Abstracts

Rationale: Traumatic brain injury is often complicated by early seizures occurring within the first week after injury. These early seizures may exacerbate the brain injury increasing the risks for development of epilepsy. The mechanisms underlying early post-traumatic seizures remain unknown. Methods: High resolution two-photon fluorescence chloride imaging and simultaneous non-invasive extracellular field potential recordings of multiple unit activity (MUA) and synchronous population activity were performed in the intact hippocampus and acute hippocampal slices in vitro of the neonatal (postnatal day (P) 5-7) transgenic mice CLM-1 expressing Clomeleon. Acute hippocampal slices were used as a model of severe traumatic brain injury. Age-matched intact hippocampal preparations from the same animals were used as controls. Simultaneous extracellular field potentials of multiple unit activity from hundreds of neurons were performed to determine whether the net responses to GABA_A receptor modulators are excitatory or inhibitory. Results: Using two-photon microscopy and the genetically expressed chloride fluorophore Clomeleon, we found that neurons in brain slices from neonatal CLM-1 mice exhibit a profound accumulation of [Cl^-]_i , resulting in long-term shift in the reversal potential for GABA_A receptor mediated responses (E_GABA). The [Cl^-]_i accumulation inverted the net operation of GABA_A-receptor from inhibition to excitation. There was a very strong correlations between neuronal [Cl^-]_i and proximity to the slice surface. The [Cl^-]_i of many morphologically normal neurons near the surface was high enough to cause E_GABA to be positive to action potential threshold. For the cells with highest [Cl^-]_i (> 100 mM) there was also a strong correlation between [Cl^-]_i and neuronal volume, with large swollen cells exhibiting [Cl^-]_i close to the chloride concentration of the extracellular solution. There was also a strong correlation between neuronal [Cl^-]_iand the probability of apoptosis as assayed by fluorescent indicator of caspase activation (FLICA). Inhibition of neuronal NKCC1-mediated inward chloride transport with the diuretic bumetanide did not reduce [Cl^-]_i in the most of large swollen neurons. Conclusions: Our results provide a possible mechanism for early pathological GABA-mediated excitation after traumatic brain injury. We are currently investigating the long-term chronic effects of neuronal trauma and intracellular chloride accumulation on development of spontaneous epileptiform discharges and anticonvulsant resistance.