NMDAR介导突触再可塑性参与麻醉药保护抑郁大鼠学习记忆功能的机制

发布时间：2018-03-23 02:05

  本文选题：麻醉药　切入点：电休克　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的研究异丙酚及小剂量氯胺酮对抑郁大鼠电休克(electroconvulsive shock,ECS)后空间学习记忆功能以及海马内谷氨酸N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor,NMDAR)和α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor,AMPAR)蛋白表达、功能和长时程增强(long-term potentiation,LTP)的影响,以探讨调控NMDAR介导的突触再可塑性改变在麻醉药异丙酚和小剂量氯胺酮保护抑郁大鼠ECS后学习记忆功能中的作用机制。方法第一部分2~3月龄健康雄性Sprague Dawley(SD)大鼠,抑郁大鼠模型的建立采用慢性轻度不可预见性应激(chronic unpredictable mild stress,CUMS)。80只抑郁模型大鼠随机分为8组(n=10):D组、DP组、DE组、DPE组、DK组、DKE组、DPKE组、DPME组。10只同期喂养的健康雄性SD大鼠为对照组(C组)。D组腹腔注射生理盐水(normal saline,NS)8ml/kg后给予假ECS处理(双耳夹电极,但不通电);DP组腹腔注射异丙酚80mg/kg,而后给予行假ECS处理;DE组腹腔注射NS8ml/kg后给予ECS处理;DPE组腹腔注射异丙酚80mg/kg后行ECS;DK组腹腔注射氯胺酮10mg/kg;DKE组腹腔注射氯胺酮10mg/kg后行ECS;DPKE组腹腔注射氯胺酮10mg/kg和异丙酚80mg/kg行ECS;DPME组腹腔注射MK801 1mg/kg和异丙酚80mg/kg后行ECS。上述处理每日1次,连续7d。C组大鼠不给予任何处理。采用糖水偏好实验(sucrose preference test,SPT)检测糖水偏好百分比(sucrose preference percentage,SPP)、旷场实验(open field test,OFT)检测直立次数、穿格次数,评估大鼠抑郁样行为;采用Morris水迷宫评价大鼠空间学习记忆功能;采用western-blot检测大鼠海马NR1、NR2A、NR2B、Glu R1蛋白表达水平。第二部分2~3月龄健康雄性SD大鼠,采用CUMS建立抑郁大鼠模型。72只抑郁模型大鼠随机分为6组(n=12):D组、DE组、DK组、DPE组、DPKE组、DPME组。12只同期喂养的健康雄性SD大鼠为C组。各组大鼠处理方法同第一部分所述,C组大鼠不予以处理。采用细胞外记录检测海马Schaffer侧枝-CA1神经通路NMDAR、AMPAR介导的兴奋性突触后场电位(field excitatory postsynaptic potential,f EPSP);检测各组大鼠海马Schaffer侧枝-CA1神经通路高频电刺激(protocol1和protocol2,protocol2刺激强度大于protocol1)的LTP诱导反应差异;检测含不同浓度异丙酚和氯胺酮的人工脑脊液灌流期间该神经通路f EPSP的变化。结果第一部分实验1:(1)实验处理前,D组、DE组、DKE组SPP、直立次数和穿格次数组间差异无统计学意义,均低于C组(P0.01)。实验处理后,与D组相比,DE组和DK组SPP、直立次数和穿格次数均升高,(P0.01);DE组SPP、直立次数高于DK组(P0.01)。(2)实验处理前,D组、DE组、DK组大鼠逃避潜伏期和目标象限探索时间组间差异无统计学意义(P0.05),其逃避潜伏期长于C组而目标象限探索时间短于C组(P0.01)。实验处理后,与C组、D组、DK组相比,DE组逃避潜伏期延长,目标象限探索时间缩短(P0.01);D组、DK组大鼠逃避潜伏期长于C组,而目标象限探索时间短于C组;D组、DK组两指标组间差异无统计学意义(P0.05)。(3)D组、DE组、DK组大鼠Glu R1蛋白表达水平均低于C组(P0.01);与D组相比,DE组、DK组大鼠海马Glu R1表达水平升高(P0.01),而DE组与DK组大鼠海马Glu R1蛋白表达水平差异无统计学意义(P0.05)。与C组相比,D组、DE组、DK组大鼠海马的NR1、NR2A、NR2B蛋白表达水平降低(P0.05);与D组相比,DE组大鼠海马NR1、NR2A、NR2B蛋白表达水平降低,而DK组大鼠NR1、NR2B亚基蛋白表达水平升高,差异有统计学意义(P0.05)。D组及DK组大鼠海马NR2A蛋白表达水平差异无统计学意义(P0.05)。各组大鼠NR2B与NR2A亚基蛋白表达水平的比例(NR2B/NR2A)差异无统计学意义(P0.05)。实验2:(1)实验处理前,D组、DP组、DE组、DPE组、DKE组大鼠SPP、直立次数、穿格次数差异无统计学意义(P0.05)。实验处理后,与D组相比,DE组、DPE组和DKE组大鼠SPP、直立次数、穿格次数明显升高(P0.01);DPE组大鼠SPP低于DE组和DKE组(P0.01),而直立次数和穿格次数与DE组差异无统计学意义(P0.05);DE组和DKE组大鼠SPP差异无统计学意义,(P0.05);DKE组直立次数和穿格次数高于DE组(P0.01)。(2)实验处理前,各组大鼠逃避潜伏期、目标象限探索时间差异无统计学意义(P0.05)。实验处理后,与D组相比,DE组、DPE组、DKE组逃避潜伏期延长,目标象限探索时间缩短(P0.01);与DE组相比,DPE组、DKE组大鼠逃避潜伏期缩短,而目标象限探索时间延长(P0.01);D组、DP组大鼠逃避潜伏期和目标象限探索时间差异无统计学意义(P0.05),DPE组和DKE组组间差异无统计学意义(P0.05)。(3)与D组相比,DE组、DPE组以及DKE组大鼠海马Glu R1蛋白表达水平明显升高(P0.01)。