PT - JOURNAL ARTICLE AU - Richard J. Burman AU - Alexandru Calin AU - Neela K. Codadu AU - Rebecca Wright AU - Sarah E. Newey AU - Maurits van den Burg AU - John Hamin Lee AU - R. Ryley Parrish AU - Joanne M. Wilmshurst AU - Colin J. Akerman AU - Andrew J. Trevelyan AU - Joseph V. Raimondo TI - Excitatory GABAergic signalling is associated with acquired benzodiazepine resistance in status epilepticus AID - 10.1101/478594 DP - 2018 Jan 01 TA - bioRxiv PG - 478594 4099 - http://biorxiv.org/content/early/2018/12/05/478594.short 4100 - http://biorxiv.org/content/early/2018/12/05/478594.full AB - Status epilepticus (SE) is defined as a state of unrelenting seizure activity. It is associated with a rapidly rising mortality rate, and thus constitutes a medical emergency. Benzodiazepines, which act as positive modulators of chloride (Cl-) permeable GABAA receptors, are indicated as the first line of treatment, but this is ineffective in many cases. We found that 48% of children presenting with SE were unresponsive to benzodiazepine treatment, and critically, that the duration of SE at the time of treatment is an important predictor of non-responsiveness. We therefore investigated the cellular mechanisms that underlie acquired benzodiazepine resistance, using rodent organotypic brain slices. Removing Mg2+ ions leads to an evolving pattern of epileptiform activity, and eventually to a persistent state of repetitive discharges that strongly resembles clinical EEG recordings of SE. We show that the persistent SE-like activity is associated with a reduction in GABAA receptor conductance and Cl- extrusion capability. We explored the effect on intraneuronal Cl- using both gramicidin, perforated-patch clamp recordings and Cl- imaging. This showed that during SE-like activity, reduced Cl- extrusion capacity was further exacerbated by activity-dependent Cl- loading, resulting in a persistently high intraneuronal Cl-. Consistent with these results, we found that optogenetic stimulation of GABAergic interneurons in the SE-like state, actually enhanced epileptiform activity in a GABAAR dependent manner. Together our findings describe a novel potential mechanism underlying benzodiazepine-resistant SE, with relevance to how this life-threatening condition should be managed in the clinic.Author contributionsRJB designed and performed experiments, collected clinical data, performed data analysis and wrote the manuscript. AC performed chloride imaging experiments. NKC, MvdB, JHL, RP and AJT setup and collected data for the extracellular recording experiments. RW and SN performed molecular biology experiments. HM provided supervision to the project. JMW assisted in collection of clinical data. CJA provided supervision for the project and assisted in the writing of the manuscript. AJT performed data analysis, provided supervision for the project and assisted in the writing of the manuscript. JVR designed experiments, performed data analysis, wrote the manuscript and provided overall supervision.