Effect And Mechanism Of Blocking TRPV4 On Methamphetamine-Induced Learning And Memory Impairment And Pathological Memory

Background:Impairment of learning and memory is a major feature of people with methamphetamine（METH）use disorders,which not only disrupts Methamphetamine life but also affects the effectiveness of treatment and is a major cause of sustained drug relapse.Therefore,the intervention of learning and memory is a potential way to reduce drug dependence,but the mechanism is not clear.In recent years,transient receptor potential vanilloid 4（TRPV4）,as a highly selective Ca²⁺channel protein,has been revealed to be a key regulator of stress injury and neuroinflammatory progression in the brain,blocking TRPV4 may alleviate drug-related learning and memory impairment in a variety of diseases.It has been proved that METH neurotoxicity is the neurobiological basis of impairment of learning and memory,which involves many mechanisms such as stress,apoptosis and inflammation.Our previous studies showed that METH induced the up-regulation of TRPV4 expression in the hippocampus of mice,but the relationship between the up-regulation of TRPV4 expression and METH-induced nerve injury was not reported.Based on this,whether the METH-induced learning and memory impairment can be ameliorated by blocking TRPV4 and exploring its possible pathway mechanism is the main starting point of this study.On the other hand,the theory of drug dependence holds that the essence of addiction is the pathological memory formed by the usurpation of normal learning and memory process by drugs.Previous studies have shown that reinforcement of normal learning and memory can help to suppress pathological memory of addiction.It has been reported that trpv4camkiiαpathway is involved in pathological memory by regulating Synaptic plasticity,and down-regulation of Camk IIαexpression can reduce drug dependence and relapse behavior,however,the direct relationship between upstream TRPV4 and drug-related pathological memory remains unclear.Therefore,on the basis of confirming that TRPV4 regulates normal learning and memory,this study further elucidates the effect of blocking TRPV4 on different stages of METH pathological memory and its possible mechanism.Objective:To clarify the role of TRPV4 in METH-induced learning and memory impairment and its possible pathway mechanism and to explore the role and mechanism of TRPV4 in METH-induced pathological memory.This study provides a theoretical basis for TRPV4 as a predictor and therapeutic target for intervention based on deepening the understanding of METH-related cognitive dysfunction and pathological memory formation mechanism.Methods:1、A mouse model of METH-induced learning and memory impairment was established.Behavioral tests such as Y maze,novel object recognition,Barnes maze,and Morris water maze were used to determine the learning and memory impairment characteristics induced by METH.Nissl staining and immunofluorescence staining were used to determine the specific brain area of METH impairing learning and memory,the effect of METH on TRPV4 expression,and the cell specificity of TRPV4expression was detected.Pharmacological intervention and gene interference technology were used to block TRPV4,and the performance of METH mice in the Y maze,novel object recognition,and Morris water maze were observed to clarify the role of TRPV4 in METH-induced learning and memory impairment.2、In mice with METH impairment of learning and memory,pharmacological methods were used to block TRPV4.Nissl staining and immunofluorescence staining were used to observe the changes in neuronal damage and apoptosis in corresponding brain regions.QPCR and Western blot were used to detect the expression of endoplasmic reticulum stress（ERS）-related apoptotic pathways in the hippocampus.Hippocampal neurogenesis was observed by immunofluorescence staining.Immunofluorescence staining,ELISA,QPCR,and Western blot were used to detect the activation of glial cells,the secretion of pro-inflammatory factors,and the expression of the CamkⅡα/NLRP3 inflammasome pathway in the hippocampus.To explore the possible pathway mechanism of TRPV4 involved in METH impairment of learning and memory.3、Gene interference technology was used to silence TRPV4 in HT22cells.Combined with cell molecular biology methods,the changes in apoptosis and ERS-related apoptosis pathways in cells were determined.The Fluo-4 AM calcium ion fluorescence probe detected the intracellular Ca²⁺level.The mechanism of TRPV4 in METH-induced hippocampal neuron injury was analyzed in vitro.4、METH-induced behavioral sensitization and conditioned place preference（CPP）models in mice were established to characterize the pathological memory of addiction.Pharmacological blocking of TRPV4was used to observe the effect on spontaneous activity and METH-induced hyperactivity in mice.And behavioral sensitization（formation,transformation,expression）and CPP effect（formation,expression,relapse）in METH pathological memory mice.Immunofluorescence staining was used to determine the changes in TRPV4 expression and neuronal activity in the Nucleus accumbens（NAc）.Western blot and Golgi staining were used to detect the changes in dendritic spine structure and synapse-related molecules in NAc.To elucidate the role and possible mechanism of TRPV4 in the pathogenic memory of METH.Results:1、（1）Repeated administration of METH（15mg/kg）impaired working memory and spatial learning and memory ability in mice.Behavioral experiments showed that the proportion of spontaneous intercountry decreased in the Y maze,the time of exploring new objects decreased in novel object recognition,the number of correct probing and the time of staying in the target opening area decreased in the Barnes maze,and the time of staying in the target platform quadrant and the number of crossing the platform decreased in Morris water maze.