Abstracts

Rationale: Stress is the most commonly reported precipitating factor for seizures. However the underlying mechanisms whereby stress triggers seizures are not yet fully understood. The goal of this study is to investigate the impact of chronic stress-induced changes in expression of the K+/Cl- co-transporter, KCC2, which is essential for effective GABAergic inhibition, on hippocampal neuronal excitability and seizure susceptibility.Methods: Adult, male C57BL/6 mice were subjected to a chronic restraint stress paradigm consisting of 30 minutes of restraint daily for 14 consecutive days and compared to minimally handled controls. Stress reactivity was determined by measuring serum corticosterone levels by immunoassay. Seizure susceptibility was measured from 2 hour electroencephalogram (EEG) recordings in a kainic acid (20mg/kg) seizure model. Changes in hippocampal KCC2 and S940 protein levels after chronic stress were measured by Western blotting and compared to mice subjected to acute stress (a single 30 minute restraint) and control mice. Alterations in GABAergic inhibition after chronic stress, was determined using gramicidin perforated patch recordings from principal neurons in acute hippocampal slices. Results: Chronic stress increases circulating corticosterone levels and is associated with increased susceptibility to kainic acid-induced seizures (20mg/kg). Following chronic stress, but not acute stress, we observe a dephosphorylation of KCC2 residue S940 and downregulation of KCC2 in the hippocampus which we hypothesize underlies changes in chronic stress induced hippocampal excitability. To determine the impact of alterations in KCC2 expression following chronic stress whilst preventing disruption of the endogenous chloride gradients, gramicidin perforated patch recordings were performed from principal hippocampal neurons. Consistent with a loss of KCC2, a depolarizing shift in the GABA reversal potential (EGABA) was observed in hippocampal CA1 pyramidal layer neurons after chronic stress which is associated with an increase in hippocampal neuronal excitability. Preliminary data suggest that restoring the chloride gradient with the sodium potassium chloride co-transporter (NKCC1) inhibitor bumetamide protects against stress exacerbated seizures.Conclusions: We show for the first time that chronic stress induces a downregulation of KCC2 in the hippocampus and a depolarizing shift in EGABA. These alterations are associated with increased neuronal excitability of principal hippocampal neurons after chronic restraint stress. These data uncover a potential, novel mechanism underlying stress-induced seizure susceptibility and implicate KCC2 as a novel therapeutic target for seizure management.