Neuronal injury evoked by depolarizing agents in rat cortical cultures

Chemical depolarization is often used to study neurotransmitter release. Three commonly used depolarizing agents, veratridine, potassium, and glutamate, were evaluated for neurotoxicity. Neuronal survival and lactate dehydrogenase efflux were measured to assay irreversible injury. In addition, video-enhanced differential interference contrast microscopy was used to measure acute neuronal swelling. We found that lactate dehydrogenase efflux and cell death associated with exposure to potassium and glutamate could be blocked by the competitive N-methyl-D-aspartate antagonist amino-phosphonovaleric acid. Neuronal swelling was observed with all three agents, and could not be blocked by amino-phosphonovaleric acid. These results suggest multiple mechanisms of neuronal injury accompanying chemical depolarization. A 60-min exposure to 100 microM veratridine increased lactate dehydrogenase appearing in the medium at the end of this exposure to 615% of control and produced a 62% loss of neurons after 20-24 h. These effects could not be blocked by amino-phosphonovaleric acid at 500 microM. Differential interference contrast imaging revealed acute neuronal swelling in response to veratridine within 5 min of exposure, and this swelling could not be blocked by amino-phosphonovaleric acid. A 60-min exposure to medium supplemented with 50 mM KCl caused a lactate dehydrogenase efflux of 204% of control and produced a 48% loss of neurons. Amino-phosphonovaleric acid blocked both the neuronal loss and the excess lactate dehydrogenase efflux. In addition, differential interference contrast monitoring showed no KCl-evoked swelling. In contrast, isotonic substitution of 50 mM KCl for NaCl resulted in acute swelling which could not be blocked by amino-phosphonovaleric acid, in addition to neuronal death and lactate dehydrogenase release. Glutamate was, as expected, neurotoxic, and as has been shown before, this toxicity could be blocked by amino-phosphonovaleric acid. Observation of neurons exposed to 300 microM glutamate revealed that this treatment was invariably associated with neuronal swelling. In the presence of amino-phosphonovaleric acid, 81% of neurons swelled to greater than 110% by 30 min exposure to glutamate. These results suggest that experimental paradigms which investigate the effects of chemical depolarization upon central neurons are likely to be associated with reversible and irreversible forms of injury. This is of special importance to any study of the mechanisms of release of substances from central neurons.