Effect Of Deoxypodophyllotoxin On Dum Of Cockroach Periplaneta Americana And Drg Of Rat

Part one: Isolation of DUM neurons from the central nervous system of the cockroach Periplaneta americana and study on the effect of DOP on ion channels of DUM neuronsDeoxypodophyllotoxin (DOP) , a active substance isolated from Sabina vulgaris Ant., is well known for its broad insecticidal activity. As a botanical pesticide, it's neurotoxicity is obviously observed. So we need do some study on it's neurotoxicity mechanism.To optimize the method of isolation of dorsal unpaired median (DUM) neurons from the central nervous system of the cockroach Periplaneta americana and identify DUM neurons. The terminal abdominal ganglions of P. am ericana were dissected, DUM neuron cell bodieswere obtained after enzymatic treatment with collagenase IA and mechanical dissociation of the median parts of the terminal abdominal ganglion, and the cells were immunocytochemically stained with anti-octopamine antibody for identifying the DUM neurons. Isolated DUM neuron cells exhibited typical morphological characteristics of neuronal cells, which are nearly pyriformand might live about twelve days. DUM neuron cells can be well obtained by using this modified method. The procedure described here provides the basis for studies of molecular biology of DUM neurons.To investigation the effect of DOP on membrane potential of DUM neurons and its correlation with sodium channel. Methods: DUM neurons were labeled with DiBAC₄(3).Laser scanning confocal microscope was used to monitor the changes of membrane potential at real time on these neurons that were treated with different concentrations of the DOP. The effect of sodium channel blocker tetrodotoxin ( TTX ) on the changes was also observed. Results: Membrane potential depolarization induced by the DOP peaked at 5 min and became stabilized after 8min. After compared with fluorescence intensity without treatment, the normalized fluorescence intensity was 69.6±3.0,72.1±2.7,77.8±3.6,86.2±3.1 in cells which were treated with 1, 5, 25, 125μmol/L DOP, respectively. These numbers are significantly lower than those from untreated control cells(P < 0.01. When DUM neurons were co-incubated with 1μmol /L TTX for 20 min, then treated with 25μmol/L DOP, the intensity changed to 63.6±5.4, which was similar to that of the control(P > 0.05). This indicated that the effect of DOP could be completely inhibited by TTX. Conclusion: DOP induced membrane depolarization of DUM neurons in the range of 1-125μmol/L and the sodium channel should be involved in this process.The effects of DOP on voltage-depent sodium currents(INa) were studied in DUM neurons using the patch-clamp tdchnique in the whole-cell configuration. The results showed: DOP could inhibit the amplitudes of INa in a concentration- and voltage-dependent manner. At the concentration of 100ug/ml DOP lowered the activation threshold and produced negative shifting of INa activation curve , induced shifting of the steady-state inactivation curve to left.The effects of DOP on [Ca²+]i in DUM neurons of rats were investigated with laser scanning confocal microscopy(LSCM). DOP elevated [Ca²+]i in Ca²⁺-containing medium in a concentration-dependent manner. The results showed that share a common mechanism with the cytotoxicity on other cells caused by DOP.Part two: Effect of DOP on ion channels in DRG neuronsTo study the effect of DOP on membrane potential of dorsal root ganglion(DRG) neurons and its correlation with sodium channel; DRG neurons were labeled with DiBAC4(3). Laser scanning confocal microscope was used to monitor the changes of membrane potential at real time on these neurons that were treated with different concentrations of the DOP. The effect of sodium channel blocker TTX on the changes was also observed; Membrane potential depolarization induced by the DOP peaked at 5 min and became stabilized after 5 min of the treatment. After comparing to fluorescence intensity without treatment,the normalized fluorescence intensity was determined to be 62.3±2.1,63.8±3.6,68.5±3.8,88.1±5.4 in cells that were treated with 1,5,25,125μmol/L DOP respectively. These numbers are significantly lower than those from untreated control cells(P < 0.01. When DRG neurons were co-incubated with 1μmol /L TTX for 20 min,then treated with 25μmol/L DOP,the intensity changed to 57.5±2.3,which was similar to that of the control (P > 0.05). This indicated that the effect of DOP could be completely inhibited by TTX; DOP induced membrane depolarization of DRG neurons in a concentration-dependent manner,suggesting that the sodium channel should be involved.The effects of DOP on high-voltage activated Ca²⁺channels (HVA Ca²⁺) were studied in DRG neurons using the patch-clamp tdchnique in the whole-cell configuration. The results showed: DOP could inhibit the amplitudes of HVA Ca²⁺ in a concentration- and voltage-dependent manner. At the concentration of 100ug/ml DOP lowered the activation threshold and produced positive shifting of HVA Ca²⁺ activation curve ,induced shifting of the steady-state inactivation curve to left and reduced the fraction of available HVA Ca²⁺ channels.The effects of DOP on [Ca²⁺]i in DRG neurons of rats were investigated with laser scanning confocal microscopy (LSCM) . DOP elevated [Ca²⁺]i in Ca²⁺-containing medium in a concentration-dependent manner. Intracellular calcium store released blocker 2-APB(2-aminoethoxydiphenyborate),DAN(2,3-diaminonaphthalene), which inhibited the elevation of intracellular calcium concentration induced by DOP. The results showed that share a common mechanism with the cytotoxicity on other cells caused by DOP. Intracellular Ca²⁺ elevation induced by DOP is due to both the release of intracellular calcium store and extracellular calcium entry.