Tutin-induced Epilepsy And Its Mechanisms In Rats

Previous studies showed that a mixture, Coriaria Lactone (CL), extracted from traditional Chinese herb Loranthus Parasiticus Mer, had a great excitatory influence on nervous system and can cause seizure. Epileptic animal models induced by CL were widely used to study the epileptogenic mechanisms and screen the anti-epileptic drugs. However, what component in the CL can cause seizure is unclear, which limits its use as a tool drug in the research of epilepsy. Tutin is a pure chemical component separated from CL. The present experiments were carried out to test if Tutin has any epileptogenic action and to investigate the mechanisms underlying that action in vivo and in vitro. Using electroencephalogram (EEG) recording, extracellularly microelectrode recording and whole-cell patch clamp technique, effects of Tutin on behavior and EEG of rats, synaptic transmission of CAl pyramidal cells in hippocampal sliceand ionic currents of acutely isolated CA1 pyramidal cells were investigated at systemic, tissular and cellular level. The results are as follows.1. Tutin microinjected through intracerebroventricle induced behavioral seizure in rats, such as immobility, blinking, vibrissae, chewing, rhythmic head nodding, scratching, forelimb or hindlimb clonus, trembling, falling, jumping, rearing, running-bouncing. EEG recording of electrographic seizure induced by Tutin showed a representative epileptic pattern, such as spikes, a typical epileptic discharge in 40 ~ 80 ms of duration, 50 ~ 150 μV (103.53 ± 36.81 μV) of amplitude occurred discretely or repetitively; discrete or continuous spike-wave complexes occurred in 3 ~ 4 Hz of frequency, 293.53 ± 41.62 μV of amplitude; multiple spikes and slow waves burst in 8 Hz of frequency and 555.54 ± 98.23 μV of average amplitude or in 3 Hz of frequency and 667.74 ± 127.24 μV of average amplitude observed in onset of grand mal seizure. The latency of epileptic onset was 5.10 ± 0.66 min and morbidity of epilepsy was 100% with type 2 of 20% and type 3 of 80% in high concentration group (25.0 μg/μl Tutin), whereas latency of epileptic onset in low concentration group (12.5 μg/μl Tutin) was longer (8.05 ± 2.79 min ) than that in high concentration group (P < 0.05, n = 5,) . Morbidity of epilepsy was 100% with typel of 20%, type 2 of 40% and type 3 of 40% in low concentration group (12.5 μg/μl Tutin). There were no difference between the two groups inmorbidity, proportion of type and motility (P>0.05, n = 5) .2. Superfusion with different concentration of Tutin, 40 μg/ml, 30 μg/ml and 20 μg/ml, caused significant increase in amplitude and number of PS waves evoked by stimulating the Schaffer collaterals. Thirty minutes after superfusion of Tutin, the amplitude of the first wave of the PSs was increased by 388.7 ± 20.1%, 317.2 ± 19.1% and 180.9 ± 11.6% for each concentration group, respectively, compared with control (P < 0.05, n = 5 for each group). With increase in amplitude, the PS number was increased to 4 ~ 11 waves from a single wave in control and manifested multiple epileptiform discharges 30 min after superfusion with Tutin. Spontaneous epileptiform discharges of CAl pyramidal cells were obtained in 9 out of 34 cases during superfusion with Tutin. The Tutin-induced multiple epileptiform discharges of the CAl pyramidal cells could be completely blocked by CNQX, in aspects of both amplitude and number of the PS. Following the application of AP-5, the increase in the wave number of the Tutin-induced epileptiform discharges was inhibited but the increase in the amplitude of the discharges was not significantly affected.3. The effects of Tutin on K+ and Na+ currents of acutely isolated CAl pyramidal neurons were observed using the whole-cell patch-clamp technique. Neurons were stepped to various potential ranging from -100 mV to +90 mV in 10 mV increment at a holding potential of -100 mV. Amplitude of outward K+ currents of CA1pyramidal neurons was a decrease of 78.81 ± 17.13% from 1342.57 ± 81.34 pA to 1066.11 ± 150.24 pA, and amplitude of inward Na+ currents was an increase of 113.93 ± 12.87% from 515.36 ± 60.51 pA to 586.40 ± 70.40 pA in the presence of Tutin (P < 0.05 , n = 10). Tutin inhibited outward K+ currents and enhanced inward Na+ currents. Sodium currents were activated by 15 ms depolarizing voltage steps ranging from -100 mV to +30 mV in 10 mV increment at a holding potential of -100 mV. The threshold for the Na+ current was around -65 mV and the largest peak amplitude of Na+current occurred at -30 mV to -20 mV. This peak amplitude of current was an increase of 113.98 ± 14.37 % from 453.25 ± 46.63 pA to 509.88 ± 45.7 pA (P < 0.05, n = 8) after Tutin application to culture dish. Current activation characterized by a Vmid of -44.27 + 0.10 mV in pyramidal neurons treated with Tutin was more negative than that of the control neurons (-34.69 ± 0.38 mV) ( P < 0.05). Vc of 2.32 ± 0.18 mV in pyramidal neurons treated with Tutin was not different from that of control neurons (2.47 ± 0.31 mV). Current inactivation characterized by a Vmid of-58.06 ± 1.29 mV and Vc of-5.69 ± 0.34 mV in pyramidal neurons treated with Tutin was not different from that of control neurons (Vmid of -60.67 ± 0.54 mV, Vc of-6.5 ± 0.39 mV) (P > 0.05, n = 6).We come to a conclusion from these results. Tutin can induce typical behavioral seizure and electrographic seizure of rats in vivoand multiple epileptiform discharges of CA1 pyramidal cells in hippocampal slices. As an efficient epileptogenic agent, it might be available component in CL and used to establish epileptic model for study of epileptic mechanisms and test of anti-epileptic drugs' efficacy. Tutin-induced epileptic model is characterized by short latency, higher morbidity, lower mortality and easy replicate. The excitable glutamate receptors, especially the non-NMDA receptors, may participate in the Tutin-induced epileptogenesis. Decreased outward K+ currents and increased inward Na+ currents occured in acutely isolated CAl pyramidal neurons treated with Tutin. Administration of Tutin made the voltage dependence of activation significantly shift to more negative potentials. Such a shift will lower the spike threshold of the neuron and thus enhance its excitability. The abnormal epileptic discharge of neurons can be then induced by Tutin.