The Effect Of Resveratrol On The Synaptic Transmission Of Cultured Hippocampal Neurons

ObjectiveTo investigate the effect of resveratrol (Res) on synaptic transmission, the in vitrocultured hippocampal neurons forming synaptic connection were used and the post-synaptic currents, including miniature inhibitory post-synaptic currents (mIPSCs) andevoked inhibitory post-synaptic currents (eIPSCs) or miniature excitatory post-synapticcurrents (mEPSCs) and evoked excitatory post-synaptic currents (eEPSCs), wererecorded with voltage clamp. The effects of Res applied by incubation or perfusion onsynapse network activity were analyzed in order to search the mechanism of the Res’sprotective functions on injured neurons.Methods1. In vitro hippocampal neuron cultureFour C57BL/6pups were dissected quickly. The hippocampus of pups was taken outand kept in4C Hank’s balanced salts medium (HBS). The hippocampus was digestedfor12min in1ml HBS containing0.25%of trypsin-EDTA at37C after washed withHBS. Then the digestion was stopped by HBS containing5%fetal bovine serum (FBS)which is pre-warmed at37C. The supernatant solution was discarded and propervolume of plating medium was added. After pipetting up and down about15~20times,the dispersed cells were dropped to plated coverslips. After the cells adhered to thecoverslips for30min at37C,1ml plating medium was added in each well. Renewalof the medium with growth medium on the1st,4thand9thdays and the recordings weredone after the neurons were cultured more than14days.2. Whole-cell recordingsAfter the neurons were cultured more than14days, the coverslip with neurons formingsynaptic connection was transferred into external solution. The whole-cell recordings of voltage clamp were performed. Alcohol and Res were applied by either perfusion orincubation as control group and experiment group, respectively.3. Data process and statistical analysisRaw data was processed by Clampfit and Igor6.0. Statistical analysis was performedusing student’s t test with Graphpad Prism5software. All data were shown in mean±SEM. The level of significance was set at P <0.05.Results1. Effects of Res on inhibitory synaptic transmission6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX,10μmol/L), an a-amino-3-hydroxy-5-methyl-4-isoxa-zolep-propionate (AMPA) receptor antagonist, and TTX (1μmol/L), asodium channel blocker, were added in the external solution to record the mIPSC oreIPSC. During the recording, Res (100μmol/L) applied by perfusion or incubation hasno significant effect on the amplitude and frequency of mIPSC (perfusion: n=10, P>0.05; incubation: n=24, P>0.05). Similarly, Res (100μmol/L) applied by perfusionhas no significant effect on the amplitude and short-term plasticity of eIPSC (n=10, P>0.05).2. Effects of Res on excitatory synaptic transmissionPicrotoxin (PTX,100μmol/L), a γ-aminobutyric acid (GABA) receptor antagonism, andTTX (1μmol/L) were added in the external solution to record the mEPSC or eEPSC.During the recording, Res (100μmol/L) applied by incubation has no significant effecton the amplitude and frequency of mEPSC (n=24, P>0.05) and the amplitude ofeEPSC (n=24, P>0.05).Conclusions1.100μmol/L of Res has no effect on inhibitory synaptic transmission in culturedhippocampal neurons.2.100μmol/L of Res has no effect on excitatory synaptic transmission in cultured hippocampal neurons.