Abstracts

Rationale: Neuronal chloride concentration ([Cl^-]_i) is an important determinant of both post-synaptic GABA_A-receptor mediated signaling and cell volume regulation.  Altered Cl^- equilibrium and corresponding reversal potential (E_Cl) result in cell swelling, accumulation of [Cl^-]_i, and GABA depolarizing responses, which foster seizures, epileptogenesis and anticonvulsant resistance via failure of inhibition.  Cl^- equilibrium is mediated by a Donnan system in which the cation-chloride co-transporters (CCCs) KCC2 (a canonical Cl^- exporter) and NKCC1 (a canonical Cl^- importer) comprise the requisite cation and chloride membrane permeability.  Under pathological conditions, this permeability may limit the rate at which Cl^- equilibrium and GABA signaling can be restored.  We investigated whether altering CCC activity affects cell swelling and [Cl^-]_i in injured neurons, and the downstream effects on GABAergic inhibition and seizures.  New high-affinity drugs that modulate CCCs activity include CLP257 that selectively activates KCC2 over other KCC family members, NKCC1 and GABA_A receptors; and the VU series, including VU 0240551 and VU 0463271 that selectively inhibit KCC2 over NKCC1. Methods: We tested the mechanism of action and specificity of the putative KCC2 up-regulator CLP257 (1-100 µM), and KCC2 inhibitors VU 0240551 (1-10 µM) and VU 0463271 (0.1-1 µM) in the organotypic hippocampal slice in vitro model of epileptogenesis. Extracellular field potential recordings and two-photon imaging of the transgenic chloride fluorophore Clomeleon were used to monitor neuronal network activity, [Cl^-]_i and neuronal cell volume.  Results: We found that: (i) CLP257 improved [Cl^-]_i homeostasis and reduced seizure activity in a concentration-dependent manner;  (ii) the GABA_A–R antagonist SR95531 prevented the anticonvulsant action of CLP257 (30 µM), and  the NKCC1 and KCC2 blocker bumetanide (10 and 200 mM) reduced the  anticonvulsant  action of CLP257 and the rate of Cl^- reduction; (iii)  the VU KCC2 antagonists reduced the  frequency  of ictal-like discharges but strongly increased  the frequency  and power of interictal epileptiform discharges and corresponding Cl^- transients in a dose-dependent manner; (iv) VU 0240551 (10 µM)  pro-convulsant  actions and corresponding Cl^- transients were not affected by block of CCCs with bumetanide (10 and 200 mM); (v) sodium channel blocker TTX abolished VU 0240551 and VU 0463271 induced epileptiform discharges and corresponding Cl^- transients without affecting E_Cl.  Conclusions: Our results indicate that: 1) the anticonvulsant actions of high dose CLP257 are likely partially mediated by increased KCC2 activity; 2) the pro-epileptic action of VU and corresponding Cl^- transients are not mediated by CCC modulation.  Our data validate CLP257 as a promising target of investigation for antiepileptic therapies, and highlight the ongoing need to develop more specific activators and inhibitors of KCC2 co-transport. Funding: NINDS (NIH) RO1 NS040109