| The emergence of LTP is considered to be a molecular model for learning and memory. Drug addiction is a kind of abnormal learning and memory and pathological processes. In our previous studies, we found that JZTX-V improved the drug withdrawal syndrome, due to the tight relationship between LTP and learning and memory. We carried out a series studies on the effects and molecular mechanism of JZTX-V on long term potentiation and cognitive behavior. The program consists of the following four parts:Part 1 The effect of JZTX-V on long term potentiation in hippocampal dentate gyrus of anesthetized ratsObjective We investigated the effect of JZTX-V on LTP in dentate gyrus of hippocampusMethods Rats were anesthetized with urethane carbamate before being fixed in a stereotaxic frame. Three holes were sequentially drilled for an outer guide cannula, a monopolar recording electrode, and a bipolar stimulating electrode, respectively. The cannula was placed into the lateral cerebral ventricle, the recording electrode was placed in the granular cell layer of dentate gyrus and the stimulating electrode was lowered into the perforant path. An evoked response was generated by stimulating the PP at low frequency (0.033 Hz) with single constant current pulses. After ensuring a steady state baseline response, which was taken about 30 min between the last two steady state response recordings, LTP was induced using a high-frequency stimuli protocol to investigate the effect of JZTX-V on different stages of LTP.Results 10 nM and100 nM JZTX-V significantly accelerated LTP induction and there was a significant main effect of the control and JZTX-V groups (P<0.01),however, they had no significant effect on basal synaptic transmission compared with control group.Conclusion JZTX-V had significant effect on the synaptic transmission in dentate gyrus of hippocampusPart 2 The effect of JZTX-V on learning and memory impairment induced by Aβ and scopolamine in miceObjective To investigate the effect of JZTX-V on Aβ and scopolamine-induced memory impairment in miceMethods After 7 days of operation, the mice were randomly divided into four groups: control, model (Aβ1-42), donepezil as a positive control, JZTX-V groups,400 pM Aβ1-42/5ul was intracerebroventricular administration to all mice (except the control group) to lead to the senile dementia model; In another experiment, the mice were randomly divided into five groups:control, model (scopolamine), donepezil as a positive control, JZTX-V (0.0024 μM and 0.024μM) groups. Scopolamine (0.5 mg/kg) was i.p. administered to all mice (except the control group) 20 min before the training of the morris water maze, with the control group receiving the same volum of saline. After 7 days of surgery, donepezil at the doses of 5 mg/kg as a positive control, was administered to the mice by oral gavage and the control and model groups received the same volum double distilled water as that of the donepezil; the JZTX-V injected into the lateral ventricle continuously for 7 days with the control and model groups receiving the same volum of saline. The morris water maze test was performed after seven days of administration.Results In Morris water maze test, we found that after administration of Aβ1-42 or scopolamine, mice in the model group showed significant long-term memory impairments, administration of Aβ1-42 or scopolamine had a significant main effect of the control and model groups (P<0.01). In hidden-platform training, donepezil improved the different indexes as indicated in morris water maze test and JZTX-V attenuated the spatial learning deficits with respect to the model group (P<0.05). On probe trial in morris water maze test, the model mice had the decreased total distance in target quadrant and the reduced quadrant percent of total time and total distance in target quadrant compared with the control group. In comparison to the model group, donepezil and JZTX-V improved the performance as indicated by a marked increase of total distance and the quadrant percent of total distance in target quadrant.Conclusion JZTX-V had significant effect on AP and scopolamine-induced memory deficits in micePart 3 Effects of JZTX-V on transient outward potassium current and delayed rectifier potassium current in hippocampal CA1 pyramidal neuronsObjective Using patch clamp recording techniques in the whole-cell mode, we investigated the effect of inhibition of IA channels in absence and the presence of JZTX-V in hippocampal CA1 pyramidal neurons to explore the mechanism of JZTX-V on long term potentiation and cognitive behavior.Methods 2 weeks Kunming mice were used to prepare hippocampal