D组大鼠海马Glu R1蛋白表达水平与DP组无明显差异(P0.05),DE组、DPE组、DKE组大鼠海马Glu R1蛋白表达水平差异无统计学意义(P0.05)。与D组相比,DE组、DPE组、DKE组大鼠海马的NR1、NR2B蛋白表达水平明显降低(P0.05);DPE组、DKE组大鼠海马NR1、NR2B蛋白表达水平高于DE组(P0.05)。与D组相比,DE组、DKE组大鼠海马NR2A蛋白表达水平降低,而DPE组大鼠海马NR2A表达水平高于D组和DKE组(P0.01)。与D组相比,DKE组NR2B/NR2A明显升高,而DPE组降低(P0.05)。D组、DP组、DE组NR2B/NR2A差异无统计学意义(P0.05)。实验3:(1)实验处理前,D组、DPE组、DPKE组、DPME组SPP、直立次数、穿格次数差异无统计学意义(P0.05)。实验处理后,与D组相比,DPE组、DPKE组和DPME组SPP、直立次数和穿格次数升高(P0.01);与DPE组相比,DPKE组和DPME组大鼠SPP、直立次数升高(P0.01),DPKE组和DPME组大鼠SPP、直立次数和穿格次数差异无统计学意义(P0.05)。(2)实验处理前,各组大鼠逃避潜伏期和目标象限探索时间组间差异无统计学意义(P0.05)。实验处理后,与DPE组相比,DPKE组和DPME组逃避潜伏期缩短、目标象限探索时间延长(P0.05);D组、DPKE组、DPME组逃避潜伏期和目标象限探索时间差异无统计学意义(P0.05)。(3)与D组相比,DPE组、DPKE组、DPME组大鼠海马Glu R1蛋白表达水平升高(P0.01);DPKE组大鼠海马Glu R1蛋白表达水平高于DPE组(P0.01);DPE组与DPME组大鼠海马Glu R1蛋白表达水平差异无统计学意义(P0.05)。各组大鼠海马NR2A亚基的蛋白表达水平差异无统计学意义(P0.05)。与DPE组相比,DPKE组、DPME组大鼠海马NR1、NR2B蛋白表达水平以及NR2B/NR2A明显升高(P0.05)。与D组相比,DPKE组和DPME组大鼠海马NR2B蛋白表达水平升高(P0.05);DPKE组和DPME组大鼠海马NR2B蛋白表达水平组间差异无统计学意义(P0.05)。D组、DPKE组、DPME组大鼠海马NR1蛋白表达水平和NR2B/NR2A差异无统计学意义(P0.05)。第二部分实验1:(1)与C组相比,D组大鼠NMDAR I/O明显降低;与D组相比,DK组大鼠NMDAR I/O明显升高,而DE组大鼠NMDAR I/O明显降低(P0.01)。与C组相比,D组大鼠AMPAR介导的f EPSP降低(P0.01);与D组相比,DK组、DE组大鼠AMPAR介导的f EPSP明显升高,而DE组高于DK组,差异有统计学意义(P0.01)。(2)protocol1高频刺激后,与C组相比,D组、DE组、DK组大鼠Schaffer侧枝-CA1神经通路LTP幅度降低;DE组大鼠LTP幅度低于D组和DK组(P0.01),D组和DK组大鼠LTP幅度差异无统计学意义(P0.05)。C组、DE组大鼠海马Schaffer侧枝-CA1通路protocol2诱导的LTP幅度高于protocol1,(P0.05);D组protocol2诱导的LTP幅度高于protocol1,但差异无统计学意义(P0.05)。实验2:(1)与DE组相比,DPE、DPKE组、DPME组大鼠NMDAR I/O明显升高(P0.01);与DPE组相比,DPKE组、DPME组大鼠NMDAR I/O升高,DPME组大鼠NMDAR I/O低于DPKE组(P0.01)。(2)与DE组相比,DPE、DPKE组、DPME组大鼠LTP幅度明显升高(P0.01);与DPE组相比,DPKE组、DPME组大鼠LTP幅度升高(P0.01);DPKE组与DPME组大鼠LTP幅度差异无统计学意义(P0.05)。(3)10μM及20μM异丙酚灌流均可显著抑制大鼠海马Schaffer侧枝-CA1通路f EPSP;20μM异丙酚抑制作用强于10μM(P0.01),起效更快,抑制程度更高。1μM氯胺酮灌流对大鼠海马Schaffer侧枝-CA1通路f EPSP无明显作用,10μM氯胺酮可轻度增加f EPSP幅度,而两剂量氯胺酮灌流期间f EPSP差异无统计学意义(P0.05)。结论(1)ECS抗抑郁机制与其上调抑郁大鼠海马Glu R1表达和AMPAR功能有关;而其损伤抑郁大鼠空间记忆功能与下调海马NMDAR表达量及Schaffer侧枝CA1神经通路NMDAR功能有关。(2)异丙酚可部分逆转ECS对NMDAR表达和功能的下调作用,减轻ECS所致的学习记忆功能损伤;异丙酚麻醉下,NMDAR阻滞剂(氯胺酮和MK801)可进一步减轻ECS对NMDAR表达和功能的下调作用,减轻ECS所致的学习记忆功能损伤。(3)激活NMDAR所介导的突触再可塑性改变是ECS下调NMDAR表达和功能的机制;异丙酚及氯胺酮阻滞ECS对NMDAR的激活,是其逆转ECS下调NMDAR表达和功能,进而发挥学习记忆功能保护作用的机制。
[Abstract]:Objective to study the effect of propofol and ketamine for electroconvulsive therapy in depressed rats (electroconvulsive shock, ECS) after the spatial learning and memory function and hippocampal glutamate N- methyl -D- aspartate receptor (N-methyl-D-aspartic acid receptor, NMDAR -3-) and alpha amino hydroxy -5- methyl isoxazole propionate by -4- (alpha -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor, AMPAR) the expression, function and long term potentiation (long-term potentiation, LTP) to