Abstracts

Rationale: Epilepsy after brain injuries caused by hypoxia-ischemia or trauma respond poorly to anticonvulsants, including those that target the inhibitory chloride-permeable GABA_A receptor (GABA_A-R). A low intracellular chloride concentration ([Cl^-]_i) is an important determinant of inhibitory postsynaptic GABA_A-R signaling. Both hypoxia and acute brain trauma are associated with large increases in [Cl^-]_i and a consequent positive shift in the reversal potential for GABA_A-R mediated responses (E_GABA). Depolarizing shifts in E_GABA in populations of injured neurons may contribute to failure of inhibition, seizures, epileptogenesis and anticonvulsant resistance. The Na⁺-K^--2Cl^- co-transporter NKCC1 and K⁺-Cl^- co-transporter KCC2 are responsible for the return of [Cl^-]_i to baseline after synaptic signaling. Immediately after injury, NKCC1 and KCC2 may also contribute to cytoplasmic volume and chloride increase. Suppressing NKCC1 activity and/or enhancing KCC2 activity may be a useful therapeutic strategy to reduce [Cl^-]_i, restore GABAergic inhibition in pathological neurons, suppress seizures and prevent epileptogenesis. Methods: We compared the acute and chronic anticonvulsant efficacy of the NKCC1 blocker bumetanide, the KCC2 antagonist VU0726722, and the putative KCC2 agonist CLP257 in in vitro model of post-traumatic epileptogenesis. Extracellular field potential recordings, two-photon imaging of Clomeleon, lactate and lactate dehydrogenase production assays were used to monitor neuronal network activity, intracellular chloride concentration ([Cl^-]_i) and neuronal cell death. Results: We found that: (i) bumetanide reduced the frequency and power of early recurrent seizures; (ii) CLP257 abolished early recurrent seizures and reduced the frequency of interictal discharges in a concentration-dependent manner; (iii) KCC2 antagonist VU0726722 strongly potentiated spontaneous interictal and ictal-like discharges; (iv) CLP257 reduced the amount of late chronic seizure activity in a concentration-dependent manner, assayed by lactate and LDH production. Conclusions: Our results validate CLP257 as a promising target of investigation for anticonvulsive and antiepileptogenic therapies, and highlight the need to investigate the mechanism of positive modulators of membrane transport.