Effect of pCREB on the apotosis of neurons in cultured hippocampal slices after seizure like activity

Abstract:

To study the effect of seizure-like activity and pCREB on the neurons in cultured hippocampal slices in vitro. Methods: Hippocampal slice cultured for 11 days were randomly divided into 3 groups:(1)control group:cultured hippocampal slices were exposed to regular media to 14 d.(2)seizure like activity model group:cultured hippocampal slices were exposed to regular media to 14 d,then returned to regular media with pilocarpine(final concentration:5 mmol/L) for another 1 h.(3)anti-pCREB antibody treated group:cultured hippocampal slices were pre-cultured with regular media with anti-pCREB antibody (final concentration: 5 μg/ml) to 14 d，then returned to regular media with pilocarpine (final concentration:5 mmol/L) for another 1 h.The apoptosis neurons of cultured hippocampal slices was detected by terminal deoxynucleotidyl dUTP nick end-labeling（TUNEL）staining in situ.Expression of pCREB protein in the neurons of cultured hippocampal slices was detected by immunocytochemistry. Results: (1)Seizure-like activity model group:the number of apoptotic neurons in CA1 of cultured hippocampal slices was（95.44±14.30） significantly increased compared with that of （41.30±9.30） in the control group(P<0.05).The expressions of pCREB in neurons in CA1 of cultured hippocampal slices were（201.36±19.31）,remarkably up-regulated compared with those of （75.12±13.71）in the control group(P<0.05).(2)Anti-pCREB antibody treated group:the number with of apoptotic of neurons in CA1 of cultured hippocampal slices was (140.88±18.32) significantly increased compared with that in the seizure like activity model group (P<0.05).The expressions of pCREB were （172.60±17.68） remarkably up-regulated compared with those in the control group (P<0.05). Conclusion: (1)Seizure-like activity can induce neuronal apoptosis and aggravate neuronal injury in cultured hippocampal slices.(2)Seizure-like activity can up-regulate the level of CREB phosphorylation to cultured hippocampal slice,and blocking CREB phosphorylation may lead to neuronal cell apoptosis accentuation.