Vulnerability to excitotoxic stimuli of cultured rat hippocampal neurons containing the calcium-binding proteins calretinin and calbindin D28K.

Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10-12 DIV. Almost all neurons (approximately 95%) died within 20 h after a transient exposure for 10 min to 50 microM glutamate, a continuous exposure to either 25 microM NMDA or 250 microM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 microM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 microM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analysed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3-5%) survived, including a subpopulation of calretinin-positive but not calbindin D28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of glutamate receptors.