Mechanisms Of Ketamine Dependence And Intervention Of Rhynchophylline

BACKGROUNDKetamine is phencyclidine,PCP(N-1-phenycyclohexy-piperidine,PCP) derivative,an N-methyl-D-aspartate(N-methyl-D-aspartate,NMDA) receptor antagonist,used to anesthetize before the operation in clinics.By now,commonly known as "K powder",it begin to first abuse pandemic in the United States since 20th century 70s.Since the 90's,with the growing problem of global scale "dance drug" abuse,ketamine quickly spread to the Asian region including mainland China.It's often abused in the entertainment places and became more and more popular as one of the new drugs.Our country is always taking strict control measures to ketamine. Ketamine was included in the national essential medicines in 1996.In recent years. Because of the increasingly serious problem of abuse,Ketamine was included in the control ofⅡpsychotropic substances in 2002.It was included in the control of one category of psychotropic psychotropic substances on January 2005.Drug-dependent complex involved in CNS central nervous system and a large number of neurons within the nucleus.So far,the formation mechanism of drug dependence has not clear.Ketamine as an NMDA receptor antagonist can non-competitive inhibit NMDA receptor activity.Because of glutamate receptors-NMDA plays an important role in the process of drug-dependent formation,NMDA receptor antagonists was considered as a way to inhibit the drug addiction for dependence symptoms.Ketamine can inhibit drug addiction and withdrawal reaction by antagonise NMDA receptor.Animal experiments show that ketamine could inhibit morphine dependence,and urging the withdrawal symptoms in mice.It's reported that ketamine was used on the treatment of opiate addiction withdrawal symptoms in clinics.However,epidemiological surveys and drug users psychological state analysis showed that joint abuse of ketamine and other mental excitatory substances,such as methamphetamine,can significantly enhance the patient's desire for addictive substances and psychological dependence.At the National Natural Science Foundation project we have finished,the original design of ketamine was as a positive control drugs to inhibit NMDA receptor and compared with traditional Chinese medicine active ingredients of anti-NMDA receptor.Our study found that ketamine and methamphetamine alone,or combined use can both induce conditioned place preference(CPP) in mice,combined use of ketamine and methamphetamine induced CPP effect was significantly prolonged than those two drugs used separately.Ketamine also increased central excitability of amphetamine.We show that ketamine has a strong psychic dependence potential on animal models.Some raleted research at domestic and abroad are basically consistent with our study results.OBJECTIVENMDA receptor and AMPA receptor exist substantial co-localization phenomenon in precursor postsynaptic density(PSD) of mature central nervous system.NMDA receptors and AMPA receptors coexist on the same synaptic.Two receptor subunits to form a specific cluster of co-localization on synaptic.This relationship of co-localization showed heterogeneity with the development of nerve cells at different stages.Compared with the NMDA receptor stability,AMPA receptor status and number are highly variable on post-synaptic membrane,which plays a very important role for regulating synaptic transmission performance.In recent years, research shows that drug dependence lead to AMPA receptor function and numbers have a series changed at early stages on neurons of mesolimbic dopamine system and related brain.Further more,it's mediated the follow-up effects and maintained it.The changes of AMPA receptors probably become a "trigger" in drug dependence.In short, for drug addiction,the function of NMDA receptors and AMPA receptors are intimately linked.We should not only study the one side while ignoring the other.Our research focus on addictive neuron-like PC12 cells caused by ketamine in vitro,studying the effect of rhynchophylline on NMDA receptor subunit(NR1)and AMPA receptor subunit(GluR2/3) on neuron-like PC12 cells,exploring the addiction mechanisms of ketamine and anti-drug addiction mechanism of rhynchophylline.METHODS AND RESULTS1.Estabishment of neuron-like PC12 cell model:PC12 rat pheochromocytoma cells(PC12 cells),separated from a rat pheochromocytoma clonal cell,which in the conditions of nerve growth factor(NGF) existence,differentiated into neuron-like cells.The morphology,structure and functions of neuron-like PC12 cells are similar to