Long-term expressional changes of Na+–K+–Cl− co-transporter 1 (NKCC1) and K+–Cl− co-transporter 2 (KCC2) in CA1 region of hippocampus following lithium-pilocarpine induced status epilepticus (PISE)

doi:10.1016/j.brainres.2008.04.047

Brain Research

Volume 1221, 24 July 2008, Pages 141-146

https://doi.org/10.1016/j.brainres.2008.04.047 Get rights and content

Abstract

NKCC1 and KCC2 are encoded by slc12 gene family and involved in the maintenance of intracellular chloride concentration which may be associated with epileptogenesis. In this study, we aimed to investigate the long-term expression profiles of NKCC1 and KCC2 in CA1 region in the mice model of lithium-pilocarpine induced status epilepticus (PISE) and their relationship with epileptogenesis. We found NKCC1 mRNA and proteins were up-regulated at 1 d, 14 d and 45 d after pilocarpine injection, while KCC2 was down-regulated. According to obtained results, there were some expressional changes of NKCC1 and KCC2. Deregulation of their expression may break the balance of intracellular and extracellular chloride concentration which contributes to the mechanism of hyperexcitability leading to seizures. Also it may provide new drug targets for development of new antiepileptic medicine.

Introduction

Epilepsy is the most common neurological disorder after stroke and mesial temporal lobe epilepsy (MTLE) is recognized as one of the most medically intractable epilepsy (Loscher, 2002). Animal models for seizure and epilepsy have played fundamental roles in understanding of the physiological and pathological pathways associated with human epilepsy and in testing novel antiepileptic drugs.

In immature neurons, GABA (γ-aminobutyric acid)_A receptor-mediated responses are often depolarizing (Ben-Ari et al., 1989, LoTurco et al., 1995), which can trigger a number of developmental phenomena, e.g. neuronal proliferation, migration, and synaptogenesis (Behar et al., 1996, Ben-Ari, 2002). Therefore, such depolarizing GABA actions shift to more negative levels during development. The accumulated evidence demonstrates that this GABA induced membrane depolarization is caused by Cl⁻ efflux due to high intracellular Cl⁻ concentration maintained in immature cells.

A variety of neurotransmitter receptors and ion channels undergo expression and functional alterations in epileptic hippocampi (Ben-Ari, 2001, Coulter, 2001). Recent data point towards a role for cation–chloride co-transporters (CCCs) in epilepsy. CCCs gene family consists of three broad groups: Na⁺–Cl⁻ co-transporter (NCC), Na⁺–K⁺–Cl⁻ co-transporters (NKCCs) and K⁺–Cl⁻ co-transporters (KCCs). To date, one NCC isoform, two NKCC isoforms [NKCC1–2] and four distinct KCC isoforms [KCC1–4] have been identified. In the nervous system, cation–chloride co-transporters (CCCs) are considered to play critical roles in the regulation of intracellular chloride concentration, and hence in the control of neuronal function (Rivera et al., 1999a, Rivera et al., 1999b). Alterations in the balance of NKCCs and KCCs may determine the switch from a hyperpolarizing to a depolarizing effect of GABA, thereby contributing to epileptogenesis in human hippocampal formation (Cohen et al., 2003, Dzhala et al., 2005, Fukuda, 2005).

Various lines of evidence correlated epileptogenesis with altered functional expression of NKCCs and KCCs transporters. Deletion of KCC2 gene expression in mice causes hyperexcitability in hippocampus, generalized seizures, and death shortly after birth (Woo et al., 2002). In an animal study, hippocampal kindling decreased KCC2 expression in the hippocampus. On the other hand, amygdala kindling of adult rats, increased NKCC1, decreased KCC1 in the dentate gyrus, but did not alter KCC2 (Okabe et al., 2003). Palma et al. showed that the excitatory effects of GABA_A receptors in cell membranes obtained from temporal lobectomies of epilepsy patients were attributed to increased NKCC1 and decreased KCC2 expression (Palma et al., 2006). Pharmacological inhibition of cation–chloride co-transporters by loop diuretics blocks epileptiform activity in hippocampal slices due to NKCC blockade (Schwartzkroin et al., 1998, Hochman et al., 1999). CCCs inhibitors also have anticonvulsant properties in humans and rodents (Dzhala et al., 2005, Fukuda, 2005). These data point towards CCCs as interesting target molecules with respect to epileptogenesis and antiepileptic pharmacotreatment and raises the question whether expression of CCCs correlates with functional aspects of MTLE, i.e. differences in epileptogenesis, pattern and extent of hippocampal damage as well as structural reorganization.

