CELLULAR NEUROSCIENCE ORIGINAL RESEARCH ARTICLE published: 01 November 2011 doi: 10.3389/fncel.2011.00023 Enhanced synaptic activity and epileptiform events in the embryonic KCC2 deficient hippocampus Ilgam Khalilov 1,2,3‡ , Geneviève Chazal 1,2,3‡ , Ilona Chudotvorova 1,2,3† , Christophe Pellegrino 1,2,3 , Séverine Corby 1,2,3 , Nadine Ferrand 1,2,3 , Olena Gubkina 1,2,3 , Romain Nardou 1,2,3 , RomanTyzio 1,2,3 , SumiiYamamoto 1,2,3 , Thomas J. Jentsch 4 , Christian A. Hübner 5 , Jean-Luc Gaiarsa 1,2,3 ,Yehezkel Ben-Ari 1,2,3 and Igor Medina 1,2,3 * 1 INSERM Unité 901, Marseille, France 2 Université de la Méditerranée, UMR S901Aix-Marseille 2, Marseille, France 3 Institut de Neurobiologie de la Méditerranée, Marseille, France 4 Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany 5 Institut für Humangenetik, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany Edited by: Enrico Cherubini, International School for Advanced Studies, Italy Reviewed by: Kai Kaila, University of Helsinki, Finland Raúl Eduardo Russo, Instituto de Investigaciones Biológicas Clemente Estable, Uruguay *Correspondence: Igor Medina, Institut de Neurobiologie de la Méditerranée/INSERM Unité 901, 163 Route de Luminy, 13273 Marseille, France. e-mail: medina@inmed.univ-mrs.fr † Present address: Ilona Chudotvorova, Brandeis University, 415 South Street, Waltham, MA 02453, USA. ‡ Ilgam Khalilov and Geneviève Chazal have contributed equally to this work. The neuronal potassium-chloride co-transporter 2 [indicated thereafter as KCC2 (for pro- tein) and Kcc2 (for gene)] is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2 +/+ ) and Kcc2 deficient (Kcc2 -/- ) mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when KCC2 is thought not to be functional in the hippocampus. Embryonic Kcc2 -/- hip- pocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABA-ergic postsynaptic currents than naïve age matched neurons. However, intracellular chloride concentration ([Cl - ] i ) and the reversal potential of GABA- mediated currents (E GABA ) were similar in embryonic Kcc2 +/+ and Kcc2 -/- CA3 neurons. In addition, KCC2 immunolabeling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collec- tively, our results show that already at an embryonic stage, KCC2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABA-ergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which KCC2 operates early as a chloride exporter or by transporter independent actions of KCC2 that are instrumental in synapse formation and networks construction. Keywords: GABA, KCC2, neuron, development, synapse, network INTRODUCTION Kcc2 is a potassium-chloride co-transporter that is exclusively expressed in neurons of central nervous system (CNS; Payne et al., 1996; Williams et al., 1999). It plays an important role in regulating intracellular chloride concentrations ([Cl - ] i ), thereby heavily impacting the magnitude of the inhibitory action of GABA in physiological processes (Rivera et al., 1999). In a wide range of animal species and brain structures, [Cl - ] i is elevated in immature neurons leading to depolarizing and often excita- tory actions of GABA (Ben-Ari et al., 1989, 2007; Leinekugel et al., 1997, 1999; Owens and Kriegstein, 2002; Caiati et al., 2010). GABA acquires progressively its hyperpolarizing actions in a time and brain structure dependent manner. In hippocam- pus of rat and mice, it is roughly completed by the second postnatal week. Extensive investigations suggest that this devel- opmental sequence is determined by a progressive reduction of the NKCC1 chloride importer and a parallel progressive enhanced operation of the KCC2 chloride exporter (Rivera et al., 1999; Gulyas et al., 2001; Stein et al., 2004). The expression of KCC2 is thought to lead to a reduction of [Cl - ] i and a shift of the actions of GABA from excitation to inhibition, although other chloride regulators – channels and transporters – take part in this sequence (Medina and Chudotvorova, 2006; Blaesse et al., 2009). Several observations suggest that KCC2 is less operational in immature than adult neurons. Thus, in the hippocampus of mice and rats KCC2 labeling is first detected at the end of embryonic development, peaking during the second postnatal week (Stein et al., 2004; Blaesse et al., 2006). KCC2 starts reducing [Cl - ] i in hippocampal pyramidal neurons 5 days after birth in rats (Khirug et al., 2005; Nardou et al., 2011). In addition, at P0–P1, KCC2 is in an inactive phosphorylated form (Rinehart et al., 2009) and forms monomers (Blaesse et al., 2006), whereas in mature struc- tures (>P20), KCC2 is dephosphorylated (Rinehart et al., 2009) and is expressed as multimers (Blaesse et al., 2006). Collectively these studies suggest that in cortical structures of mice and rats the KCC2 is not functional as a transporter during embryonic devel- opment, starting to contribute to the neuronal ion homeostasis during the first postnatal week and becoming fully operational at P10–P15. Frontiers in Cellular Neuroscience www.frontiersin.org November 2011 |Volume 5 | Article 23 | 1