sympathetic nerve cells.In recent years,it has become a model to neural scientists to study nerve cell about neurotransmitter synthesis,storage and release,ion channels and receptors.PC12 cells containing T,L,N-type calcium channel and NMDA receptor.And NMDA receptors on PC12 cells are same with hippocampal neurons. PC12 cells also expressed AMPA receptor subunit GluR2 mRNA(GIuR-B flip mRNA) before and after differentiation.Compared to primary cultured neurons, PC12 cells easy to acquire,with high propagation speed,and the nature of stability for cell subculture.In our research,we use NGF stimulate PC12 cells,induced it differentiate into neuron-like cells,neuron-like PC12 cells not only show the various morphological characteristics of neurons,but also express NMDAR and AMPR which detected by immunohistochemical method.So we established a suitable cell model successfully.2.The neurotoxic effect of ketamine and rhynchophylline:It takes about 48 hours to couse nerve cells addicted by use opiates,amphetamines in vitro.Inducing time is important,too short the addiction model can not be established but too long nerve cells will die because of the cytotoxicity of drugs.Our experiment design inducing time is 48 hours to cause cell addicted by use ketamine.We impose different doses of ketamine in the neuron-like PC12 cells, studying the damage of cells caused by drugs,while comparing different doses of rhynchophylline.The neurotoxic effect of ketamine and rhynchophylline were measured with MTT cell viability test.From the experiment results we can see:after 48 hours,cell viability of PC12 cells showed a downward trend with the rising concentration of ketamine.When ketamine doses up to more than 1.0mmol/L,the cell viability began to decrease. 1.5mmol / L and 2.0mmol / L concentration group has significant difference compared to control group(P＜0.01),note the maximum safe dose of ketamine is 1.0mmol / L.Compared with ketamine treatment group,cell viability of all rhynchophylline treatment groups have no significant differences comared to control group.That tell us rhynchophyUine are safe for PC12 Cells at the range of 0.25～1.00 mmol / L.3.Detecting NMDA receptor subunit NR1 and the AMPA receptor subunit GluR2 / 3 protein expression on neuron-like PC12 cells by immunofluorescence technique:Immunofluorescence cytochemistry was based on the principle of antigen-antibody reaction.Firstly,marks known antigen or antibody on fluorescein and made them became fluorescence marker,then make use of such a fluorescent antibody(or antigen) as a molecular probe to check inside the cell or tissue corresponding antigen(or antibody).This experiment detected NMDA receptor subunit NR1 and the AMPA receptor subunit GluR2/3 protein expression on neuron-like PC12 cells by immunofluorescence technique.Analyzing the relationships between NMDA receptors and AMPA receptors on ketamine-addicted cells,using Chinese herbal medicinal ingredients rhynchophylline to intervene,exploring the anti-addiction mechanism of rhynchophyllineAcorrding to the different treatment for PC12 cells,we set eight groups for test: one control group;one ketamine treatment group;three rhynchophylline treatment groups,and three ketamine combine with rhynchophylline treatment groups.PC12 cells inoculated in 96-well plates with NGF-induced,when it differentiate into neuron-like cells,remove the complete medium in each holes,using PBS washing holes clean and than add basic medium as control group;Ketamine(final concentration of 1.00mmol/L) one group;Rhy three groups(final concentration of 0.25mmol/L,0.50mmol/L,1.00mmol/L).In combined three groups,Rhy was added 15 min prior to ketamine treatment.(Rhy final concentration in each group was 0.25mmol/L,0.5mmol/L,1mmol/L;Ketamine final concentration was 1mmol/L). The whole plate was divided into eight groups,each groups occupy 10 holes.Put the 96-well plates at nurturing incubator(temperature 37℃,5%CO2) after dosed with. 48 hours later,take out the plates and do immunofluorescent staining.Observed and captured images with fluorescence microscope.Using Image-Pro Plus 6.0 analysis, statistics for average optical density(Mean Density) value of each positive vision. The expression of NR1 and GluR2/3 protein get stronger as the value get higher.As it can be seen from the experimental results:①For AMPA receptor,the expression of GluR2/3 was a significant difference(P <0.01) compared with control group after exert a 1mmol/L ketamine to cells.Expression of GluR2/3 protein was significantly enhanced.The three Rhy treatment groups(Rhy of 0.25,0.50,1.00 mmol / L) were all no significantly different compare to control group.For three conbined treatment groups,data shows the expression of GluR2/3 was significantly different(P＜0.01) compared with control group in Rhy 1.00mmol / L + KETA 1.00mmol / L group,while the other two groups had no significant differences.