| ?Purpose?Recent studies have documented that antagonizing 5-HT6 R can reduce epileptic discharge and improve the learning and memory ability of chronic epileptic rats.However,the underlying mechanism is still not clear.In this study,the rat model of epilepsy induced by lithium chloride-pilocarpine(Lithium Chloride-Pilocarpine,Li Cl-PILO)was established and RNA-sequencing(RNA-Seq)was used to screen for potential 5-HT6 receptor downstream targets.Then,we used the 5-HT6 R antagonist SB271046,as well as the agonist WAY181187,to investigate the mechanism of the 5-HT6 R,which is involved in the regulation of epileptic seizures as well as learning and memory.?Method?1.The rat model of epilepsy was established by intraperitoneal injection of Li ClPILO.2.To screen the differentially-expressed genes(DEGs)among the control group,epilepsy group,and SB271046 intervention group,the RNA-sequencing(RNA-Seq)technique and the R software "LIMMA" package were used.P <0.05 and log2| Fold Change | > 2 were set to screen the DEGs.3.Western blot(WB)was used to detect the expression of 5-HT6 R and KCNQ2/3in hippocampus of Li Cl-PILO induced epilepsy rats at different time points from acute phase,latent period to chronic phase(0d,30 min,1d,3d,7d,14 d,30d)after status epilepticus.4.In chronic epileptic rat brain slices,the whole cell patch clamp technique was used to detect changes in action potential(AP),spontaneous excitatory postsynaptic current(s EPSC),spontaneous inhibitory postsynaptic current(s IPSC),and M-current in hippocampal neurons,and the effects of 5-HT6 R and M-channel activity on AP,s EPSC,s IPSC and M-current of hippocampal neurons in chronic epileptic rat brain slices.The changes in hippocampal long-term potentiation(LTP)in chronic epileptic rats,as well as the effects of 5-HT6 R and M-channel activity on hippocampal LTP in chronic epileptic rats,were detected using a brain slice electrophysiological technique.?Result?1.The results of WB showed that the expression of 5-HT6 R in the hippocampus of chronic epileptic rats was significantly higher than that in the control group,and the expression of 5-HT6 R in the hippocampus of Li Cl-PILO induced epilepsy rats was gradually up-regulated from acute phase,latent phase to chronic phase(0d,30 min,1d,7d,14 d and 30d).2.The whole-cell patch clamp test results showed that the neuron excitatory /inhibitory neurotransmitter system in the hippocampal neurons of chronic epilepsy rats is imbalanced,and the neurons changed to hyperexcited state;5-HT6 R antagonist SB271046 improved the excitatory / inhibitory nervous system imbalance and hyperexcited state of hippocampal neurons in chronic epileptic rats.3.The electrophysiological results of brain slices showed that the basic synaptic transmission function of the hippocampus of chronic epilepsy rats was impaired,and the hippocampal synaptic plasticity was significantly decreased in chronic epileptic rats;the5-HT6 R antagonist SB271046 could improve the basic synaptic transmission function and synaptic plasticity in the hippocampus of chronic epileptic rats.4.RNA-Seq results showed that there were 116 differentially expressed genes between the control group and the epilepsy group,and 235 differentially expressed genes between the epileptic group and the SB271046 intervention group.The Venn diagram results revealed that there were 32 overlapping genes,including KCNQ2.5.WB results showed that after Li Cl-PILO induced status epilepticus in rats,the expressions of KCNQ2 and KCNQ3 in the hippocampus were gradually down-regulated from the acute phase,the latent phase to the chronic phase(0d,30 min,1d,3d,7d,14 d,30d).6.The results of whole-cell patch clamp and WB showed that the hippocampal neuron M-current of chronic epilepsy rats was significantly decreased,and the expression of KCNQ2/3 was significantly down-regulated;5-HT6 R antagonist SB271046 could increase the M-current of hippocampal neurons of chronic epileptic rats and up-regulate the expression of KCNQ2 in hippocampus of chronic epileptic rats,but did not upregulate the expression of KCNQ3.7.The results of whole-cell patch clamp test showed that the excitatory / inhibitory neurotransmitter system of hippocampal neurons in chronic epileptic rats was imbalance and the neurons were transformed into hyperexcited state,M-channel opener NEM and5-HT6 R antagonist SB271046 could significantly improve the excitatory / inhibitory neurotransmitter system imbalance and hyperexcited state of hippocampal neurons in chronic epileptic rats,while M-channel inhibitor XE991 could block the above effects of SB271046.8.The electrophysiological results of brain slices showed that hippocampal synaptic plasticity significantly decreased in chronic epileptic rats,M-channel openers NEM and5-HT6 R antagonist SB271046 could significantly improve hippocampal synaptic plasticity in chronic epileptic rats,while M-channel inhibitor XE991 could block the above effects of SB271046.?Conclusion?The excitatory/inhibitory neurotransmitter system in the hippocampal CA1 area of chronic epileptic rats is disrupted,the neurons become hyperexcited,and hippocampal synaptic plasticity is significantly impaired;SB271046,a 5-HT6 R antagonist,can improve the balance of excitatory / inhibitory neurotransmitter systems in hippocampal neurons,reduce neuronal excitability,and improve long-term potentiation in chronic epileptic rats.The mechanism may be through the activation of M-channels. |
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