Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

Abstract

Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4"-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 microM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In a parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 microM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large-conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD-induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs.

Figure 1. OGD induced neuronal death in the hippocampal CA1 region was largely reduced by RVT

(A) Representative photomicrographs of hippocampal CA1 regions show TO-PRO-3-I fluorescence immediately after 20 min exposure to different treatments: OGD, OGD +100 μmu;M Resveratrol, OGD + 100 μM PQQ and control bath solution. Quantification of TO-PRO-3-I staining from 20 min OGD treatment and with the presence of DMSO, RVT, PQQ are shown in B. Bars represent the mean ±SEM, * compare between the different OGD treatment groups with control * P<0.05, ** P<0.01; ‡ compare between the OGD with OGD + RVT group, ‡ P<0.05, ‡‡ P<0.01.

Figure 2. RVT attenuates OGD-induced impairment of pyramidal neuron excitability

A. OGD-induced changes in pyramidal neuron excitability recorded in current clamp mode. During the pre-OGD control period, OGD period and recovery from OGD period, the Vm and excitability changes were periodically measured every 20 seconds by delivering a 200–300pA/100 ms duration current pulse to the recorded cell. Representative traces from the control and after 15 min. OGD and 5 min. recovery phases, as indicated by the arrows, are displayed in the expanded scale in the lower panel in A, showing a marked Vm depolarization and loss of spike number over a 15 min. OGD insult. B. Shows that in another pyramidal neuron recording that when 100 μM RVT was present during OGD Vm and spike number was maintained (a–c in the lower panel in B).

Figure 3. Quantifications of acute effect of RVT and PQQ on OGD induced excitability changes in pyramidal neuron

The neuronal membrane potential and spike number before, during and after OGD treatment were compared between OGD and OGD plus 100 μM RVT groups (A–B) or OGD and OGD plus 100 μM PQQ groups. (A) 15 min OGD causes a time-dependant membrane potential depolarization in the control OGD group which was significantly reduced in the present of RVT. Similarly, RVT also inhibited the OGD-induced the spikes decrease (B). In the same condition, however, PQQ failed to show similar effects (C, D). .* and ** indicate that the differences in between OGD and OGD + RVT groups or OGD and OGD + PQQ group were statistically significant at the levels of p<0.05 and p<0.01, respectively.

Figure 4. RVT attenuates the OGD-induced increased spontaneous EPSCs in pyramidal neurons

A. Shows the OGD-induced increased sEPSCs (downward current deflections) for different periods of OGD. B. In the presence of 100 μM RVT, both the OGD-induced increased frequency and amplitude increase of the sEPSCs were essentially inhibited. C. Comparison of cumulative probability of sEPSC amplitudes between the control OGD (dashed line) and OGD + RVT (solid line). The data used were taken from A and B during the 15 min. OGD treatment. D. Summary of all the experiments showing that the OGD-induced increase in sEPSC frequency is greatly inhibited by 100 μM RVT. For the analyses presented in C and D, only downward current deflections greater than −4 pA from the baseline in all the recordings were considered as acceptable sEPSC events and were included for these analyses.* and ** indicate that the differences in between OGD and OGD + RVT groups were statistically significant at the levels of p<0.05 and p<0.01, respectively.

Figure 5. The acute effect of RVT on pyramidal neuron during early OGD is mediated by potentiation of BK channels

Voltage-clamp recordings of CA1 pyramidal neurons exposed to a 15 min.OGD treatment at −60 mV holding potential under different conditions as indicated (A). The traces represent the changes in holding current (down indicates negative current reflecting the Vm depolarization shown in Fig. 2A) for OGD, OGD+RVT (100 μM) and OGD + RVT + PAX (10 μM), as noted on the figure. The relative speed and degree of Vm depolarization under these recording conditions were proportional to the slope and amplitude of the negative holding currents. The OGD induced currents were reduced in the presence of RVT; which was completely blocked by PAX). The negative holding current was greatly increased with PAX. B. The inhibition by RVT of the OGD induced sEPSCs frequency was reduced by 10 μM PAX to greater than control levels. At each time point, the sEPSC events in each minute during OGD were summed and presented as sEPSC frequency/per min as indicated on the y-axis. C. Summary of all the experiments including additional controls of recording in the bath alone, PAX alone in the bath aCSF and PAX+OGD; the holding currents in of recordings in the bath control was −2.7±3.0 pA, the values of holding currents shift at the end of OGD were −10.9 ± 6.1 pA for PAX alone, −71.6 ± 9.0 pA for OGD alone, −50.6 ± 4.0 pA for OGD+RVT and −128.6 ± 22.7 pA for OGD+RVT+10 μM PAX.. All p values were determined by one-way ANOVA test. **. Indicates p < 0.01 of the difference between OGD and OGD+RVT. ‡‡. Indicates p < 0.01 of the difference between OGD+RVT and OGD+RVT+PAX. No difference was found between OGD+PAX and OGD+PAX+RVT groups.