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. Since its inception five decades ago, many variations of the brain slice preparation method have emerged. While all variations are currently used by many labs throughout the world, no study has comprehensively examined the impact of these variation on the quality of the acute brain slice preparation. In this study, we comprehensively examined the effect of animal sacrifice methods (decapitation or transcardial perfusion) and cutting solution (normal or sucrose artificial cerebrospinal fluid) on brain slice preparation. 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. To modulate the stress incurred in acute brain slice preparation, we administered antioxidants or HIF-1 inhibitor, chrysin, to the cutting solution. 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. This is evidenced by a preservation of inhibitory GABAergic neurons in slices prepared with this combination. We subsequently found that this loss of inhibitory GABAergic neurons significantly influenced the genesis and dynamics of induced acute epileptiform activity. The slices with preserved inhibition had less successful induction of acute epileptiform activity and a seizure-like event that is typical of those induced in brain with preserved inhibition. Finally, we 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 preservinginhibitory GABAergic neurons and the degree of inhibition in ex vivo acute brain slice preparation.