As neurons need much energy to maintain cellular calcium homeostasis and abnormal influx of calcium ions is neurotoxic w24x, it’s possible that neurons that are hypoactive within their energy state Alogliptin selleckchem become vulnerable to mild stimuli of calcium influx, which is less harmful to normal neurons. To look at this possibility, we addressed neurons with large KCl, low KClqZ Asp CH DCB 100 mM., or low KClqactinomycin N 1 mgrml. for 24 h. The nerves were therefore treated with minimal KCl medium or high KCl medium for yet another 6 h, and LDH activities produced over the past 6 h were measured. As shown in Fig. 5A, neurons rescued by Z Asp CH DCB produced more LDH task once the neurons were treated 6 h in-the high KCl method, than 2 neurons rescued with high KCl or low KClqactinomycin N. Six time treatment with low KCl method did not produce this result. Similar results were obtained in neurons recovered with 30 mM Boc Asp FMK Fig. 5B.. More over, we considered the effect of glutamate, still another inducer of Ca2q influx via NMDA receptor. Under the conditions utilized in the existence of minimal KCl and Mg2q., glutamate at 1 mM was less dangerous to the neurons maintained in high KCl method or neurons saved with actinomycin D. On the other hand, nerves rescued with 30 mM Boc Asp FMK Lymphatic system were susceptible to subsequent therapy with low KClq1 mM glutamate for 6 h Fig. 6.. As still another indicator of cell death, the disintegration of cell membranes was examined using PI which is taken up in dead cells and becomes fluorescent by intercalating into DNA. As shown in Fig. 7, neurons rescued from low KCl induced apoptosis by 100 mM Z Asp CH DCB or 30 mM Boc Asp FMK were at risk of subsequent treatment with large KCl 2 Fig. 7A. and glutamate Fig. 7B. for 6 h. Applying this criterion, about half the neurons died and became permeable to PI. These findings were verified by morphological examination Fig. 8.. Neurons were initially maintained with medium containing high KCl, low KClq30 mM Boc Asp FMK, and GW0742 low KClq100 mM Z Asp CH DCB for 2-4 h, then changed 2 to medium containing low KCl, low KClq1 mM glutamate, and high KCl. Many neurons were still living when the medium was changed to that containing low KCl for 6 h Fig. 8A,B,G.. Nevertheless, when nerves rescued by 30 mM Boc Asp FMK Fig. 8D. and by 100 mM Z Asp CH DCB Fig. 8H. were treated with the medium containing large KCl for 6 h, many 2 nerves stained red with PI, revealing extensive neuronal death. Likewise, nerves rescued by 30 mM Boc Asp FMK were vulnerable to treatment with the medium containing low KClq1 mM glutamate for 6 h Fig. 8F.. Many neurons preserved with high KCl medium were still alive when turned to the medium containing minimal KClq1 mM glutamate for 6 h Fig. 8E., though their neurites became somewhat diminished.