Glutamate load fosters spreading depolarization under osmotic stress in brain slices

Rita Frank^1*Stephane Marinesco²Ferenc Bari¹Ákos Menyhárt^1*Eszter Farkas¹

• ¹University of Szeged, Szeged, Hungary
• ²Universite Claude Bernard Lyon 1, Villeurbanne, France

Cerebral edema is a hallmark of lesion progression after acute ischemic stroke (AIS) and a key contributor to the evolution of spreading depolarizations (SDs). SD waves trigger extracellular glutamate accumulation and excitotoxic injury, but the cellular mechanisms linking edema, glutamate release, and SD dynamics remain incompletely understood. Here, we examined how inhibition of glial swelling and volume-regulated glutamate release, or blockade of neuronal ionotropic glutamate receptors, alters SD under hypo-osmotic stress in rat brain slices. Using local field potential recordings, intrinsic optical signal imaging, and enzyme-based glutamate biosensors, we quantified the spatiotemporal dynamics of SDs and extracellular glutamate levels. Pharmacological inhibition of aquaporin-4 water channels, the Na⁺-K⁺-Cl⁻ cotransporter 1, or volume-regulated anion channels restricted the SD-affected cortical area, shortened SD duration, and reduced extracellular glutamate accumulation, yet paradoxically promoted SD spread. In contrast, blockade of NMDA or AMPA/kainate receptors markedly decreased SD propagation and glutamate buildup, while slowing propagation velocity and enhancing SD amplitude. Notably, both astrocytic (aquaporin-4 water channels, Na⁺-K⁺-Cl⁻ cotransporter 1) and neuronal interventions disrupted typical SD initiation patterns, producing atypical, multifocal SDs. These findings demonstrate that astrocyte volume regulation and ionotropic glutamate receptors synergistically govern SD features under osmotic stress. Taken together, these results highlight astrocytic water and ion homeostasis, along with neuronal glutamatergic signaling, as central therapeutic targets to mitigate excitotoxic injury in acute cerebrovascular disease.