（2）In METH-induced learning and memory impairment mice,there were significant neuronal damage characteristics in the frontal cortex and hippocampal CA1and DG regions,with pathological manifestations of pyknosis,unclear structure,and loss of Nissl bodies in some neurons,and prominent apoptosis neurons in the frontal cortex and hippocampal CA1 region.（3）METH increased the expression of TRPV4 in the CA1 and DG regions of the hippocampus in mice,and TRPV4 was abundant in neurons,astrocytes,and microglia,especially in neurons.（4）Intraperitoneal injection of TRPV4 specific inhibitor HC067047 and si RNA silencing TRPV4 in the hippocampus in vivo significantly increased the proportion of spontaneously altered country in the Y maze,the time to explore the novel object in the novel object recognition,the time to stay in the target platform quadrant and the number of crossing the platform in the Morris water maze.These results suggest it can improve the learning and memory impairment induced by METH in mice.2、（1）Blocking TRPV4 by HC067047 alleviated METH-induced neuronal damage in mouse hippocampal CA1 and DG regions,and the apoptosis of hippocampal CA1 neurons was significantly inhibited.（2）METH up-regulated the expression of GRP78,PERK,IRE1,ATF6,CHOP,cleaved caspase-12 and cleaved caspase-3 in ERS-related apoptosis pathways,and blocking TRPV4 inhibited the expression of these molecules.These results suggest that TRPV4 may be involved in METH-induced apoptosis of hippocampal neurons by mediating ERS.（3）METH reduced the expression of DCX in the hippocampal DG region of mice,and blocking TRPV4 could increase the level of DCX in the hippocampal DG region,suggesting that the inhibited hippocampal neurogenesis in mice was restored.（4）METH increased the levels of pro-inflammatory factors（IL-6,IL-1β,TNF-α）in the hippocampus and serum of mice,and blocking TRPV4 could inhibit the secretion of these pro-inflammatory factors.（5）METH increased the number of GFAP-positive astrocytes and IBA-1positive microglia in the hippocampus of mice,reduced the process complexity of microglia,and increased the expression of GFAP,IBA-1,CD68,and CD11B,which were reversed by blocking TRPV4.It is suggested that the activation of glial cells in the hippocampus is inhibited.（6）METH increased the expression of CamkⅡα,p-Camk IIα,NLRP3,ASC and caspase-1 in the CamkⅡα/NLRP3 inflammasome pathway,and blocking TRPV4 inhibited the expression of these molecules.TRPV4 may be involved in METH-induced hippocampal neuroinflammation by mediating the CamkⅡα/NLRP3 inflammatory signaling pathway.3、（1）METH had significant cytotoxicity and induced apoptosis of HT22 cells in a concentration-dependent manner.（2）METH up-regulated the expression of GRP78,p-PERK,p-IRE1,ATF6,CHOP,cleaved caspase-12,cleaved caspase-9 and cleaved caspase-3 in ERS-related apoptosis pathways in HT22 cells.The specific ERS inhibitor 4-PBA alleviated the apoptosis and inhibited the activation of the ERS-related apoptotic pathway,indicating that ERS was involved in METH-induced apoptosis.（3）METH increased the expression of TRPV4 in HT22 cells and promoted Ca²⁺influx.（4）Silencing TRPV4 by si RNA inhibited METH-induced apoptosis and ERS-related apoptosis pathway activation;（5）After silencing TRPV4,intracellular Ca²⁺accumulation was also significantly reduced in HT22 cells,suggesting that TRPV4 may be involved in METH induced apoptosis by regulating Ca²⁺to mediate ERS.4、（1）Treatment with HC067047 could inhibit the enhancement of spontaneous activity in mice induced by a single dose of METH,while single or multiple doses of HC067047 alone did not induce sensitization.（2）Blocking TRPV4 at the sensitization formation stage could inhibit METH-induced behavioral sensitization in mice,but blocking TRPV4 at the sensitization transition stage and expression stage had no effect.（3）Blocking TRPV4 at the stage of CPP formation could inhibit the METH-induced CPP effect,but intervening at the stage of CPP expression and reactivation had no effect.（4）During METH-induced CPP formation,the number of c-fos labeled neurons in the NAc of mice was significantly increased,and the activity of the neurons in the NAc was significantly inhibited after blocking TRPV4.（5）METH-induced increases in the density,length,and branch number of dendritic spines in the NAc neurons of CPP mice.Blocking TRPV4 can inhibit these increases in dendritic spine complexity,suggesting that TRPV4 may affect the formation of pathological memory in METH by regulating NAc neural plasticity.Conclusions:TRPV4 may be involved in METH-induced learning and memory impairment in mice by regulating the ERS-related apoptosis pathway and Camk IIα/NLRP3 inflammasome signaling pathway,promoting hippocampal neuron apoptosis and neuroinflammation inhibiting hippocampal neurogenesis.In addition,TRPV4 may be involved in the formation of METH pathological memory by regulating NAC Neuroplasticity.