Roles Of Tau And MAP-2in CCK-8Alleviating Spatial Memory Impairment Induced By Morphine And Its Mechanism

Long-term application of opioids can lead to tolerance and adiiction.Meanwhile, cognitive and learning and memory was impaired by opioidstreatment. Spatial learning and memory is a kind of scenes and events memory,and morphine dependence or acute morphine treatment could induce spatialmemory impairment. Cholecystokinin octopeptide (CCK-8), one of the mostefficient anti-opioid peptides, is widely spread in the central and peripheralnervous system, which play an important role in modulating memory processsuch as avoidance reaction, spatial identification and so on. However, theeffect of CCK-8on morphine induced memory impairment and its relatedmechanism is unclear.Synaptic plasticity is the physiological basis of learning and memory, andspatial learning and memory impairment is closely related to the synapticplasticity change in hippocampus region. The skeletal system plays animportant role in regulating synaptic plasticity. Tau and MAP-2regulates thepre-synaptic and postsynaptic plasticity and steady-state plasticity of neuronsby affecting the stability of microtubules which are located in the axon anddendrites respectively. Study showed that phosphorylation state of tau andMAP-2was also participated in the learning and memory process. But,whether tau and MAP-2were responsible for the effect of CCK8on morphineinduced synaptic plasticity change is not clear.We use Morris water maze to observe the effect of exogenous CCK8onspatial reference memory impairment in acute and chronic morphine-treatedmice. Then we aimed to clarify the role of skeleton associated protein ineffects of CCK-8on synaptic plasticity change induced by morphine toexplore the mechanism of CCK-8on memory impairment by morphine.Part1: Effects of CCK-8on spatial memory in morphine-treated mice Objective: We use Morris water maze to observe the effect of morphinedependence and acute morphine on spatial reference memory; different dosesof exogenous CCK-8were given before morphine to evaluate effect of CCK-8on spatial memory in morphine-treated mice; effect of exogenous CCK-8treatment alone on spatial reference memory was also observed.Methods:①Morphine dependent model was established by increasingdoses manner (1-6days, morphine was subcutaneous injected at dose followed:20,40,60,80,100,100mg/kg, respectively,2times a day,12hours interval);different doses (0.001,0.01,0.1and1ug) of CCK-8was given by lateralventricle injection,2times a day at12hours interval. Morris water maze inthe next7days to adapt, training and testing, training and tests are conducted30minutes after drug injection. During this period, morphine100mg/kg orCCK-8were still given to mice; to observe effect of exogenous CCK-8onspatial reference memory in chronic morphine-treated mice, CCK-8was given30minutes before every morphine injection.②we adopted a2-day Morriswater maze model to assess acute drug treatment on spatial memory.1,5andor CCK-8(0.001,0.01,0.1and1μg, i.c.v)30minutes before training and testto assess effect of morphine or CCK-8on spatial reference memoryacquisition and retrieval respectively;10mg/kg morphine(i.p) was given30minutes before training, and different doses of CCK-8(0.001,0.01,0.001and1μg) were given30minutes before morphine or test to observe exogenousCCK-8on acute morphine induced spatial memory acquisition impairment.Results:①552mice were included in the final statistical analyses.②Chronic morphine significantly impaired spatial reference memory onthe probe test but did not affect the performance in place navigation trails;Spatial reference memory impairment by chronic morphine could delay to7days after morphine spontaneous withdrawal; CCK-8(0.1and1μg) alonesignificantly improved the spatial reference memory; CCK8(0.01,0.1and1μg) significantly improved the impairment of spatial reference memoryinduced by chronic morphine.③Morphine (5and10mg/kg) treatment30minutes before training significantly impaired spatial reference memory acquisition in mice, whichshowed a poor performance in place navigation trails and probe test in watermaze experiment, but morphine (1,5, and10mg/kg) treatment30minutesbefore test had no effect on memory retrieval; CCK-8(0.01,0.01and1μg)treatment30minutes before training or CCK-8(0.1and1μg) before test couldpromote the acquisition and retrieval of spatial reference memory respectively;CCK-8(0.1and1μg) before training or test could significantly improveimpairment of spatial memory acquisition by morphine (10mg/kg) in mice,but CCK-8(0.001and0.01μg) had no obvious effect.Summary:①This part of study found that chronic morphine obviouslyimpaired spatial reference memory, and the damage can be delay to7daysafter morphine withdrawal; CCK-8(0.01,0.1,1μg) could improve the spatialreference memory damage induced by chronic morphine; at the same time,CCK-8(0.1and1μg) alone could significantly improved the spatial referencememory in mice.