ABSTRACT
Seizures and brain injury lead to water and Cl- accumulation in neurons. The increase in intraneuronal Cl- concentration ([Cl-]i) depolarizes the GABAA reversal potential (EGABA) and worsens seizure activity. Neocortical neuronal membranes have a low water permeability due to the lack of aquaporins necessary to move free water. Instead, neurons use cotransport of ions including Cl- to move water. Thus, increasing the extracellular osmolarity during seizures should result in an outward movement of water and salt, reducing [Cl-]i and improving GABAA receptor-mediated inhibition. We tested the effects of hyperosmotic therapy with a clinically relevant dose of mannitol (20â¯mM) on epileptiform activity, spontaneous multiunit activity, spontaneous inhibitory post-synaptic currents (sIPSCs), [Cl-]i, and neuronal volume in layer IV/V of the developing neocortex of C57BL/6 and Clomeleon mice. Using electrophysiological techniques and multiphoton imaging in acute brain slices (post-natal day 7-12) and organotypic neocortical slice cultures (post-natal day 14), we observed that mannitol: 1) decreased epileptiform activity, 2) decreased neuronal volume and [Cl-]i through CCCs, 3) decreased spontaneous multi-unit activity frequency but not amplitude, and 4) restored the anticonvulsant efficacy of the GABAA receptor modulator diazepam. Increasing extracellular osmolarity by 20â¯mOsm with hypertonic saline did not decrease epileptiform activity. We conclude that an increase in extracellular osmolarity by mannitol mediates the efflux of [Cl-]i and water through CCCs, which results in a decrease in epileptiform activity and enhances benzodiazepine actions in the developing neocortex in vitro. Novel treatments aimed to decrease neuronal volume may concomitantly decrease [Cl-]i and improve seizure control.
