NMDA receptor activation inhibits neuronal volume regulation after swelling induced by veratridine-stimulated Na⁺ influx in rat cortical cultures

Neurons and glia experience rapid fluctuations in transmembrane solute and water fluxes during normal brain activity. Cell volume must be regulated under these conditions to maintain optimal neural function. Almost nothing is known, however, about how brain cells respond to volume challenges induced by changes in transmembrane solute flux. As such, we characterized the volume- regulatory mechanisms of cultured cortical neurons swollen by veratridine- stimulated Na⁺ influx. Exposure of cortical neurons to 100 μM veratridine for 10-15 min caused a 1.8- to 2-fold increase in cell volume that persisted for at least 90 min. This volume increase was blocked by extracellular Na⁺ removal or by exposure to 5 μM tetrodotoxin, indicating that swelling is a result of Na⁺ entry via Na⁺ channels. Treatment of cells with veratridine together with various NMDA receptor antagonists had no effect on the magnitude of swelling. NMDA receptor antagonist-treated cells, however, underwent nearly complete volume recovery within 50-70 min after veratridine exposure. This recovery suggests that NMDA receptor activation disrupts neuronal osmoregulatory pathways. Volume regulation was blocked by Ba²⁺, quinidine, or 5-nitro-2-(3-phenylpropylamino) benzoic acid, indicating that swelling activates volume regulatory K⁺ and Cl^- channels. Veratridine also caused a rapid, transient increase in intracellular Ca²⁺. Extracellular Ca²⁺ removal or intracellular Ca²⁺ chelation prevented or dramatically reduced veratridine-induced increases in intracellular Ca²⁺ and completely blocked volume recovery. These findings indicate that increases in Ca²⁺ during cell swelling induced by Na⁺ influx are required for activation of neuronal volume-regulatory pathways.