②Acute morphine significantly affected the spatial reference memoryacquisition in mice, but did not affect memory retrieval; CCK-8before thetraining or test significantly improved impairment of memory acquisitioninduced by morphine; CCK-8treatment alone could improved spatialreference memory acquisition or retrieval.Part2: mechanism of CCK-8alleviating spatial memory impairment bymorphine1effect of CCK-8on acetylcholine content and dendritic spine density inhippocampus of morphine-treated miceObjective: Acetylcholine content and dendritic spine density change inhippocampus were observed to clarify the role of synaptic plasticity change inCCK-8on spatial memory in morphine-treated mice.Methods: We established acute or chronic morphine mice model first,then LC-MS and Golgi staining were adopted to detect acetylcholine levelsand density of dendritic spines in morphine or CCK-8treatment.Results:48mice were included in the final statistical analyses. Acute morphine significantly decreased the content of acetylcholine in thehippocampus, but acetylcholine content increased after chronic morphinetreatment; Acute or chronic CCK-8(1μg) could significantly increaseacetylcholine content in hippocampus; pre-injection of CCK-8(1μg) couldobviously reverse acetylcholine content decreased by acute morphine, but hadno obvious effect on chronic morphine. Chronic morphine significantlyreduced density of dendritic spines in the hippocampus; Chronic CCK-8(1μg)alone obviously increased density of dendritic spines in the hippocampus;Pre-injection of CCK-8(1μg) could obviously reversed effect of hronicmorphine on density of dendritic spines in hippocampus.2effect of CCK-8on tau and MAP-2expression in hippocampus ofmorphine-treated miceObjective: Microtubule associated protein tau and MAP-2expression inhippocampus were observed, in order to investigate the role of skeletonprotein system in synaptic plasticity changes.Methods: Tau and MAP-2expression in hippocampus by morphine andCCK-8treatment were detected by RT-PCR and Western Blot.Results:48mice were included in the final statistical analyses. Acutemorphine or CCK-8treatment alone did not affect tau and MAP-2expressionin hippocampal, pre-injection of CCK-8(1μg) could obviously reverse theeffect of acute morphine induced phosphorylation of tau Ser396, MAP-2Ser136and Thr1620/1623; phosphorylation of tau Ser262had no obviouschange after acute morphine treatment, and was increased by acute CCK-8treatment.Pre-injection of CCK-8(1μg) could obviously reverse the effect chronicmorphine on tau and MAP-2expression-downregulation and phosphorylationup-regulation in hippocampal; chronic CCK-8(1μg) treatment had no effecton the expression of tau, but obviously up-regulated the expression of MAP-2,and deceased phosphorylation level of tau and MAP-2.3mechanism of CCK-8downregulating morphine induced tau and MAP-2phosphorylation Objective: On the basis of acute morphine and morphine chronic cellmodel is established, we focused on activity of kinase and phosphorylaserelated to tau and MAP-2phosphorylation, in order to clarify the mechanismsof tau and MAP-2phosphorylation by morphine or CCK-8treatment.Methods: We observed intracellular cAMP level by time distinguishfluorescence detection, in order to confirm the acute and chronic morphinecell model was success established. Western Blot and enzyme activity kitswere used to detected tau and MAP-2phosphorylation and PKA, GSK-3βandPP2A activity changes.Results: cAMP level in SH-SY5Y cells is decreased within3h andincreased after24h of morphine treatment; cAMP level obviouslyup-regulated by10M naloxone in SH-SY5Y cell treaed with100Mmorphine for48h. SH-SY5Y treated with100M morphine for30minutes or48hours as acute or chronic morphine cell model.Cosistant with animal experiment, pre-treatment of CCK-8obviouslydecreased phosphorylation level of tau and MAP-2induced by acute orchronic morphine; to explore the mechanism of tau and MAP-2phosphorylation, PKA, GSK-3β and PP2A activity were detected. Resultsshowed that PKA, GSK-3β and PP2A activity were significantly decreased orincreased in acute or chronic morphine treatment SH-SY5Y cell; the PKAspecific inhibitors H-89could significantly reversed tau Ser262phosphorylation by chronic morphine, and GSK-3β specific inhibitorsSB216763significantly reversed phosphorylation of tau in Ser396and MAP-2in Ser136and Thr1620/1623.Acute CCK-8could increase activity of PKA and decrease activity ofGSK-3β respectively, PP2A activity showed no obvious change; ChronicCCK-8could obviously decreased the activity of PKA and GSK-3β butincreased PP2A activity significantly. Pre-injection with10-6M of CCK-8could obviously reverse PP2A activity by acute morphine treatment, andreverse PKA and GSK-3β activity by chronic morphine treatment. Summary: This part of the study indicated that exogenous CCK-8couldrelieve morphine induced changes in synaptic plasticity by regulatingacetylcholine content and dendritic spines density in hippocampus; the rationof phosphorylation and dephosphorylation level of tau and MAP-2might beparticipated in effect of CCK-8on morphine induced synaptic plasticitychange.Conclusions: This study reveals the effect of exogenous CCK-8on acuteand chronic morphine-induced spatial reference memory impairment andsynaptic plasticity mechanism, effect of CCK-8on morphine-inducedphosphorylation of tau and MAP-2and its mechanism were observed inanimal and SH-SY5Y cell. We can reach the conclusion as follows:1CCK-8(0.01,0.1,1μg) could improve the spatial reference memorydamage induced by chronic morphine; CCK-8(0.1and1μg) before thetraining or test significantly improved impairment of memory acquisitioninduced by acute morphine. High dose of CCK-8may improve spatialmemory loss by morphine through anti-opiod way and/or the effect onmemory regulation itself.2Exogenous CCK-8(1μg) could relieve morphine induced changes insynaptic plasticity by regulating acetylcholine content and dendritic spinesdensity in hippocampus, and phosphorylation level of tau and MAP-2might be participated in effect of on morphine induced synaptic plasticitychanges.3Acute morphine increased phosphorylation level of tau and MAP-2bydecreasing PP2A activity, PKA and GSK-3β might be major responsiblefor tau and MAP-2phosphorylation by chronic morphine treatment;10-6Mof CCK-8might decreased tau and MAP-2phosphorylation throughinhibiting PKA and/or GSK-3β activity; pre-injection with10-6M ofCCK-8could obviously reversed PP2A activity by acute morphinetreatment, and reversed PKA and GSK-3β activity by chronic morphinetreatment.