investigate the effects of NMDAR mediated regulation of synaptic plasticity and mechanisms of learning and memory function in the anesthetic propofol and ketamine protect depression after ECS in rats. Methods the first part of the 2~3 month old male Sprague (Dawley SD) rats, establishment of rat model of depression by chronic unpredictable mild stress (chronic unpredictable mild stress, CUMS).80 suppression Depression model rats were randomly divided into 8 groups (n=10): D group, DP group, DE group, DPE group, DK group, DKE group, DPKE group, healthy male SD rats DPME group.10 rats fed over the same period as control group (group C).D group received intraperitoneal injection of saline (normal saline, NS 8ml/kg) sham ECS treatment (ear clip electrodes, but no electricity); DP group received intraperitoneal injection of propofol 80mg/kg, and then give the sham ECS treatment; group DE received intraperitoneal injection of NS8ml/kg after treated with ECS; DPE group received intraperitoneal injection of propofol 80mg/kg ECS after intraperitoneal injection of ketamine 10mg/kg; DK group; DKE group received intraperitoneal injection of ketamine 10mg/kg after ECS; DPKE group received intraperitoneal injection of ketamine and propofol for 10mg/kg 80mg/kg ECS; DPME group intraperitoneal injection of MK801 1mg/kg and 80mg/kg ECS. after the treatment of propofol 1 times a day, continuous 7d.C group were not given any treatment. The sucrose preference test (sucrose preference test SPT (s) detection of sucrose preference percentage Ucrose preference percentage, SPP), open field test (open field, test, OFT) detection of vertical frequency, the number of crossing line, to evaluate the depression like behavior of rats; to assess learning and memory function of rats by Morris water maze; using Western-blot detection of rat hippocampal NR1, NR2A, NR2B, Glu expression level of R1 protein. The second part 2~3 month old healthy male SD rats, using CUMS to establish the rat model of depression.72 depression model rats were randomly divided into 6 groups (n=12): D group, DE group, DK group, DPE group, DPKE group, healthy male SD rats group DPME.12 synchronous feeding for C group. Each group in the same way as the first part of the treatment, C group rats were treated by extracellular recording. Detection of hippocampal Schaffer collateral -CA1 neural pathway NMDAR, excitatory postsynaptic field potentials mediated by AMPAR (field excitatory postsynaptic potential, f; EPSP) were measured in rat hippocampal Schaffer collateral -CA1 God 缁忛，

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