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

## TL;DR

This paper shows that how brain slices are prepared affects neuron survival and epileptic activity, with oxidative damage playing a key role.

## Contribution

The study identifies oxidative stress as a key cause of inhibitory neuron loss during brain slice preparation and shows its impact on epileptiform activity.

## Key findings

- Transcardial perfusion with sucrose aCSF best preserves inhibitory neurons and reduces epileptiform activity.
- Oxidative damage is the main cause of GABAergic neuron loss during slice preparation.
- Loss of inhibitory neurons significantly alters network dynamics and epilepsy models in brain slices.

## Abstract

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.

Ex vivo brain slice preparation is a popular technique in neuroscience research with many variations in preparation method. We compared two popular variations in brain slice preparation and found that the choice of brain slice preparation technique significantly affects the viability of neuronal preparation. We found that inhibitory interneurons are particularly affected during brain slice preparation due to oxidative stress damage. The loss of inhibitory interneurons during brain slice preparation significantly affected the brain slice network and the epileptiform activity it generates in the acute epilepsy induction model. Neurochemistry research should account for the impact of brain slice preparation on neuronal physiology.