Ahi1基因敲除造成小鼠脑内神经递质释放改变

  本文关键词：Ahi1基因敲除造成小鼠脑内神经递质释放改变，由笔耕文化传播整理发布。

        目的观察Ahi1基因敲除(Ahi1KO)小鼠是否存在抑郁样表型，进一步探讨神经递质改变在抑郁样行为发生机制中的作用。方法采用Western blot法测定Ahi1KO小鼠以及对照小鼠(Ahi1杂合子)脑组织中Ahi1蛋白的表达水平；利用悬尾实验(TST)、强迫游泳实验(FST)、糖水偏好实验等行为学实验观察Ahi1KO小鼠是否存在抑郁样表型；采用高效液相色谱法(HPLC)测定脑组织中五羟色胺(5-HT)、五羟基吲哚乙酸(5-HIAA)、多巴胺(DA)、高香草酸(HVA)、二羟基苯乙酸(DOPAC)、谷氨酸(glutamate)、γ-氨基丁酸(GABA)等神经递质的含量；采用ELISA法测定脑组织单胺类神经递质代谢相关酶单胺氧化酶(MAO)的活性。为研究抗抑郁药对这种抑郁样行为是否存在治疗作用，将Ahi1KO小鼠随机分为2组，实验组、盐水组。实验组给予每天腹腔注射丙咪嗪(20mg/kg体重)，盐水组给予腹腔注射相同剂量生理盐水，3周后观察行为学改变，并应用HPLC测定脑组织中神经递质的改变。结果Western blot结果显示：与对照组相比，Ahi1KO小鼠基本无Ahi1蛋白表达。悬尾实验和强迫游泳实验表明Ahi1KO小鼠的不动时间较对照组明显增长(P＜0.05)。在糖水偏好实验中，Ahi1KO小鼠的总饮水量以及糖水占总饮水量的百分比是明显降低的(P＜0.05)。出生4天的小鼠脑组织中，Ahi1KO小鼠与对照组的5-HT的含量没有明显差别，出生10天、1月、2月的小鼠脑组织中，对照组的5-HT的含量明显增加(P＜0.05)，而Ahi1KO小鼠增加不明显；同样DA的水平在出生4天的Ahi1KO小鼠脑组织中也没有明显差别，出生10天及1月的小鼠脑组织中的DA的含量Ahi1KO小鼠较对照组降低(P＜0.05)。出生4天和10天的小鼠脑组织中的谷氨酸和GABA的含量，Ahi1KO小鼠与对照组相比却无明显差别(P>0.05)。取1月龄小鼠不同部位脑组织(杏仁核、海马、皮层、下丘脑、脑干)进行5-HT和DA等神经递质检测，发现Ahi1KO小鼠的脑组织的5-HT及其代谢产物5-HIAA较对照组含量明显降低(P＜0.05)，，5-HIAA与5-HT的比值（代谢率）则比对照组增高(P＜0.05)。同5-HT类似，DA及其代谢产物HVA、DOPAC也观察到同样结果。ELISA法检测脑组织中脑干MAO的活性结果显示Ahi1KO小鼠的MAO的活性与对照组相比明显升高(P＜0.05)。Ahi1KO小鼠注射丙咪嗪3周后，在TST、FST中不动时间较盐水组明显缩短(P＜0.05)，五羟色胺的水平较盐水组升高(P＜0.05)，而多巴胺的含量却没有明显变化(P>0.05)。结论敲除Ahi1基因可在小鼠中造成抑郁样行为。Ahi1KO小鼠不同脑组织中抑郁相关单胺类神经递质如5-HT、DA含量降低，但氨基酸类神经递质如谷氨酸、GABA的含量无明显改变。在Ahi1KO小鼠脑组织中，MAO的活性增高可能是引起单胺类神经递质含量降低的一个原因。丙咪嗪治疗可以减轻抑郁样症状，Ahi1KO小鼠脑组织中5-HT的含量可以部分恢复，因此Ahi1KO小鼠是研究抑郁机制的一个理想模型，并可用于抗抑郁药物筛选。