Although the distributions of KCC2 and NKCC1 have been reported using immunohistochemistry and in situ hybridization, little information is available on the long-term expressional changes of NKCC1 and KCC2 in CA1 region of hippocampus in the mice model of lithium-pilocarpine induced status epilepticus (PISE). Therefore we aim to investigate the long-term expression changes in KCC2 and NKCC1 expression in the mice model of PISE.

Section snippets

Behavioral investigations

In this mouse model of epilepsy, the onset of continuous status epilepticus was found at 25.8 ± 7.3 min after pilocarpine injection and interrupted by injection of diazepam after 1 h. Spontaneous behavioral seizures were observed in all pilocarpine-treated animals of 14 d and 45 d except one in the 14 d group. The spontaneous seizures typically consisted of periods of freezing, clonic movements of the forelimbs, rearing, and even falling. The total duration of the observed behavioral seizure was

Discussions

The pilocarpine model of epilepsy in rodents is a well-characterized model that employs status epilepticus to induce epileptogenesis and has many features similar to MTLE, i.e. spontaneous recurrent seizures, hippocampal cell loss, supra-granular mossy fiber sprouting, dentate gyrus cell dispersion and can be used as an animal tool to understand the basic mechanisms of epileptogenesis (Honchar et al., 1983, Turski et al., 1983, Liu et al., 1994, Mathern et al., 1995).

The role of cation–chloride

Experimental procedures

In total, 36 male adult Balb/c mice were used. The mice were 6–8 weeks of age, 18–24 g of body weight. Animals were housed under standardized environmental conditions (12 h light/dark cycle, 21 ± 1 °C and 55 ± 5% humidity) and allowed free access to food and water for at least 5 d acclimatization. All experiments were performed in accordance with the guidelines of the Animal Care Committee of Sun Yat-sen University. Efforts were made throughout the study to minimize animal suffering and to use the

Acknowledgments

We wished to thank Dr. Pei Zhong for assistance in manuscript writing. This study was supported by the National Natural Science Foundation of China (10671213).

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Citation Excerpt :
For instance, in hippocampal brain slices from adult patients with temporal lobe epilepsy (TLE), downregulation of KCC2 and upregulation of NKCC1 leads to depolarizing GABAA receptor responses in a subpopulation of subicular principal neurons (Cohen et al., 2002; Huberfeld et al., 2007; Munoz et al., 2007). We and others have reported similar findings in the kindling and pilocarpine rat models of TLE (Rivera et al., 2002; Okabe et al., 2003; Pathak et al., 2007; Li et al., 2008; Brandt et al., 2010; Barmashenko et al., 2011; Kourdougli et al., 2017). The potential role of upregulated neuronal NKCC1 in epilepsy has led to the suggestion that the NKCC1 inhibitor bumetanide may exert anticonvulsant and antiepileptogenic effects (Kahle and Staley, 2008; Kahle et al., 2008; Ben-Ari et al., 2012; Ben-Ari, 2017; Kharod et al., 2019; Auer et al., 2020; Liu et al., 2020).
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Research ReportLong-term expressional changes of Na+–K+–Cl− co-transporter 1 (NKCC1) and K+–Cl− co-transporter 2 (KCC2) in CA1 region of hippocampus following lithium-pilocarpine induced status epilepticus (PISE)

Abstract

Introduction

Section snippets

Behavioral investigations

Discussions

Experimental procedures

Acknowledgments

Biol. Cell

Int. Rev. Neurobiol.

Neurosci. Res.

Epilepsy Res.

Neuron

Neurosci. Res.

Brain Res.

J. Biol. Chem.

Epilepsy Res.

Epilepsy Res.

Epilepsy Res.

GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms

J. Neurosci.

Cell death and synaptic reorganizations produced by seizures

Epilepsia

Excitatory actions of gaba during development: the nature of the nurture

Nat. Rev., Neurosci.

Giant synaptic potentials in immature rat CA3 hippocampal neurones

J. Physiol.

NKCC1 transporter facilitates seizures in the developing brain

Nat. Med.

Research Report
Long-term expressional changes of Na⁺–K⁺–Cl⁻ co-transporter 1 (NKCC1) and K⁺–Cl⁻ co-transporter 2 (KCC2) in CA1 region of hippocampus following lithium-pilocarpine induced status epilepticus (PISE)