| Epilepsy is a common neurological disease in clinic,whose main manifestation is temporary dysfunction of the brain due to the abnormal discharge of neurons in the brain.Its causes are complex.In recent years,the changes of the structure and function of the brain,such as increased excitatory neurotransmitter and change of synaptic function,have been found in the area of epileptic seizures.And these changes can make the epilepsy show the characteristics of repeated attacks,which is one of the causes of frequent seizures and refractory.Dysfunction of glutamate receptor may be one of the causes.Neuropeptide Y(NPY)is a polypeptide composed of 36 amino acid residues which widely distribute in peripheral nervous system and central nervous system.And in the central nervous system,it is mainly in the thalamus,hypothalamus,hippocampus,cerebral cortex and other parts,especially the highest density in the hippocampus(Ledri M et al.,2011).In recent years,studies have shown that NPY is a potent endogenous anti epileptic factor(Cardoso A et al.,2010),and it is reported that NPY plays an important role in the regulation of neuronal excitability and inhibition of seizures.But until now,the mechanism of NPY inhibiting seizures is still not well known(S?rensen AT et al.,2009).In the past,most of the studies on the mechanism of action were limited to the protective effect of NPY on neurons.It is suggested that NPY inhibits the N type calcium channel of presynaptic membrane mainly through Y2 and Y5 receptors,which can reduce the calcium influx,then to reduce the release of excitatory neurotransmitter glutamate and to inhibit the excitatory transmission of postsynaptic membrane and play a role in inhibiting seizures(Silva AP et al.,2007).There are few studies on the middle part of how to induce calcium channel change and whether there are other mechanisms to exert its antiepileptic effect.The change of glutamate receptor function may lead to the imbalance between excitability in vivo and inhibitory neurotransmitters,which can induce epileptic seizures and neuronal damage.AMPA receptor is one of three receptors of ionotropic glutamate,which is an important class of excitatory amino acid receptors besides NMDA receptors.It mainly mediates the transmission of the fast excitatory transmitter and widely distributes in the central nervous system.Its over-activation can enhance the excitability of neurons and play an important role in the pathogenesis of epilepsy(Yue Hu et al.,2012).We hypothesize whether NPY might inhibit the function of epileptic seizures by inhibiting the function of AMPA receptor.In this study,we select model of epileptiform discharges in rat hippocampal neurons,detect the effects of NPY on epileptiform discharges in hippocampal neurons of rats by using the Patch clamp technique,detect the changes of AMPA receptor glu2 subunit mRNA and total protein and protein phosphorylation in hippocampal neurons under epileptiform discharge and effect of NPY on it,and detect the NPY related apoptosis index after epileptiform discharges in hippocampal neurons.At last,we verify whether NPY can act on the hippocampal neurons of AMPA receptor to inhibit the seizure of animals by animal experiment,and to explore the mechanism of NPY in regulating the biological activity of AMPA receptor,reducing the damage of excitatory neurotransmitter to neurons and epileptic seizure.This experiment is divided into four parts.Part one Effects of neuropeptide Y on AMPA current in ippocampal neurons of epileptic dischargeObjective:Use the primary cultured hippocampal neurons of rats as the research object,and treatment with extracellular solution of Mg2+ free for 3h.And then we continued to culture them with normal extracellular fluid,and proceeded with whole cell patch clamp experiment,to compare the effects of neuropeptide Y on AMPA currents in hippocampal neurons of rats in state of epileptic discharges of hippocampal neurons and to investigate the effect and mechanism of neuropeptide Y on the function of AMPA receptor.Method:We selected neonatal SD rats within 24 hours as the experimental object,and had the hippocampal neurons for primary culture.Hippocampal neurons were divided into control group,model group(Mg2+ free group)and NPY(1?m)intervention group when culture in vitro for 12 d.Control group was treated for 3h after changed with normal extracellular fluid,model group was treated for 3h with magnesium free extracellular fluid,and NPY group first was incubated for 0.5h with NPY(1?mol/L)cell culture medium,and then was treated for 3h with magnesium free