Role Of L-Type Calcium Channels In Delayed Neuronal Death Of Hippocampus After Transient Forebrain Ischemia

The vulnerability of neurons to cerebral ischemia varies widely among different regions in the CNS. Different neuronal populations within a brain region also show different susceptibility to ischemic insult. Studies have shown that CA1 pyramidal neurons in hippocampus are particular vulnerable to ischemic insult and die 3-7 days after transient forebrain ischemia, whereas CA3 neurons are relatively resistant to transient ischemia and remain viable after such a insult. The mechanisms of this selective neuronal damage are not fully understood.To CA1 pyramidal neurons, the cell death a few days after a transient insult in human stroke and in rodent ischemia models is hypothesized to follow from Ca2± overload and it plays a critical role. This hypothesis has received its best support from evidence limited to the period of ischemia and immediately following reperfusion. However, the short-lived elevation of Ca2± is difficult to reconcile with the onset of CA1 neuronal death that manifestes itself days later. Moreover, the notion that excitotoxicity with attendant neuronal calcium overload is the predominant mechanism underlying ischemic neural injury faces challenge owing to negative results form several recent clinical trials with antagonists of NMDA-type glutamate receptors and L-type Ca2± channels. Moreover, it has been shown that apoptosis of cultured neurons can be attenuated by increasing [Ca2±]i.Importantly, a recent study on CA1 hippocampal neurons 2-3 days after transient forebrain ischemia shows that no Ca2± overload exists and even the resting calcium levels is lower than that of normal.Voltage-gated Ca2± channels have a central role in neuronal function and are essential for converting electrical activity into biochemical events. Specially, activation of the L-type Ca2± channel increases the expression of a group of Ca2±-regulated genes, including those encoding c-Fos, brain-derived neurotrophic factor (BDNF), and Bcl-2, that have been shown to be required and important for neuronal survival. L-type Ca2± channels are prevalent in hippocampal pyramidal neurons, contributing -30-50% of total calcium current. Therefore, modulation of L-type Ca^channel activity may have dramatic effects on pyramidal cell.So, we speculate that reduced voltage-dependent Ca2± influx in CA1 neurons at some stage of reperfusion may play a crucial role and be involved in the delayed neuronal death of hippocampus after ischemia.The aim of our present study is to examine the temporal changes in L-type calcium channel currents in CA1 and CAS pyramidal neurons of rat hippocampus after transient forebrain ischemia and its possible cellular mechanisms, and futher to mimic the in vivo ischemia-induced changes by using pharmacological manipulation in cultured hippocampal neurons for determining the role of L-type calcium channels in neuronal death.We recorded the single L-type calcium currents in cell-attached patches of actually dissociated hippocampal CA1 neurons prepared from controls (n=25), 30min (n=19), 6h (n=23), 24h (n=23) and 48h (n=16) after 15 min ischemia. Ischemia of this degree and duration consistently produced selective cell death in the CA1 region of rat hippocampus. TotalL-type Ca2"1" channel current in patches was found increase at 30min after reperfusion and then persistent decrease between 24-48h of reperfusion (VH= -50 mV to VC = -10 mV; controls, 0.439±0.058pA(n=25); 30min, 1.517±0.312pA(n=19); 6h, 0.419±0.099pA(n=23); 24h,0.154±0.024pA(n=23), 48h: 0.080±0.021pA(n=16); mean±SEM). No significant changes were observed in the single channel conductance between control and each of postischemic group. In consistent with the changes in total patch current, open probability of the channels also displayed a biphasic change, an transient increase at 30 min and a persistent decrease between 24h and 48h of reperfusion, in CAl neurons after ischemic insult compared with that of control(P<0.01) (VH = -50 mV to VC = -10 mV; controls, 0.125±0.015; 30min, 0.