CORTICAL-NEURONS CONTAINING CALRETININ ARE SELECTIVELY RESISTANT TO CALCIUM OVERLOAD AND EXCITOTOXICITY IN-VITRO

Calbindin and the more recently identified protein calretinin are stru cturally related calcium-binding proteins having a broad distribution in the brain. Recent evidence supports a neuroprotective role for calb indin in regulating calcium homeostasis during periods of heightened C a2+ influx. It is not known if calretinin might have a similar functio n. We investigated if calretinin-containing neurons have a survival ad vantage in rat neocortical cultures treated with a calcium ionophore o r excitatory amino acids. Neuronal cultures were challenged with the c alcium ionophore A23187 at different concentrations to produce a broad range of cell death. Cell loss was quantified for both the calretinin immunopositive and the calretinin immunonegative populations of neuro ns. We found that 3 h after exposure to 2 mu M A23187 there was a 48% loss of the calretinin immunonegative population of neurons whereas th e calretinin immunopositive set of neurons was reduced by only 18%. Ca lretinin positive neurons were still relatively spared after treatment with 3 mu M A23187. The ionophore had no cytotoxic effect when calciu m ions were removed from the extracellular medium. We also studied glu tamate excitotoxicity by treating the neuronal cultures with the excit atory amino acids glutamate, N-methyl-D-aspartate or kainate for 5 min and examining survival three hours later. We found again that calreti nin-containing neurons were relatively spared after exposure to the ex citatory amino acids; at doses of N-methyl-D-aspartate and kainate tha t produced a 32-40% loss of calretinin immunonegative neurons, only 2- 10% of calretinin immunopositive neurons died. Similar results were ob tained for glutamate. These results demonstrate that neurons containin g calretinin are better able to survive disturbances in calcium homeos tasis than cells not containing this calcium-binding protein. The fact that this effect was observed with ionophore treatment, as well as ex citatory amino acids, suggests that neither the density nor distributi on of glutamate receptors on the different cell types was a factor in determining selective vulnerability. We hypothesize that the neuroprot ective effect of calretinin is due to the buffering capacities of the protein in a manner analogous to that suggested for calbindin.