Abstracts

Rationale: Severe pediatric brain injuries are frequently followed by seizures in the first week after injury, with the youngest infants suffering the highest incidence of seizures. One mechanism of acute seizures induced by brain injury may be inversion of signaling by the inhibitory neurotransmitter GABA. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl^-]_i) and neuronal activity associated with the widespread neural shear injury induced during preparation of acute brain slices.Methods: Two-photon fluorescence chloride imaging and extracellular field potential recordings of multiple unit activity (MUA) were performed in the intact hippocampus and acute hippocampal slices in vitro of postnatal days (P) 6-7 CLM-1 mice expressing Clomeleon. Acute hippocampal slices were used as a model of severe traumatic brain injury and age-matched intact hippocampi were used as controls. 16-sites probe extracellular recordings of MUA at different depths through the hippocampal slices were used to determine whether the net responses to GABA_A receptor (GABA_A-R) modulators are excitatory or inhibitory. Results: We found that neurons in hippocampal slices, unlike in the intact hippocampi, from P6-7 mice exhibit profound accumulation of [Cl^-]_i, resulting in long-term shift in the reversal potential for GABA_A receptor-mediated responses (E_GABA). There were strong correlations between neuronal [Cl^-]_i, biomarkers of neuronal damage and proximity to the slice surface. There was also a significantly higher probability of neuronal network synchronization in acute slice preparations. This raises the possibility that trauma-induced [Cl^-]_i accumulation may cause a positive shift in E_GABA in large population of damaged neurons that remain synaptically active, which could alter network activity and contribute to post-traumatic seizures. In the presence of AMPA, NMDA and GABA_B-R antagonists, exogenous GABA_A-R agonist isoguvacine induced responses on MUA frequency were both age- and depth-dependent. In P7-14 hippocampal slices, isoguvacine increased MUA frequency in superficial layers indicating excitatory GABA_A-R mediated signaling, and decreased MUA frequency in the inner layers indicating inhibitory GABA_A-R mediated signaling. In contrast, in slices from mature (P27-29) animals, isoguvacine decreased MUA frequency in deep areas while no MUA could be recorded from superfisial areas, indicating inhibitory action of GABA in adult slices, and inactivity of high [Cl^-]_i neurons. Traumatic increases in [Cl^-]_i in P6-7 slices were significantly reduced by antagonist of NKCC1 but not KCC2 cotransporters.Conclusions: These findings indicate that following trauma, developing neurons with high [Cl^-]_i remain synaptically active, inverting the net effects of the inhibitory neurotransmitter GABA and increasing the frequency of neuronal network activity. Excessive [Cl^-]_i accumulation following severe neuronal damage correlates with neuronal inactivation and death. The sustained synaptic activity of traumatized neurons with high [Cl^-]_i may provide the basis for post-traumatic hyperexcitability and seizures.