The Role And Mechanism Of NMDA Receptors In Stress-induced Impairment Of Synaptic Plasticity And Cognition

Stress refers to the non-specific neuro-endocrine-immune response when the body is stimulated by the internal and external environment,severe stress can impair synaptic plasticity and cognition.N-methyl-D-aspartic acid(NMDA)receptors is an important glutamate receptor that mediates synaptic plasticity and cognitive function.However,there are still contradictions in the research on NMDA receptors in stress-induced synaptic plasticity and cognitive impairment.Some researches have shown that hyperfunction of NMDA receptors is an important factor in stress-induced impairment of synaptic plasticity and cognitive dysfunction,while other researches suggest that the function of NMDA receptors is insufficient under stress.D-serine is one of the important factors that regulate the function of NMDA receptors and its role in stressinduced synaptic plasticity and cognitive impairment remained unclear.Therefore,this study first explored whether over-activation or hypofunction of NMDA receptors is involved in impairment of synaptic plasticity and cognition by stress,and then observed the role of D-serine metabolic pathway in dysfunction of NMDA receptors;finally,based on synaptic plasticity,compound 297 was screened from natural compounds which can improve stress-induced impairment of synaptic plasticity,and further investigated its regulation on the function of NMDA receptors and D-serine metabolic pathway in its improvement on stress-induced emotional disorders and cognitive impairment.Chapter 1.The role and mechanism of NMDA receptors in stress-induced impairment of synaptic plasticity and cognitionSynaptic plasticity is the neurobiological basis of learning and memory and is widely used in research on learning and memory.Long-term synaptic plasticity mainly includes long-term potentiation(LTP)and long-term depression(LTD).In order to clarify the effects of acute and chronic stress on the synaptic plasticity and cognitive function,several acute(acute restraint and single injection of CORT)and chronic stress(chronic restraint and chronic injection of CORT)models were first established.In vivo electrophysiological experiments were used to record population spikes(PS)to reflect changes in hippocampal synaptic plasticity(LTP and LTD),novel object recognition test,novel location recognition test and nesting test were used to evaluate the cognition of mice.Then NMDA receptors antagonists or NMDA receptors co-agonists were adopted to inhibit or enhance the function of NMDA receptors and observe their effects on hippocampal LTP in mice,aiming to explore whether over activation or hypofunction of NMDA receptors is involved in impairment of LTP and cognition by stress.Finally,high performance liquid chromatography(HPLC)was used to determine the level of neurotransmitters such as D-Serine and Western Blots(WB)was used to determine the expression of proteins such as NMDA receptors,aiming to discover the possible mechanism of stress-induced synaptic plasticity and cognitive impairment.Section 1 The effects of stress on synaptic plasticity and cognition in miceElectrophysiological results showed that both acute(single injection of CORT,acute restraint)and chronic stress(21 days chronic injection of CORT and 21 days chronic restraint)significantly impaired hippocampal LTP of mice,Acute stress(single injection of CORT)significantly impaired hippocampal LTD,indicating that both acute and chronic stress can impair synaptic plasticity.The results of location and object recognition experiments showed that the discrimination index of novel location was significantly reduced after single injection of CORT,the discrimination index of novel object were significantly lower than those of normal mice after 21 days chronic injection of CORT and 21 days chronic restraint,suggesting that both acute and chronic stress can cause cognitive impairment.Nesting scores were significantly decreased after 21 days chronic injection of CORT,suggesting that chronic stress can impair nesting ability.The results of CORT determination showed that both single and and chronic injection of CORT significantly increased the levels of serum corticosterone in mice,which is consist with the increased level of corticosterone by behavioral stress.These above results suggest that acute and chronic stress can impair synaptic plasticity and cognition.Subsequent experiments adopted single injection of CORT as an acute stress model and 21 days chronic injection of CORT as a chronic stress model to observe the effects and mechanisms of acute and chronic stress on the function of NMDA receptors.Section 2 The effects of CORT on the function of NMDA receptorsCORT models were adopted and NMDA receptors antagonists or NMDA receptors agonists were used to inhibit or enhance the function of NMDA receptors to observe their effects on hippocampal LTP,aiming to explore the changes of the function of NMDA receptors after stress.Results showed that the hippocampal glutamate level was significantly higher than that of normal mice after single and chronic injection of CORT,suggesting that glutamate may over-activate NMDA receptors to mediate excitotoxicity.Therefore,we first observed whether different NMDA receptors antagonists could alleviate impairment of LTP by CORT.Results showed that intracerebroventricular(i.c.v.)injection NMDA receptors antagonist D-AP5,selective GluN2A antagonist PEAQX and selective GluN2B antagonist Ro25-6981 had no significant effect on impairment of LTP by CORT and there is a tendency to further aggravate the impairment of LTP by chronic injection of CORT.These above results suggest that CORT may not cause excessive activation of NMDA receptors and excitotoxicity.Then we observed whether using different vehicles to enhance the function