Contribution of downregulation of L-type calcium currents to delayed neuronal death in rat hippocampus after global cerebral ischemia and reperfusion

Abstract

Transient forebrain ischemia induces delayed, selective neuronal death in the CA1 region of the hippocampus. The underlying molecular mechanisms are as yet unclear, but it is known that activation of L-type Ca2+ channels specifically increases the expression of a group of genes required for neuronal survival. Accordingly, we examined temporal changes in L-type calcium-channel activity in CA1 and CA3 pyramidal neurons of rat hippocampus after transient forebrain ischemia by patch-clamp techniques. In vulnerable CA1 neurons, L-type Ca2+-channel activity was persistently downregulated after ischemic insult, whereas in invulnerable CA3 neurons, no change occurred. Downregulation of L-type calcium channels was partially caused by oxidation modulation in postischemic channels. Furthermore, L-type but neither N-type nor P/Q-type Ca2+-channel antagonists alone significantly inhibited the survival of cultured hippocampal neurons. In contrast, specific L-type calcium-channel agonist remarkably reduced neuronal cell death and restored the inhibited channels induced by nitric oxide donor. More importantly, L-type calcium-channel agonist applied after reoxygenation or reperfusion significantly decreased neuronal injury in in vitro oxygen-glucose deprivation ischemic model and in animals subjected to forebrain ischemia-reperfusion. Together, the present results suggest that ischemia-induced inhibition of L-type calcium currents may give rise to delayed death of neurons in the CA1 region, possibly via oxidation mechanisms. Our findings may lead to a new perspective on neuronal death after ischemic insult and suggest that a novel therapeutic approach, activation of L-type calcium channels, could be tested at late stages of reperfusion for stroke treatment.

Figure 1.

Selective reduction of whole-cell L-Ca²⁺-channel currents in CA1 neurons after ischemia–reperfusion. A, C, Whole-cell L-Ca²⁺-channel currents in CA1 (A) and CA3 (C) neurons from control (top) and 24 h postischemic (bottom) animals. The cell was voltage clamped at −50 mV and the traces were obtained by stepping the voltage to potentials from −40 to +50 mV for 100 ms. B, D, I–V curves constructed from peak currents in control and 24 h postischemic CA1 (B) and CA3 (D) neurons. Shown are mean values obtained for 15 cells, and vertical bars indicate SEM. Note that voltage dependence is not shifted after ischemia. Calibration: 20 ms, 200 pA.

Figure 2.

Persistent modification of single L-Ca²⁺-channel gating in CA1 but not CA3 neurons after ischemia–reperfusion. A–D, Five representative leak-subtracted recordings from cell-attached patches on CA1 (A, B) and CA3 (C, D) neurons of control and ischemic animals. A, C, Control group. B, D, 48 h of reperfusion group during repetitive depolarizations (−50 to −10 mV). All perfectly recorded patches were used to create an average total current for each group (shown below the 5 single traces). Voltage protocol is shown at the bottom. Calibration: 60 ms, 1 pA. E, Changes in average total patch current calculated by division of the integral of each ensemble average by the pulse or window duration in CA1 neurons after ischemia. Mean ± SEM for current amplitude during the 300 ms depolarization from holding potential −50 mV to testing potential −10 mV. *p < 0.01 compared with control. F, Unitary current amplitudes to multiple test voltages in patches from control and postischemic CA1 neurons. Individual values represent the mean amplitude of all clearly resolvable L-type openings during the pulse for each patch at each voltage (n = 10 patches per group at each voltage). Mean slope conductance for each group was calculated from the average of individual patch slope conductance. G, Comparisons of voltage-dependent activation of L-type calcium channels in control and postischemic CA1 neurons. Voltage dependence of activation represented by P/P_max plotted against membrane potential. Smooth curves derived from the Boltzmann relation are given by P/P_max = 1/{1 + exp[(V_1/2 − V)/k]}. H, Unitary current amplitudes to multiple test voltages in patches from control and 48 h postischemic CA3 neurons (n = 10 patches per group at each voltage).

Figure 3.

