| Cholinergic regulation is the most important neuroregulatory mechanism in the central nervous system.The neurotransmitter acetylcholine(Ach)acts on its target alkaloid bacteria receptors and nicotinic receptors for cholinergic regulation.Nicotine receptors,also known as nicotine receptors(nAChR),exist in homomer or heteromer forms,of which the most abundant homomer is ?7-nAChR,which is one of the major nAChR subtypes in the brain,especially in the hippocampus.?7-nAChR belongs to the pentamer-ligand gated ion channel superfamily,which responds to its agonists(such as Ach,choline,etc.)by opening the internal channels of permeable cations,i.e.rapid depolarization of the membrane opens the ?7-nAChR channel,leading to the influx of sodium and calcium ions.A large number of compounds can modulate the function of ?7-nAChR by binding to the allosteric sites of the receptor other than the normal sites of binding agonists and antagonists.Among these compounds,?7-nAChR PAM is a compound that enhances the current of ?7-nAChR in the presence of an agonist such as Ach opening the receptor channel.?7-nAChR PAMS is a more promising therapeutic tool than agonists.As an important central target,?7-nAChR mediates many physiological functions and is involved in the occurrence and development of many neurological diseases.However,whether the enhancement of ?7-nAChRs expression and function can trigger neuroprotection or induce neurotoxicity is still controversial.The conditions of neuroprotection or neurotoxicity are still unclear.This would greatly impede the development of drugs that target the regulation of ?7-nAChRs.Solving this problem will enable people to have a new understanding of the function and pharmacology of ?7-nAChRs as well as its role in disease pathogenesis and treatment,which is of great significance.This topic has carried on the thorough discussion to this scientific problem.The main objective of this study was to investigate the effect of enhanced ?7-nAChR on the function of hippocampal neurons.NS1738,A positive allosteric modulator of ?7-nAChR,and PNU120596 plus a selective agonist(choline)of ?7-nAChR were used to investigate the effects of chronic A? treatment on primary cultured hippocampal neurons.In this experiment,three different concentrations of ?7-nAChR(1 ?M),10 ?M,and 100?M)were set,and the experimental conditions were controlled under the condition that the?7-nAChR channel was not open or open,respectively.Experimental indicators include single-cell patch clamp recording,determination of cellular Lactate Dehydrogenase(LDH)release,Western blotting,immunofluorescence,and flow cytometry.The experimental results are as follows:1.MAP2 and Hochest immunofluorescence staining were used to identify the degree of maturity of primary cultured hippocampal neurons.The results showed that the primary hippocampal neurons were mature after 7 days of culture and could be used in experiments.2.Single cell patch clamp recording showed that the threshold concentration(10 ?M)of choline could not record the current response,and the upper threshold concentration(100?M)of choline could record a small inward current response,and 10000 ?M choline could induce a typical inward current response.3.Compared with the control group(non-treated group),LDH secretion of primary cultured hippocampal neurons was decreased after treated with 1 ?M choline and 5 ?M NS1738 for 7 days(P<0.05).The protein expression ratio of Bax/Bcl-2 in primary cultured hippocampal neurons was decreased(P<0.05).The mitochondrial membrane potential(??m)of primary cultured hippocampal neurons increased(P<0.05).Similar results were obtained after 7 days of treatment with 1 ?M choline and another PAM,PNU120596(1?M).These results indicated that choline reduced the natural apoptosis of primary cultured hippocampal neurons in rats when the concentration of choline below the threshold could not open the ?7-nAChR channel.PAM doesn't work at this point.4.Compared with the control group(non-treated group),LDH secretion of primary cultured hippocampal neurons was significantly decreased after treated with 10 ?M choline and 5 ?M NS1738 for 7 days(P<0.01).Primary cultured hippocampal neurons ??m was significantly increased(P<0.001).Similar results were obtained when 10 ?M choline was treated with another PAM,PNU120596(1 ?M),for 7 days.These results showed that chronic treatment of 10 ?M choline did not produce neurotoxicity in normal cultured neurons,but reduced neuronal apoptosis,which was similar to the effect of choline plus PAM,and there was no statistical difference between the two groups.The neuroprotective effect of choline was not enhanced by PAM even at the threshold(10 ?M).5.Compared with the control group(untreated group),no significant changes in LDH levels were observed in the primary cultured hippocampal neurons treated with 100 ?M choline for 7 days,but LDH secretion was significantly increased in the primary cultured hippocampal neurons treated with 100 ?M choline and 10 ?M NS17382 for 7 days(P<0.001).The enhancement effect was blocked by ?7-nAChR blocker(MLA).LDH secretion of primary cultured hippocampal neurons was significantly increased after treatment with 100 ?M choline and 1 ?M PNU120596 for 7 days(P<0.01).These results indicate that PAM can induce significant neurotoxic effects by enhancing the function of?7-nAChR in the presence of ?7-nAChR channel at a concentration above the choline threshold.6.The effect of PAM was further examined using chronic A? neurotoxic cell model.Treatment with 100 n M A? for 10 days resulted in hyperexcitability of cultured primary cultured hippocampal neurons,which was inhibited by the ?7-nAChR inhibitor MLA.However,no hyperexcitation was observed in ?7-nAChR knockout mice(P<0.05).The results showed that the hyperexcitability of primary cultured hippocampal neurons induced by A? treatment was mediated by ?7-nAChRs.7.After chronic treatment with A? for 7 days,LDH secretion was increased in A?treatment group compared with the control group(P<0.05).A? and PAM treated for 7 days,both choline(10 ?M)+NS1738+A? group and choline(10 ?M)+PNU120596+A? group significantly increased the secretion of LDH in primary cultured hippocampal neurons(P<0.05).Compared with choline+A? group,the secretion of LDH of primary cultured hippocampal neurons in choline+NS1738+A? group and choline+PNU120596+A? group was significantly increased(P<0.01).The results showed that the function of ?7-nAChRs was enhanced by chronic treatment of A?,and the toxic effect of PAM occurred.8.When treated with 10 ?M choline and PAM for 7 days,the number of action potential(AP)in choline+NS1738 and choline+PNU120596 groups increased compared with the control group(non-treated group)(P<0.05).Compared with the choline group,the number of AP in the choline+NS1738 group was increased at 50 p A depolarization,and that in the choline+PNU120596 group was increased at 40-50 p A depolarization(P<0.05).The results showed that 10 ?M choline co-treated with PAM could improve neuronal excitability.These results suggest that the use of PAM to enhance ?7-nAChR function produces neuroprotection or neurotoxicity depending on whether the ?7-nAChR channel is turned on.Activation of ?7-nAChR(at low concentrations of agonists)in the absence of channel activation reduced the natural apoptosis of primary cultured rat hippocampal neurons,but PAM did not appear to have an effect.The mechanism of this neuroprotective effect may be caused by the activation of ?7-nAChR-coupled intracellular signaling pathway,which deserves further investigation.Activation of ?7-nAChR(at higher concentrations of agonists)in the presence of open channels does not,by itself,induce neurotoxic reactions,possibly due to the rapid closure kinetics of activated ?7-nAChR channels.However,increased expression and function of ?7-nAChR with PAM or chronic treatment with A?resulted in neurotoxic effects,which involved enhanced ?7-nAChR mediating neuronal hyperexcitability and neurotoxic effects. |
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