②For NMDA receptors,only Rhy 1mmol / L group was significantly different(P＜0.05) compared with control group outside the other seven groups.③Contrasted AMPA receptor subunit GluR2/3 with NMDA receptor subunit NR1,we can see that apart from ketamine 1mmol / L group,in the other treatment groups,expression of NR1 protein at the overall level are above those of GluR2/3.The expression of GluR2 / 3 protein was significantly increased after treat cells with Ketamine 1mmol / L,which even higher than the level of NR1.CONCLUSION1.It was verified from the experiment that the neuron-like PC12 cells is a very effective tool to study glutamate receptors.2.The reasonable dose range of ketamine for PC12 Cells was noted.The security of Rhynchophylline was evaluated by it's cytotoxicity.3.AMPA receptors played a crucial role in ketamine addiction.And the anti-addiction effect of rhynchophylline are showed by influencing on AMPA receptors.DISCUSSIONFrom our research results,we give such a theory to explain:the "selective inhibitor effect" of ketamine and the "occupier effect" of Rhynchophylline.Usually,ketamine only inhibit the NMDA receptor binding sites on postsynaptic membrane(but we inferred that these binding sites must be positioned in a co-localization clusters of AMPA receptor and NMDA receptor,such as NR1-GluR2 /3 co-localization cluster),that show the effect of anti-addiction to morphine,Meth and so on.However,when stimulation reach for certain extent(such as time and dose,etc.), the dormant AMPA receptor in cells are activated again,resulting in a series of dynamic distribution.On one hand AMPA receptors have been anchored to the postsynaptic membrane continuously,on the other hand it get into the cells continuously through the reverse pinocytosis.Further more,it's produce a chain reaction leading to the so-called "silent synapses" activated(only NMDA receptors on post-synaptic membrane,but no functional AMPA receptors).So these NMDA receptors were "waken up" by "recruitment" the AMPA receptor,and possibly formed a new kind of co-localization clusters of AMPA receptor and NMDA receptor.By this way,ketamine show it's effect of addiction.Why rhynchophylline as a non-competitive NMDA receptor antagonist is same with ketamin,but not show addictive and hallucinogenic effects like it?This is because of the non-selectivity occupied by rhynchophylline.When rhynchophylline attack these binding sites,it will accupy any site as long as these sites performance characteristics of the NMDA receptor,Including the binding site on the "silent synapse"(maybe it can explain the reason for what Rhy reduced the expression of NR1).That's very different from "the ketamin style",which only accupied the sites positioned in a co-localization clusters.So when these sites have all been occupied,the dormant AMPA receptor in cells can not be recovered anymore(which explains why rhynchophylline treatment can not increase expression of GluR2/3 protein),Even if there is still a "free AMPA receptors",but no backup room of the NMDA receptor binding sites.With the Rhy intervene,ketamine can not activite AMPA receptors to induce depolarization effect of NMDA receptors,so that a series of nerve conduction signals are block,which is rhynchophylline intervention mechanism to ketamine and its mechanism of anti-addictionIn short,the addictive effect of ketamine has a great relationship with the interaction of NMDA receptors and AMPA receptors.The offset of AMPA / NMDA ratio after treated with ketamine can not be simply defined absolute increase or decrease.But we still can draw a conclusion that AMPA receptors played a crucial role in ketamine addiction.And the anti-addiction effect of rhynchophylline through such ways which by regulate the NMDA-AMPA receptor complex pathway to achieve.What kind of means by ketamine to affect the AMPA receptors? What's the difference between ketamine and rhynchophylline to the binding site of AMPA receptors and NMDA receptors on molecular conformation? All this questions await further study.Note:The statistical analysis in this thesis were processed with SPSS software. One-way ANOVA followed by least significant different test.(or Dunnett's T3 test,if equal variances not assumed)...