Inhibitory GABAergic Neuron Loss due to Oxidative Damage During Ex Vivo Acute Brain Slice Preparation Influences Genesis and Dynamics of Epileptiform Activity.

Ex vivo acute brain slice is a popular technique in neuroscience research with many variations. While many variations are currently used by labs around the world, no study has comprehensively examined the impact of these variations on the quality of the acute brain slice preparation. In this study, we compared different animal sacrifice methods (decapitation or transcardial perfusion) and cutting solution (normal or sucrose artificial cerebrospinal fluid). Brain slices were prepared from 10 to 12 weeks old male Wistar rats (Rattus norvegicus). Neuronal population was quantified by immunohistochemistry against various neuronal markers. Neuronal dynamics was evaluated by in vitro electrophysiology using two acute epilepsy models-zero-magnesium and 4-aminopyridine. We found that the method of brain slice preparation significantly affected the quality of the brain slice preparation. In general, the combination of transcardial perfusion and sucrose artificial cerebrospinal fluid produces the optimal brain slice preparation. The slices prepared with transcardial perfusion and sucrose aCSF had higher preservation of inhibitory interneurons and subsequently less successful induction of acute epileptiform activity. We also found that loss of inhibitory GABAergic neurons during brain slice preparation is primarily due to oxidative damage. Limiting oxidative stress is an effective neuroprotection strategy to prevent loss of inhibition in brain slice preparation. In conclusion, consideration of brain slice preparation method is crucial in preserving inhibitory GABAergic neurons and the degree of inhibition in the slice. Loss of inhibitory interneuron due to oxidative stress significantly affects quality of brain slice preparation and subsequent ex vivo epileptiform activity induction and dynamics.