of NMDA receptors could alleviate impairment of LTP by CORT.Results showed that injection(i.c.v.)of endogenous NMDA receptors co-agonist D-serine,L-serine(the precursor of D-serine)and glycine(NMDA receptors co-agonist)significantly alleviated impairment of LTP by CORT;intraperitoneal(i.p.)injection of D-amino acid oxidase(DAAO)inhibitor MPC which can reduce the clearance of endogenous D-serine,also alleviated impairment of LTP caused by single injection of CORT.Injection(i.p.)of 1 mg/kg NMDA receptors glycine site antagonist L-701,324 blocked the improvement of Dserine and MPC on impairment of LTP by CORT.The above results suggest that hypofunction of NMDA receptors may be an important reason of impairment of LTP by CORT.In order to further verify whether whether CORT would specifically cause the hypofunction of GluN2A or GluN2B,we observed the combined use of D-serine and PEAQX or Ro25-6981 selectively enhances the effect of GluN2A or GluN2B on the impairment of LTP by CORT.The results showed that the combined use of D-serine and Ro25-6981 or PEAQX significantly alleviate hippocampal impairment of LTP by CORT and the improvement has a decreasing trend compared with D-serine alone without statistical difference,suggesting that the function of GluN2A and GluN2B are significantly reduced after injection of CORT.These above results suggest that hypofunction of NMDA receptors may be involved in impairment of hippocampal LTP by stress,the hypofunction NMDA receptors after chronic stress is more serious than acute stress,suggesting the mechanisms of hypofunction of NMDA receptors may be differenent after acute and chronic stress.Section 3 The role of D-serine metabolic pathway in hypofunction of NMDA receptors by corticosteroneBy observing the protein expression of NMDA receptors and the changes of D-serine metabolic pathway,we explored the role of NMDA receptors and D-serine metabolic pathway in hypofunction of NMDA receptors after injection of CORT.After single injection of CORT,the protein expression of hippocampal GluN2A and GluN2B were not changed compared with normal mice.After chronic injection of CORT,the protein expression of GluN2B in the hippocampus was significantly increased compared with that of normal mice and the protein expression of GluN2A has a higher tendency than normal mice.In the D-serine metabolic pathway,single injection of CORT had no significant effect on the levels of D-serine and its precursor L-serine,serine racemase(SR)and DAAO,while the protein expression of vesicle-associated membrane protein 2(VAMP2)and Na’-dependent alanine-serine-cysteine transporter-1(ASC-1)were significantly decreased in the hippocampus;injection(i.c.v.)of Disoleucine,the selective activator of ASC-1 antiporter activity which can promote the release of D-serine,significantly alleviate hippocampal impairment of LTP caused by single injection of CORT.Chronic injection of CORT had no significant effect on the protein expression of VAMP2,ASC-1 and DAAO while the content of D-serine and the protein expression of SR were significantly decreased.These above results suggest that hypofunction of NMDA receptors in response to decreased release of D-serine may be the main reason for impairment of LTP caused by aucte stress,while hypofunction of NMDA receptors in response to decreased content of D-serine may be the main reason for impairment of LTP caused by chronic stress.Chapter 2.The role of NMDA receptors in the improvement of 297 on stressinduced cognitive impairmentAntidepressant can improve some mental symptoms caused by stress,but it has no significant effect on the cognitive impairment caused by stress.Therefore,there is an urgent need to develop drugs that can simultaneously improve emotional disorders and cognitive impairment caused by stress.The main ingredients of Uncaria rhynchophylla,a traditional Chinese medicine,are indole alkaloids,former researches showed that indole alkaloids could alleviate cognitive impairment caused by many factors,such as amyloid β-protein(Aβ)and aluminum chloride,it was also reported that indole alkaloids has antidepressant effects in stressed mice,suggesting that indole alkaloids may be potential anti-stress drugs.Our previous electrophysiological experiments also found that compound 297 from indole alkaloids alleviate impairment of LTP and LTD caused by CORT.However,whether compound 297 could improve stress-caused emotional disorders and cognitive impairment by regulating the function of NMDA receptors and D-serine metabolic pathway still remained unclear.A 28 days chronic unpredictable mild stress(CUMS)model was established,and 20 mg/kg compound 297 from indole alkaloids,its isomers 315 and positive control fluoxetine were administered by administrated(i.g.)one week before modeling.All drugs were administrated until the end of behavioral experiments.The physical strength of the mice was evaluated by rotarod test;open field test and elevated plus maze evaluation were adopted to evaluate the anxiety-like behavior of mice,sucrose preference test was adopted to evaluate depression-like behavior of mice,novel object recognition test and Morris water maze were adopted to evaluate the function of learning and memory of mice.Finally,HPLC was used to determine the level of neurotransmitters such as D-Serine and WB was used to determine the expression of proteins such as NMDA receptors,aiming to explore whether compound 297 could improve CUMS-caused emotional disorders and cognitive impairment by regulating the function of NMDA receptors and D-serine metabolic pathway.Section 1 The effects of natural compounds on impairment of hippocampal synaptic plasticity by corticosteroneThis part of the experiment first observed the effects of natural compounds on impairment of LTP and LTD caused by single injection of CORT,and then observed the dose-effect relationship