A novel, flexible, and accessible method for the ex vivo induction and quantification of excitotoxicity

Excitotoxicity is a key driver of neuronal death in diverse brain conditions, yet most toxicity assays rely on in vitro models that remove cells from their complex native environment within the brain parenchyma. Here, we present a novel ex vivo method to quantify N-methyl-d-aspartate (NMDA)-induced excitotoxicity using acute brain slices from male and female young adult c57bl/6 and aged B6129SF2J mice, similar to those used for conventional electrophysiological recordings. Acute hippocampal slices were recovered in an N-methyl-D-glucamine (NMDG)-based recovery solution, then treated with low-magnesium artificial cerebrospinal fluid (aCSF) containing the co-agonist glycine to promote receptor activation, with or without exogenous NMDA. Following treatment, slices were fixed, cryoprotected, and cryosectioned to 20 μm for immunohistochemistry. Apoptotic cell death was assessed by staining for cleaved caspase-3, and was combined with the percentage of dead space to calculate a toxicity index for overall excitotoxic cell death. Importantly, exposure to low-magnesium aCSF with glycine alone was sufficient to elevate active caspase-3 levels, an effect that was further enhanced by exogenous NMDA application and prevented by NMDAR antagonism. Our ex vivo method largely preserves the cytoarchitecture and local microenvironment of brain tissue, enabling the assessment of cell-specific vulnerabilities to excitotoxic damage in select brain regions at defined ages. It is particularly well-suited for use in neurodegenerative disease models, where excitotoxic susceptibility may evolve over time. In all, the approach described here provides a reliable and accessible alternative to dissociated cell cultures, bridging the gap between in vitro and in vivo systems for studying glutamate-induced cell death.