slices and the patch clamp technique in the whole cell mode was used,-60 mV, the holding voltage. Currents were evoked by depolarizing voltage with variable prepulse voltages and interpulse intervals. Thick-walled patch electrodes had tip resistances of 2-8 MΩ. Hippocampal slices were bathed in artificial cerebrospinal fluid well-bubbled with 95% O2 and 5% CO2, the tool drug and JZTX-V included in external solution were applied through gravity perfusion administration. Recording of effects of JZTX-V on transient outward potassium current and delayed rectifier potassium current in hippocampal CA1 pyramidal neurons was performed using an EPC10 patch-clamp amplifier controlled by pulse software. Curves of the amplitude, activation, deactivation of the transient outward potassium current(IA)and delayed rectifier potassium current (IK) in hippocampal CA1 pyramidal neurons were obtained and analysed.Results Both 500 nM and 1 uM JZTX-V could inhibit IA channels in the presence of JZTX-V in hippocampal CA1 pyramidal neurons in KM mouse and had significant variation compared with the control group. However,1 uM JZTX-V had no significant effect on IK channels measured in hippocampal pyramidal neurons; The application of 1 uM JZTX-V compared with the control group affected the activation and inactivation characteristics of IA channel measured in hippocampal pyramidal neurons and the curve of IA shifted in the direction of depolarization to different degree. But it had no effect on the activation characteristics of Ik channel.Conclusion 1 uM JZTX-V could inhibit IA channels in the presence of JZTX-V in hippocampal CA1 pyramidal neurons in KM mice and affected the activation and inactivation characteristics of IA channel, the curve of IA shifted in the direction of depolarization. So the effect of JZTX-V of its improving effects on long term potentiation may be correlative with the regulation of IA channel.Part 4 Effects of JZTX-V on expression and trafficking of Kv4.2 and KChIP2 in rat brainObjective We investigated the effect of JZTX-V on expression and trafficking of Kv4.2 and Kv channel-interacting proteins (KChIP2) to explore the mechanism of JZTX-V on long term potentiation and cognitive behavior.Methods Gradient centrifugation was used to generate a good separation of plasma membranes and endosomes to investigate the effect of JZTX-V on Kv4.2 expression and trafficking and Kv channel-interacting proteins (KChIP2) expression. Enrichment of the plasma membranal compartment was confirmed by probing the different fraction with antibodies for plasma membranal maker proteins:Na/K-ATPase; EEA1 for endosomes. An injection cannula was inserted stereotaxically into the lateral ventricle used to inject JZTX-V continuously for 10 days. Rat brain tissues including the cortex and hippocampus were collected 10 days after JZTX-V administration. The western blot technique was used to determine whether JZTX-V altered the expression and the subcellular distribution of Kv4.2 and KChIP2 protein.Results The result was shown in Western Blotting:in the present study, JZTX-V was injected into the lateral ventricle continuously for 10 days with the control group receiving the same volum of saline, had failed to alter expression and the subcellular distribution of Kv4.2 and KChIP2 in the hippocampal area (P>0.05, n=3). In contrast, in the cortex of rats, JZTX-V did not alter the expression and redistribution of Kv4.2 compared with the control group (P>0.05,n=3), JZTX-V increased the expression of KChIP2 (P<0.01, n=3) and had no effect on the trafficking in the cortex (P>0.05, n=3).Conclusion In the hippocampus of rats, JZTX-V has failed to alter expression and the subcellular distribution of Kv4.2 and KChIP2(P>0.05, n=3). In contrast, in the cortex of rats, JZTX-V did not alter the expression and redistribution of Kv4.2. Compared with the control group (P>0.05, n=3), JZTX-V increased the expression of KChIP2 (P<0.01, n=3) and had no effect on the trafficking in the cortex (P>0.05,n=3). compared with the control group (P>0.05,n=3), JZTX-V increased the expression of KChIP2 (P<0.01, n=3) and had no effect on the trafficking in the cortex (P>0.05, n=3).Conclusion In the hippocampus of rats, JZTX-V has failed to alter expression and the subcellular distribution of Kv4.2 and KChIP2(P>0.05, n=3). In contrast, in the cortex of rats, JZTX-V did not alter the expression and redistribution of Kv4.2. Compared with the control group (P>0.05, n=3), JZTX-V increased the expression of KChIP2 (P<0.01, n=3) and had no effect on the trafficking in the cortex (P>0.05,n=3). |
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