Abstracts

Rationale: Traumatic brain injury is a major cause of acquired epilepsy, but despite a substantial latent period no effective prophylactic therapy exists. Alterations in neuronal chloride transport in both acute trauma (Dzhala et al. J Neurosci 2012) and chronically epileptic tissue (Huberfeld et al. J Neurosci 2007) raise the possibility that altered chloride homeostasis ad GABA signaling contribute to post-traumatic epileptogenesis. We determined anticonvulsant and antiepileptic efficacy of diuretics, including bumetanide (10 M and 100 M), furosemide (100 M and 1 mM) and acetazolamide (10 M) in organotypic hippocampal slice cultures, a rapid in vitro model of post-traumatic epileptogenesis (Berdichevsky et al. J Neurosci 2013). Methods: 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 acute and persistent neuronal chloride accumulation after the widespread neuronal shear injury that occurs during slice preparation, as well as short-term neuronal chloride accumulation associated with recurrent post-traumatic seizures. Corresponding changes in the chloride reversal potential and GABA_A receptor-mediated signaling are likely to underlie the progressively reduced efficacy of phenobarbital and phenytoin in post-traumatic seizures. We found a positive correlation between the anticonvulsant efficacy of diuretics and reduction of neuronal chloride concentration. Bumetanide, a sodium-potassium-chloride (NKCC1) co-transporter blocker, significantly increased the anticonvulsant efficacy of phenobarbital. High concentrations of furosemide (NKCC1 and potassium-chloride (KCC2) co-transporter blocker) abolished spontaneous seizures at all stages of epileptogenesis, but often failed to reduce short interictal discharges and prevent epilepsy. Conclusions: Multiple mechanisms contribute to post-traumatic increases in neuronal chloride concentration, which in turn contribute to anticonvulsant resistance, presumably as a consequence of alterations in GABA-mediated inhibition. New strategies for more effective reductions in traumatic and seizure-induced intracellular chloride accumulation could provide the basis for effective treatments for post-traumatic edema and seizures.