhibitory to excitatory might be a mechanism contributing to excitotoxicity in injured neurons.
Nabekura
Junichi
J
Department of Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan. nabekura@physiol2.med.kyushu-u.ac.jp
Ueno
Tsuyoshi
T
Okabe
Akihiko
A
Furuta
Akiko
A
Iwaki
Toru
T
Shimizu-Okabe
Chigusa
C
Fukuda
Atsuo
A
Akaike
Norio
N
D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D019771:Axotomy; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D005456:Fluorescent Dyes; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism; D009118:Muscimol / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013449:Sulfonamides; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D014628:Vagotomy; D000096922:K Cl- Cotransporters
D002118:Calcium; D002712:Chlorides; D004232:Diuretics; D005456:Fluorescent Dyes; D018755:GABA Agonists; D018756:GABA Antagonists; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D009118:Muscimol; D009532:Nickel; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D013449:Sulfonamides; D027981:Symporters; D013779:Tetrodotoxin; D000096922:K Cl- Cotransporters