创伤后应激障碍样行为异常大鼠海马钙依赖性反应紊乱

目的探讨情感行为异常大鼠海马钙离子及其相关的钙依赖性反应,进一步认识创伤后应激障碍(PTSD)样行为异常的神经生物学基础。方法将240只雄性Wistar大鼠随机分组为海马阈下电刺激组(SE,n=96)、海马电极埋植对照组(CE,n=96)和正常对照组(NC,n=48),采用频率25Hz、波宽1ms、串长10s、串隔7min、强度100μA的恒流、单脉冲电流,反复刺激大鼠海马以建立PTSD样行为异常动物模型;采用神经生化、流式细胞仪、荧光标记术及Westernblotting等方法,定量观测了实验大鼠海马Na+-K+-ATP酶与Ca2+-ATP酶活性,细胞内游离钙离子含量与钙调素(CaM)相对活性平均通道荧光,以及海马组织总CaM表达的动态变化规律。结果电刺激停止后12h阈下刺激大鼠海马细胞线粒体Na+-K+-ATP酶活性即明显下降犤(0.56±0.17)mmol/(kg·s),F=4.438,P<0.05犦,48h仍显著低于NC组犤(0.61±0.17)mmol/(kg·s),P=0.026犦;24h线粒体Ca2+-ATP酶活性亦明显降低犤(0.53±0.14)mmol/(kg·s),F=4.999,P<0.05犦,72h仍显著低于NC组犤(0.61±0.17)mmol/(kg·s),P=0.027犦。海马细胞内游离钙离子含量于电刺激停止后12～48h明显高于两对照组(F=16.355,P<0.01),72h仍显著高于NC组犤(290±70)nmol/L,P=0.03犦,游离CaM平均通道荧光则同步降低(F=10.655,P<0.05),而海马组

The disturbance of hippocampal calcium-dependent responses in rats with PTSD-like behavior

Aim To investigating hippocampal calcium and calcium-dependent responses in rats with posttraumtic stress disorder(PTSD)-like behavior and to further understand the neurobiological mechanisms involved in the long-term neuropsychological sequelae in PTSD.Methods 240 male Wistar rats were divided randomly into 3 groups.Group SE (n=96) for rats with PTSD-like behavior by constant pulsating current of 100 μ A with intratrain frequencies of 16 Hz, pulsating duration of 1 ms,train duration of 10 s and interstimulus interval of 7 min for 5 days with 8 times per day.Group CE (n=96) for control of electrode implanted in hippocampus without stimulation, and Group NC (n=48) for normal control.Neurochemistry,flow cytometer,fluorescence labeling technique and Western blotting were adopted to detect activities of Na+-K+-ATPase and Ca2+-ATPase, levels of intracellular calcium and free calmodulin (CaM),and the total CaM expression.Results The Na+-K+-ATPase activity in mitochondria of hippocampal cells in Group SE rats was significantly decreased at 12 h ( 0.56± 0.17) mmol/kg@ s,F=4.438,P< 0.05],and still lower than group NC at 48 h after the last stimulation ( 0.61± 0.17) mmol/kg@ s, P=0.026],while the Ca2+-ATPase activity was also significantly decreased at 48 h (0.53± 0.14) mmol/kg@ s, F=4.999, P< 0.05] and 72 h post-stimulation (0.61± 0.17) mmol/kg@ s, P=0.027 vs group NC].The intracellular free calcium levels were markedly increased than control groups from 12 to 48 hours (F=16.355,P< 0.01),and still elevated at 72 hour after the final stimulation (290 ± 70) nmol/L,P=0.03 vs group NC].Meanwhile,the mean channel fluorescence of intracellular free CaM decreased remarkably synchronously (F=10.655,P< 0.05), while the Western analysis demonstrated the total hippocampal CaM expression was significantly elevated at 48 hour after the last stimulation in group SE rats (F=16.247,P< 0.05).Conclusion The lasting increased levels of intracellular free calcium and expression of Ca2+ /CaM in hippocampus, as well as the dysfunction of Na+-K+ pump and Ca2+-ATPase in hippocampal mitochondria may play important roles in the long-term neuropsychological sequelae in PTSD.