of compuound 297,which simultaneously alleviated impairment of LTP and LTD,on impairment of LTP caused by single injection of CORT when given by different doses and ways.Results showed that single injection(i.c.v.)of compound 297 significantly alleviated hippocampal impairment of LTP and LTD,suggesting that compound 297 had a better improvement on impairment of synaptic plasticity by CORT.Results showed that injection(i.c.v.)of compound 297 and 327 significantly alleviated hippocampal impairment of LTP by CORT;single injection(i.p.)of compound 297 and 315 significantly alleviated hippocampal impairment of LTP by CORT;single administration(i.g.)of compound 297,315 and 327 significantly alleviated hippocampal impairment of LTP by CORT;seven consecutive days administration(i.g.)of compound 297,315,327 and 307 significantly alleviated hippocampal impairment of LTP by CORT;on the 7th day of drug administration,compound 297 and 315 had no significant effect on the body weight of mouse while compound 327 and 307 significantly reduced the body weight of mice,suggesting that compound 297,315,327 and 307 alleviated hippocampal impairment of LTP by CORT while compound 327 and 307 may have toxic effects when administered chronically.These above results showed that central and peripheral administration of compound 297 alleviated impairment of hippocampal synaptic plasticity by CORT,suggesting that compound 297 from indole alkaloids may be a potential anti-stress drug.Section 2 The role of NMDA receptors in the improvement of compound 297 on stress-induced cognitive impairmentA 28 days CUMS model was established to verifying whether compound 297,which can alleviate impairment of synaptic plasticity caused by single injection of CORT,could improve CUMS-induced emotional disorder and cognitive impairment by regulating the function of NMDA receptors and D-serine metabolic pathway.1.The effect of compound 297 on the emotion and cognition of CUMS miceResults showed that the body weight of CUMS mice was significantly decreased compared with the normal mice,297 significantly alleviated the weight loss caused by CUMS;in the rotarod test,the retention time of CUMS mice was significantly decreased compared with the normal mice,297 significantly increased the retention time of CUMS mice;suggesting that 297 significantly alleviated the decrease in body weight and physical strength caused by CUMS.In the open field test and elevated plus maze test,the exploratory behavior of CUMS mice was significantly decreased compared with the normal mice,297 significantly enhanced the exploratory behavior of CUMS mice,suggesting that 297 significantly alleviated the anxiety-like behaviors caused by CUMS.In the sucrose preference test,the sucrose preference of CUMS mice was significantly decreased compared with the normal mice,297 and fluoxetine significantly increased the sucrose preference of CUMS mice,suggesting that 297 significantly alleviated depression-like behaviors caused by CUMS.In the Morris water maze,the escape latency was significantly increased while the time in the target quadrant and number of crossing the plate were significantly decreased in CUMS mice,297 significantly reduced the escape latency of CUMS mice,297 and fluoxetine significantly increased time in the target quadrant of CUMS mice,297 had a tendency to increase number of crossing the plate of CUMS mice,suggesting that 297 significantly improved the impairment of spatial memory caused by CUMS.These above results suggest that compound 297 from indole alkaloids simultaneously improved emotional disorders and cognitive impairment caused by CUMS.2.The regulation of compound 297 on the function of NMDA receptors in CUMS miceResults showed that the level of serum CORT of CUMS mice was significantly increased compared with the normal mice,297 significantly reduced the level of serum CORT of CUMS mice;suggesting that 297 alleviated the stress response caused by CUMS.The level of hippocampal glutamate of CUMS mice was significantly increased compared with the normal mice,297 significantly reduced the level of hippocampal glutamate of CUMS mice.The protein expression of hippocampal GluN2A and GluN2B of CUMS mice were increased compared with the normal mice,297 and fluoxetine could significantly reduce the protein expression of hippocampal GluN2A and GluN2B of CUMS mice.The hippocampal content of D-serine and the protein expression of its synthetase SR were significantly reduced of CUMS mice compared with the normal mice while the protein expression of ASC-1,VAMP2 and DAAO remained unchanged,297 significantly increased the hippocampal protein expression of SR of CUMS mice and promoted synthesis of D-serine.These above results suggest that compound 297 from indole alkaloids recovered the function of NMDA receptors and D-serine metabolic pathway of CUMS mice.In summary,the main conclusions of this study are as follows:1.Both acute and chronic stress significantly impaired hippocampal synaptic plasticity and cognition in mice,hypofunction rather than hyperfunction of NMDA receptors may be involved in stress-induced impairment of synaptic plasticity and cognition.2.Abnormal D-serine metabolic pathway may be involved in the hypofunction of NMDA receptors by stress.aucte stress caused decreased release of D-serine While chronic stress caused decreased content of D-serine.Regulation of NMDA receptor dysfunction caused by abnormal D-Serine metabolic pathway may be an effective strategy to prevent and treat cognitive impairment caused by stress.3.compound 297 can alleviate impairment of synaptic plasticity by CORT and also simultaneously improved emotional disorders and cognitive impairment caused by CUMS,which may be associated with restoring the function of NMDA receptors and D-serine metabolic pathway;suggesting that compound 297 may be a potential antistress drug.