Dissociated cell cultures of dopaminergic neurons from embryonic rat mesencephalon were grown in completely defined serum-free medium. We are using these cultures to describe and investigate the separate stages involved in excitotoxic cell death of neurons. Changing the medium of these cultures with medium containing 4.0 mM glutamine leads to complete destruction of the cultures. This damage is blocked by an antagonist (APV) of the NMDA-type of glutamate receptors. Excitatory amino acids (EAA) operating via NMDA receptors are responsible for culture damage. A low level of glutamate (micromoles) contaminating the large amount of glutamine is responsible for initiating damage; however, this NMDA-receptor event is finished in minutes after the medium change. Over the next 3-6 h the cells are damaged by unknown mechanisms; following this damage the level of glutamate in the extracellular medium rises to several hundred micromoles over the next 12 h. Most of this glutamate is generated extracellularly since it also appears if the medium is removed and recultured in wells with no cells. The most likely explanation is that damaged cells release glutaminase into the culture medium which converts the extracellular glutamine to glutamate. If similar mechanisms operate in vivo, disintegrating neurons in a damaged area could generate large quantities of glutamate from the abundant glutamine in the cerebrospinal fluid; if healthy neurons were in the vicinity (as is found in vivo around a damaged area) this glutamate would subsequently damage these cells starting a cascade of ever widening cell damage and death.
