Reduction of KCC2 Expression and GABAAReceptor-Mediated Excitation after In Vivo Axonal Injury

Abstract

After axotomy, application of muscimol, a GABA_Areceptor agonist, induced an increase in intracellular Ca²⁺([Ca²⁺]_i) in dorsal motor neurons of the vagus (DMV neurons). Elevation of [Ca²⁺]_i by muscimol was blocked by bicuculline, tetrodotoxin, and Ni²⁺. 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.<i>In situ</i> 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.