Nitrobenzylthioinosine mimics adenosine to attenuate the epileptiform discharge of hippocampal neurons from epileptic rats

Abstract

Nitrobenzylthioinosine (NBTI), a specific inhibitor of type 1 equilibrative nucleoside transporter, could regulate the extracellular adenosine concentration and have protective roles in seizures. However, the protection mechanism of NBTI in seizures remains poorly understood. Here, the expression pattern and subcellular distribution of adenosine A1 receptor were detected by Western blot analysis and double-labeling immunofluorescence staining in Lithium Chloride-Pilocarpine induced epileptic rat model. At 24 h after pilocarpine induced rat seizures, hippocampal slices were prepared and the evoked excitatory postsynaptic currents (eEPSCs) amplitude of pyramidal neurons in hippocampus CA1 region was recorded using whole-cell patch clamp. In vivo, compared to control group, Western blotting analysis showed that the expression of adenosine A1 receptor protein was increased at 24 h and 72 h after seizure, didn’t change at 0 min and 1 w, and decreased at 2 w. Double-label immunofluorescence revealed that adenosine A1 receptor was mainly expressed in the membrane and cytoplasm of neurons. In Vitro, adenosine decreased the eEPSCs amplitude of pyramidal neurons in hippocampus CA1 region, NBTI also had the same effect. Meantime, NBTI could further inhibit eEPSCs amplitude on the basis of lower concentration adenosine (50µM), and adenosine A1 receptor inhibitor DPCPX partially reversed this effect. Taken together, we confirmed that the expression of adenosine A1 receptor protein was increased in the early seizures and decreased in the late seizures. At the same time, NBTI mimics adenosine to attenuate the epileptiform discharge through adenosine A1 receptor, which might provide a novel therapeutic approach toward the control of epilepsy.