NKCC1 and KCC2 prevent hyperexcitability in the mouse hippocampus
Introduction
The hippocampus is an important CNS structure involved in temporal lobe epilepsy (Aronica et al., 2007, Liu et al., 2007). Inhibition provided by ionotropic GABAA receptors is essential in maintaining normal brain function and protecting against seizures. Indeed, the GABAergic tone, by hyperpolarizing postsynaptic membranes and increasing membrane conductances (shunting), dampens excessive glutamatergic excitation and prevents synchronization of neuronal networks into epileptiform activity. GABAA receptors mediate the hyperpolarization effect through the opening of an anion channel, allowing Cl− to move into the cell (and HCO3− to move out under physiological conditions with much less permeability), and leading to plasma membrane hyperpolarization. The inward driving force for Cl− ions is due to the low intracellular Cl− concentration which is maintained by a secondary active transport mechanism, the neuronal-specific K–Cl cotransporter, KCC2. This overall process matures concomitant with the development of glutamatergic excitation (Stein et al., 2004, Ben-Ari et al., 2007, Ben-Ari, 2007, Represa and Ben-Ari, 2005), which in rodents translates to the first 2 weeks of postnatal life. At birth, however, neuronal Cl− is elevated and the equilibrium potential of Cl− (ECl) is above the resting membrane potential (Luhmann and Prince, 1991, Owens et al., 1996, LoTurco et al., 1995). This implies that the absence of KCC2 expression at birth, alone, cannot account for the high neuronal Cl−.
The formation of neuronal connections during early brain development also depends on the delicate balance between inhibition and excitation (Ben-Ari, 2002). As the rodent brain switches from GABA excitation to GABA inhibition during the first 2 weeks of postnatal life, this period constitutes a critical moment as excessive GABAergic excitation might lead to increased seizure susceptibility (Dzhala and Staley, 2003, Khazipov et al., 2004) while excessive GABAergic inhibition might prevent growth or synapse formation (Ben-Ari, 2002). Based on the observation that NKCC1 expression is high in immature CNS neurons and down-regulated during postnatal development (Plotkin et al., 1997, Wang et al., 2002, Dzhala et al., 2005), several studies have examined the possibility that Cl− accumulation in immature neurons is mediated by the inward Na–K–2Cl cotransporter (Fukuda et al., 1998, Sipila et al., 2006, Yamada et al., 2004, Chub et al., 2006, Achilles et al., 2007, Rohrbough and Spitzer, 1996, Ikeda et al., 2003). By using bumetanide to inhibit the Na–K–2Cl cotransporter, these studies show a role for NKCC1 in accumulation of intracellular Cl−. Furthermore, a report indicates that NKCC1 may facilitate seizures in the developing brain (Dzhala et al., 2005) by accumulating intracellular Cl− in the hippocampal pyramidal neurons and thus attenuating the GABAA receptor-mediated inhibition. However, high external [K+] was used in this study to increase brain hyperexcitability, which is a concern when studying the role of cation-chloride cotransporters, since they are K+-dependent transport pathways, and elevated external K+ increases inward Cl− transport. Interestingly, in a recent study where l μM kainate was used as a model, inhibition of NKCC1 by bumetanide was shown to have opposite effect on seizure activities (Kilb et al., 2007). Thus, whether NKCC1 increases excitability by raising Cl− in hippocampal pyramidal cells is still unresolved. In fact, several other studies have questioned the role of the cotransporter in Cl− accumulation in developing neurons of the brainstem (Balakrishnan et al., 2003) and retina (Zhang et al., 2007). Furthermore, there is evidence that NKCC1 may not be ubiquitously down-regulated in the developing hippocampus, but instead undergoes a change of localization from the soma of interneurons and pyramidal neurons to dendritic compartments (Marty et al., 2002), or from neuronal layers to glial formations (Hubner et al., 2001). These observations are significant since NKCC1-mediated elevation of Cl− in GABAergic interneurons may reduce their inhibition by GABA, resulting in increased inhibitory output to pyramidal neurons and higher network activity. Furthermore, glial cells also play an important role in epileptic activity by regulating the K+ ion concentration in the extracellular space (Lux et al., 1986, D’Ambrosio, 2004) and glial NKCC1 may be important for clearance of extracellular K+ (Chen and Sun, 2005). Thus, we wanted to re-address the role of the Na–K–2Cl cotransporter using control saline conditions versus 4-aminopyridine (4-AP) as a convulsive agent, control conditions versus bumetanide as an inhibitor of the cotransporter, and wild-type versus NKCC1 knockout (Delpire et al., 1999) hippocampal slices. To test the 4-AP seizure model, we also use brain slices from older animals, where KCC2 plays a key role in preventing hyperexcitability and where reduction of KCC2 expression by half leads to hyperexcitability in the slice model and increased seizure susceptibility in the whole animal (Woo et al., 2002). In summary, our data show that NKCC1, as KCC2, prevents hyperexcitability and the development of seizures in the hippocampus. However, as NKCC1 was shown to not accumulate Cl− in young P9–P13 pyramidal neurons, the protective effect of NKCC1 is likely to occur through a mechanism or mechanisms independent of Cl− regulation in the pyramidal cells, such as working as an extracellular K+ clearance pathway or increasing intracellular [Cl−] of interneurons.
Section snippets
Animals
Mice used in these experiments were housed in microisolators in a standard animal care facility with a 12 h light/dark cycle, and free access to food and water. All procedures were approved by the Vanderbilt University Animal Care and Use Committee in agreement with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Genotyping
Wild-type, heterozygous, and homozygous KCC2 and NKCC1 mice were generated from heterozygous matings. Genotyping was performed by
Results
NKCC1 expression is highest in immature CNS and decreases during postnatal development towards adulthood (Plotkin et al., 1997, Wang et al., 2002, Kanaka et al., 2001, Mikawa et al., 2002, Li et al., 2002). Due to the large inward Na+ and Cl− driving force, NKCC1 is a good candidate for intracellular Cl− accumulation in immature neurons and GABA excitatory effects. However, other evidences suggest that NKCC1 expression may not be ubiquitously down-regulated in all types of neurons during CNS
Discussion
Recent studies have uncovered multiple roles for the Na–K–2Cl cotransporter, NKCC1, in volume and ion homeostasis in the brain, and in diverse pathologies of the CNS. For instance, the endothelial NKCC1 is involved in the increased salt and fluid movement into the brain that is associated with ischemic brain injury (Pedersen et al., 2006, Chen and Sun, 2005). Furthermore, the cotransporter is also involved in glial cell swelling and glutamate release, leading to neuronal excitotoxicity (Chen
Acknowledgement
This work was supported by a grant from the National Institutes of Health (R01 NS36758) to E. Delpire.
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