Enhanced synaptic activity and epileptiform events in the embryonic KCC2 deficient hippocampus

Abstract

The neuronal potassium-chloride co-transporter 2 [indicated thereafter as KCC2 (for protein) 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−/− hippocampi 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 (EGABA) 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. Collectively, 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.

A variant of KCC 2 from patients with febrile seizures impairs neuronal Cl − extrusion and dendritic spine formation

“…A strong mechanistic link from the above observations on the defects of KCC2-R925H to the human febrile seizure phenotype is provided by studies which have shown that genetic deficits in mKCC2 expression result in increased network excitability 22 and higher susceptibility to seizures 10,23. However, it is not known whether the enhanced seizure susceptibility in the animal models is attributable to reduced neuronal Cl − extrusion and/or to impairments in spine formation.…”

Homeostatic regulation of KCC2 activity by the zinc receptor mZnR/GPR39 during seizures

“…9). As the inhibitory GABA A receptor-mediated drive is critically dependent on sufficient KCC2 activity (Rivera et al 1999; Woo et al 2002; Zhu et al 2005; Viitanen et al 2010; Khalilov et al 2011), an adaptive process that upregulates KCC2 activity may restore the homeostatic balance between excitation and inhibition. In fact, as previous observations suggest that GABA signaling is depolarizing at the onset of seizure activity (Li et al 2008; Miles et al 2012), the mechanism described here may be critically important for restoring inhibitory drive.…”

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

“…The major finding of our study is that KCC2, a protein instrumental in the development and plasticity of GABA A receptormediated signaling (Blaesse et al, 2009) and in the maturation and function of dendritic spines and glutamatergic synapses (Li et al, 2007;Gauvain et al, 2011;Khalilov et al, 2011;Fiumelli et al, 2012), is a substrate of the calcium-activated protease calpain.…”

“…KCC2 is the principal Cl Ϫ extruder in mature central neurons and is responsible for maintaining [Cl Ϫ ] i below electrochemical equilibrium, a necessary condition for generation of "classical" hyperpolarizing IPSPs (Rivera et al, 1999;Farrant and Kaila, 2007). Moreover, KCC2 plays a major role already at the early stages in the development of excitatory synapses (Khalilov et al, 2011). Notably, cortical pyramidal neurons lacking KCC2 display an aberrant spine morphology in vitro (Li et al, 2007), and overexpression of KCC2 has been shown to induce functional spines in vivo (Fiumelli et al, 2012).…”