No change in the expression of Ca_V1.2 and Ca_V1.3 subunits in CA1 region after ischemia–reperfusion. A, Western blots showing changes in Ca_V1.2 and Ca_V1.3 subunits levels after 24 h of reperfusion. β-Actin was loading control. N, Negative control. B, Quantitative analysis of the normalized signal intensities in Western blots (p > 0.05; n = 3). C, Representative immunostaining of the CA1 region showing the distribution of Ca_V1.2 and Ca_V1.3 subunits in the cell body of pyramidal neurons from a control animal and an experimental animal 48 h after ischemia. Scale bar, 10 μm. D, Change in the percentage of patches with channel openings in CA1 neurons after ischemia–reperfusion.

Figure 4.

Effects of extracellular redox couple SNP and Bay K 8644 on whole-cell Ca²⁺-channel currents. A–D, Example of time-series plot of Ca²⁺-channel current amplitudes in control (A, B) and postischemic (C, D) CA1 neurons; each plotted point represents the current amplitude evoked by repeated step depolarizations (100 ms, 0.1 Hz) to −10 mV from a holding potential of −50 mV. For the period indicated by the horizontal bar, the perforate was exchanged for one containing 2 mm DTNB (A, C) or 2 mm DTT (B, D). Inset shows example currents from this recording before and during exposure to agent as indicated. Calibration: 50 ms, 150 pA (A), 200 pA (B), and 100 pA (C, D). E–G, Comparison of the effects of redox couples DTT and DTNB, GSH and GSSG on whole-cell Ca²⁺-channel currents in control and postischemic CA1 neurons. Values are the mean ± SEM of measurements made in 10–15 neurons from at least four different rats. The asterisk indicates a statistically significant difference (p < 0.05 or 0.01; see Results). E, Control. F, 24 h after ischemia. G, Summary of percentage change caused by redox couples normalized to control in Ca²⁺ currents from control and postischemic neurons. H, Time course for the effects of Bay K 8644 on the SNP-induced inhibition of the Ca²⁺ current in the cultured hippocampal neurons. Inset shows example of Ca²⁺-channel currents elicited by a step to +10 mV. a, Control; b, 200 μm SNP; c, 200 μm SNP plus 0.3 μm Bay K 8644. Calibration: 50 ms, 400 pA. I, Comparison of the effects of SNP and Bay K 8644 on the average Ca²⁺ currents at +10 mV (n = 8). *p < 0.05 versus control group; ^#p < 0.05 versus SNP group.

Figure 5.

Downregulation of L-type Ca²⁺ channels contributes to hippocampal neuronal cell death in vitro. A, B, Summary of the effects of nifedipine (Nfdp;10 μm), nimodipine (Nmdp;10 μm), ω-conotoxin GVIA (CgTx; 2 μm), ω-agatoxin IVA (AgTx; 0.2 μm), SNP (200 μm), SNP (200 μm) plus nimodipine (10 μm), SNP (200 μm) plus Bay K 8644 (0.3 μm), and Bay K 8644 (Bay K; 0.3 μm) on the survival rate of cells in the cultured hippocampal neurons. The solvent alcohol (Alco) was used as control. Values are the mean ± SEM of 18 measurements from three experiments. *p < 0.05 versus control group; ^#p < 0.05 versus SNP group. C, D, Reduced cell death after oxygen-glucose deprivation (OGD) in hippocampal neurons treated with Bay K 8644 after 1 h of reoxygenation. C, Primary hippocampal neurons were treated with solvent alcohol (controls) or 0.3 μm Bay K for 23 h. Cells were exposed to 4 h of OGD, and apoptotic nuclei were quantified after staining with Hoechst 33342. Neurons treated with Bay K 8644 showed less pyknosis after OGD compared with control neurons. D, Quantification of experiments described in C. Treatment with Bay K 8644 significantly reduced apoptotic cell death compared with control cultures. *p < 0.05 versus control group; ^#p < 0.05 versus OGD group.

Figure 6.

Significant reduction of ischemic neuronal injury in rats treated with L-type Ca²⁺-channel agonist Bay K 8644 after 12 h of reperfusion. A, Effect of Bay K 8644 on the ischemic damage of CA1 pyramidal neurons in rats. Bay K 8644 (0.2 mm) was delivered intracerebroventricularly with osmotic minipumps at 0.5 μl/h after 12 h of reperfusion for 3 d. Scale bar, 20 μm. B, Neuronal density (number of intact pyramidal cells per 1 mm linear length) in the CA1 field. n = 6 for each group. *p < 0.01 compared with sham-operated control. ^#p < 0.01 compared with ischemic control.