    Objective To investigate whether Ahi1KO mice have depressive phenotype andfurther explore the role of neurotransmitters in the mechanisms of depressive behaviors.Methods Western blot analysis was used to determine the expression levels of Ahi1inAhi1KO mice and control mice (Ahi1heterozygote). Tail suspension test, forcedswimming test, and sucrose preference test were used to examine depressive phenotype inmice. The contents of neurotransmitters including serotonin (5-HT),5-hydroxy indoleacetic acid (5-HIAA), dopamine (DA),4-hydroxy-3-methoxyphenylacetic (HVA),dihydroxyphenylacetic acid (DOPAC), glutamate and gamma amino acid butyric acid(GABA) were measured by high performance liquid chromatography (HPLC). Wemeasured the monoamine oxidase activity (MAO) in the brainstem of control and Ahi1KOmice by an enzyme linked immunosorbent assay method (ELISA). In order to studywhether these mice responded to known anti-depressant impramine, Mice were treatedwith imipramine or normal saline intraperitoneally for3weeks. Three weeks later, thechange of behaviors was evaluated by forced swimming test and tail suspension test, thechanges of neurotransmitters were examined by HPLC.Results Western blot analysis revealed that there was almost no Ahi1expression inAhi1KO mice (P＜0.05).Tail suspension test and forced swimming test shown thatsignificant decrease of immobility time was found in Ahi1KO mice compared with controlmice (P＜0.05). In sucrose preference test, total consumed liquid and the percentage ofconsumed sucrose water was markedly decreased in Ahi1KO mice (P＜0.05).At postnatalday4, there was no difference for the content of serotonin in control and Ahi1KO mice(P>0.05). However, after postnatal day10、1month and2month，serotonin level wassignificantly increased in control mice, while only a slight increase of serotonin wasobserved in Ahi1KO mice. Similarly, dopamine level was not significantly elevated inAhi1KO mice as in control mice after postnatal day4, but dopamine level was muchlower in Ahi1KO mice after postnatal day10d、1m compared with control mice (P＜0.05). Glutamate and GABA levels were not influenced in control and Ahi1KO mice at4d,10d(P>0.05). We further measured serotonin levels in the cortex, hippocampus, hypothalamus,brainstem, and amygdale at1-m-old mice, the level of serotonin and the metabolite ofserotonin5-HIAA was decreased in Ahi1KO mice compared with control mice (P＜0.05).However, the ratio of5-HIAA to serotonin, namely serotonin turnover, was markedlyelevated in Ahi1KO mice(P＜0.05). Similar tendency was also observed in dopamine, themetabolite of dopamine (DOPAC and HVA).MAO activity was significantly increased inAhi1KO mice compared with control mice(P＜0.05).After imipramine treatment for3weeks, immobility time of Ahi1KO mice in forced swimming test and tail suspension testwas significantly decreased(P＜0.05). Serotonin level was elevated(P＜0.05), however,dopamine level was not changed (P>0.05).Conclusion Ahi1KO caused depressive behaviors in mice.In Ahi1KO mice, thecontent of depression-related neurotransmitter such as5-HT and DA was decreased,however, the level of amino acid neurotransmitters (such as glutamate and GABA) werenot influenced. MAO activity was elevated in Ahi1KO mice, which may result in thedecrease of monoamine in brains. Imipramine alleviated the depressive phenotype;moreover, the content of5-HT in Ahi1KO mice was partly restored. Therefore, Ahi1KOmice are a genetic model of depression for the mechanistic study and a useful tool toscreen anti-depressants.

Ahi1基因敲除造成小鼠脑内神经递质释放改变

中文摘要4-6Abstract6-7前言9-11第一部分 AHI1 基因敲除对小鼠行为学的影响11-20    材料和方法11-16    结果16-19    小结19-20第二部分 AHI1 敲除小鼠脑内神经递质水平及单胺氧化酶活性的改变20-31    材料和方法20-25    结果25-29    小结29-31第三部分 抗抑郁药对 AHI1 KO 小鼠抑郁样症状以及神经递质水平的影响31-36    材料和方法31-32    结果32-35    小结35-36讨论36-39结论39-40参考文献40-43综述：五羟色胺与抑郁治疗43-50    参考文献47-50英文缩写词表50-51攻读学位期间公开发表的论文51-52致谢52-53

  本文关键词：Ahi1基因敲除造成小鼠脑内神经递质释放改变，由笔耕文化传播整理发布。