The K+/Cl- cotransporter KCC2 is the main chloride extruder in neurons and it exerts an essential role in determining the polarity of GABAA receptor-mediated chloride currents. The increase of KCC2 expression after birth is responsible of the “switch of GABA” effect from excitatory to inhibitory. KCC2 also covers a critical role in dendritic spine morphogenesis and in maintenance of glutamatergic synapses. Alteration in its expression could lead to an imbalance between excitation and inhibition and to the pathogenesis of diseases such as epilepsy. We studied KCC2 expression and distribution in a murine model of Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE), conditionally expressing the ADNFLE-linked nicotinic β2V287L mutation (TG), for comparison with control mice (CTRL) in prefrontal cortex (PFC), somatosensory cortex (SS) and thalamus (TH). By immunocytochemical approaches, we carried out densitometric analyses of KCC2 expression at different postnatal ages (P8, P21, P60) and we observed a significant increase of KCC2 expression in PFC layer V and a decrease of this cotransporter in the thalamic reticular nucleus (RT) in TG P60 mice compared to age-matched controls. Further analyses of some GABAergic markers showed no substantial alterations. Next, we estimated GABAA reversal potential (EGABA) by perforated patch-clamp recordings on acute dissociated neurons from TG and CTRL mice during postnatal development. EGABA progressively hyperpolarized up to the second postnatal week, but no difference was found between TG and CTRL mice. On the other hand, in mature (older than P28) cortical slices, a potentiation of both EPSCs and IPSCs stimulation was detected in response to nicotine in TG compared to control neurons and, especially in the case of EPSCs, leading towards excitation. Our results suggest that KCC2 and the GABAergic system could be implicated in the pathogenesis of ADNFLE and that β2V287L mutation could alter the excitatory/inhibitory balance in murine forebrain.
Expression of the K+/CL- cotransporter KCC2 in a conditional murine model of autosomal dominant nocturnal frontal lobe epilepsy / D. Modena, P. Aracri, M. Ascagni, D. Iannantuoni, C.E. Donati, S. Brusco, S. Meneghini, A. Coatti, M.E. Pasini, A. Becchetti, A. Amadeo. ((Intervento presentato al 26. convegno G.I.S.N. tenutosi a Verona nel 2016.
Expression of the K+/CL- cotransporter KCC2 in a conditional murine model of autosomal dominant nocturnal frontal lobe epilepsy
D. Modena;P. Aracri;M. Ascagni;S. Brusco;M.E. Pasini;A. Amadeo
2016
Abstract
The K+/Cl- cotransporter KCC2 is the main chloride extruder in neurons and it exerts an essential role in determining the polarity of GABAA receptor-mediated chloride currents. The increase of KCC2 expression after birth is responsible of the “switch of GABA” effect from excitatory to inhibitory. KCC2 also covers a critical role in dendritic spine morphogenesis and in maintenance of glutamatergic synapses. Alteration in its expression could lead to an imbalance between excitation and inhibition and to the pathogenesis of diseases such as epilepsy. We studied KCC2 expression and distribution in a murine model of Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE), conditionally expressing the ADNFLE-linked nicotinic β2V287L mutation (TG), for comparison with control mice (CTRL) in prefrontal cortex (PFC), somatosensory cortex (SS) and thalamus (TH). By immunocytochemical approaches, we carried out densitometric analyses of KCC2 expression at different postnatal ages (P8, P21, P60) and we observed a significant increase of KCC2 expression in PFC layer V and a decrease of this cotransporter in the thalamic reticular nucleus (RT) in TG P60 mice compared to age-matched controls. Further analyses of some GABAergic markers showed no substantial alterations. Next, we estimated GABAA reversal potential (EGABA) by perforated patch-clamp recordings on acute dissociated neurons from TG and CTRL mice during postnatal development. EGABA progressively hyperpolarized up to the second postnatal week, but no difference was found between TG and CTRL mice. On the other hand, in mature (older than P28) cortical slices, a potentiation of both EPSCs and IPSCs stimulation was detected in response to nicotine in TG compared to control neurons and, especially in the case of EPSCs, leading towards excitation. Our results suggest that KCC2 and the GABAergic system could be implicated in the pathogenesis of ADNFLE and that β2V287L mutation could alter the excitatory/inhibitory balance in murine forebrain.File | Dimensione | Formato | |
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