extracellular fluid.BIBP3226+NPY intervention group first were incubated for 0.5h with cell culture medium containing NPY Y1 receptor blocker BIBP3226(1?mol/L),and then incubated for 0.5h after adding NPY with a final concentration of 1?mol/L.Finally,they were treated for 3h after adding the magnesium free extracellular fluid.Whole cell patch clamp technique was used to detect the action potential of control group,model group(Mg2+ free group)and NPY(1?m)intervention group.It was also used to detect the neuron IAMPA of control group,model group(Mg2+ free group),NPY(1?m)intervention group and BIBP3226+NPY intervention group,and then calculated the peak current density.Result:Patch clamp experiments showed that the cells in the model group had stable abnormal action potentials,the frequency and amplitude of action potential were significantly higher than those in control group,and the frequency and amplitude of action potential of NPY(1?m)control group were significantly lower than those in model group(P<0.05).Neuron IAMPA peak density of model group was significantly lower than it in the control group(P<0.05),Neuron IAMPA peak density of NPY(1?m)control group is significantly higher than it in the model group(P<0.05),Neuron IAMPA peak density of BIBP3226 +NPY intervention group was significantly lower than it in the NPY control group(P<0.05).Conclusion:The cells in the model group shows abnormal action potential after the treatment for 3h with magnesium free extracellular fluid,which can simulate the epileptiform discharge,and hippocampal neurons are pretreated by NPY could inhibit this discharge.The epileptiform discharges of hippocampal neurons can appear IAMPA decrease of the neuronal,and NPY can reduce the inhibition of epileptiform discharges on neuronal IAMPA,to avoid the excessive decline of the neuronal IAMPA.NPY may play an important role in antiepileptic by regulating the function of AMPA receptor in hippocampal neurons.The effect of NPY is inhibited by the addition of NPY receptor blocker BIBP3226,which suggests that NPY can play an important role in the regulation of AMPA receptor function by activating Y1 receptor.Part two Effects of neuropeptide Y on the AMPA receptor GluR2 subunit of epileptiform discharges state in hippocampalneuronsObjective:Regard Primary culture hippocampal neurons of rats as the research objectand making theepileptiform discharges in hippocampal neurons models,we detected the effect of AMPA receptor GluR2 subunit function in NPY hippocampal neurons by using RT-PCR and Western blot.Method:Selected neonatal SD rats within 24 hours as the experimental object,and have the hippocampal neurons for primary culture.When they were cultured in vitro for 12 d,detected the expression of AMPA receptor subunit GluR2 in hippocampal neurons by immunofluorescence assay.And the expression of GluR2 mRNA in hippocampal neurons and the changes of total protein and phosphorylation of GluR2 subunit were detected by RT-PCR and Western blot.Result:Immunofluorescence staining showed normal appearance of neurons in control group,and the expression of GluR2 in cell axon was obvious.The expression of GluR2 was significantly decreased in the model group,and the expression of GluR2 in NPY group was higher than that in model group.RT-PCR detection showed that the GluR2 mRNA of model group was significantly lower than that of control group(P<0.05);the GluR2 mRNA of NPY control group was significantly higher than that of model group(P<0.05);the GluR2 mRNA of BIBP3226+NPY control group was significantly lower than that of NPY group(P<0.05).Western blot showed that the protein content of GluR2 subunit in model group was slightly lower than that in control group(P>0.05);the phosphorylation level of GluR2 subunit in model group was significantly higher than that in control group(P<0.05);the phosphorylation level of GluR2 subunit in NPY control group was significantly lower than that in model group(P < 0.05);the phosphorylation level of GluR2 subunit in BIBP3226+NPY control group was significantly higher than that in NPY group(P<0.05).Conclusion:The cause for the inhibition of AMPA receptor function in hippocampal neurons during epileptiform discharges may be that the replication of GluR2 mRNA was inhibited and protein phosphorylation.This is due to the decrease of the AMPA receptor of the normal GluR2 subunit on the surface of neurons and the function changes.Neuropeptide Y can inhibit the excessive inhibition of epileptiform discharges on AMPA receptor GluR2 subunit in hippocampal neurons;this may be one of the mechanisms of its inhibition of epilepsy.Neuropeptide Y may regulate AMPA receptor GluR2 function by activating Y1 receptors in hippocampal neurons.Part three Effects of neuropeptide Y on AMPA receptor to reduce neuronal apoptosis in hippocampal neurons after epileptiform dischargesObjective:Use the primary cultured hippocampal neurons of rats as the experimental object to investigate whether the apoptosis of hippocampal neurons occurred after epileptiform discharges and whether neuropeptide Y could inhibit the apoptosis of hippocampal neurons by AMPA receptor.Method:Selected neonatal SD rats within 24 hours as the experimental object,detected the apoptosis of neurons in each group by using TUNEL method,and detected the expression of caspase-3 protein and mRNA levels in neurons of each group.Result:TUNEL test showed that the model group being compared with the control group,the positive neurons of hippocampus neurons was significantly increased,and NPY intervention group was significantly lower than the model group(P<0.05).The protein content of Caspase-3 in the model group was significantly higher than that in the control group,and the mRNA was significantly higher than that in the control group(P<0.05).The level of caspase-3 protein and mRNA expression in NPY intervention group were significantly lower than those in the model group(P<0.05).The level of caspase-3 protein and mRNA expression in CNQX+NPY intervention group were significantly higher than those in the NPY group(P<0.05).Conclusion:Neuronal apoptosis may occur after epileptiform discharges in hippocampal neurons,which is consistent with the performance of animal model of acute epilepsy.Neuropeptide Y can inhibit neuronal apoptosis induced by epileptiform discharge which has a protective effect on neurons.The effect of neuropeptide Y is inhibited after the AMPA receptor antagonist CNQX is inhibited,it is suggested that neuropeptide Y may play an important role in the protection of neurons against apoptosis by affecting the function of AMPA receptor in hippocampal neurons.Part four Effects of neuropeptide Y on GluR2 subunit after acute seizure in rats and behavioral changesObjective: Select SD rats as the experimental object;by observing the acute seizure in rats induced by injecting kainic acid in the intracerebroventricular after injecting NPY and BIBP3226+NPY separately in CA3 area in hippocampal,we studied the behavioral changes of rats and the changes of GluR2 subunit function in CA3 area of hippocampus,and investigated the effects of neuropeptide Y on epileptic seizures in rats by having the AMPA receptor GluR2 subunit of hippocampal neurons in rats as the target spot.Method: SD rats were divided into four groups(10 rats in each group of)and we observed the behavior of rats in each group.According to Racine grading standard,the degree of epileptic seizure was evaluated,such as ?-?level for mild episodes and ?or more as severe episodes.After intraventricular injection for 12 hours,the total protein and the phosphorylation of GluR2 subunit in hippocampal CA3 area of 5 rats in each group were detected by Western blot method.RT-PCR method was used to detect the expression of GluR2 mRNA in hippocampal CA3 area of each group.Result: Within 20 minutes,the rats in the model group had epileptic severe seizures;the NPY intervention group had mild episode;the level of epileptic seizures of the BIBP3226+NPY intervention group was significantly higher than that of NPY group(P<0.05);no seizure occurred in group control.After 12 hours of injection,the protein content of GluR2 subunit in the model group was not statistically different from the control group(P>0.05),the phosphorylation level of GluR2 subunit was significantly higher than that of control group,and GluR2 mRNA was significantly lower than that of control group(P<0.05).The phosphorylation level of GluR2 subunit in NPY intervention group was significantly lower than that of model group;GluR2mRNA was significantly higher than that of model group(P<0.05).The phosphorylation level of GluR2 subunit in BIBP3226+NPY intervention group was significantly higher than that of NPY group;GluR2mRNA was significantly lower than that of NPY group(P<0.05).Conclusion: Intracerebroventricular injection of kainic acid can induce epileptic seizure,and can inhibit the expression of AMPA receptor subunit GluR2 in hippocampus.NPY can inhibit the epliepsia outbreak.The mechanism mayi inhibit the function of GluR2 subunit by activating the Y1 receptor of hippocampal neurons to inhibit the occurrence of epilepsy. |
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