L型钙通道介导突触可塑性参与外侧缰核深部脑刺激的抗抑郁机制

发布时间：2018-05-01 17:30

本文选题：可塑性 + 深部脑刺激　；参考：《广州医科大学》2017年硕士论文

【摘要】：【研究背景】抑郁症严重影响人类健康与社会发展,世界卫生组织估计至2020年抑郁症将成为全球第二大疾病。当前针对抑郁症的治疗以药物治疗以及心理治疗为主,然而仅有三分之一的患者取得较满意疗效。临床上把久治不愈的的抑郁症病人称为难治性抑郁(treatment-resistant depression,TRD),对于这类患者,临床上往往采用深部脑刺激(Deep Brain Stimulation,DBS)的治疗方式,并取得了显著的疗效。外侧缰核(Lateral Habenula,LHb)是连接基底节与边缘系统的核团,参与调控奖赏系统及单胺类神经递质的分泌。深部脑刺激外侧缰核(LHb-DBS)是治疗难治性抑郁的主要方式之一,但其机制目前仍不明确。Malinow.R、Hongmei Meng等认为,DBS通过调控LHb神经元的放电,上调脑内单胺类递质的释放从而改善抑郁症状,然而这一提高单胺类神经递质的假说与DBS运用于对单胺类药物不敏感患者的临床现象以及DBS快速起效的临床特点不相符合,提示LHb-DBS治疗抑郁存在其它机制。目前研究发现快速抗抑郁效果是由哺乳动物雷帕霉素位点(mTOR,mammalian target of rapamycin)激活介导的,而在下边缘皮层进行深部脑刺激能上调mTOR磷酸化水平,提示LHb-DBS可能通过激活mTOR达到治疗抑郁。Duman等发现L型电压依赖钙通道(L-type voltage-dependent calcium channel,L-VDCC)阻断剂能有效阻断mTO R的激活与KET的快速抗抑郁作用,提示L-VDCC也可能参与了LHb-DBS治疗抑郁的过程。同时其它研究表明,DBS能诱导长时间的突触改变,而mTOR的激活常常伴随着树棘突发生、突触重构等突触改变。进一步提示LHb-DBS可能通过激活mTOR改善突触可塑性达到抗抑郁效果的机制。本研究采用免疫印迹、行为学和膜片钳等技术,揭示并探讨mTOR激活以及相关突触可塑性在LHb-DBS治疗抑郁的机制。【研究目的】验证激活mTOR与改善神经可塑性是LHb-DBS治疗抑郁在重要途径,明确L-VDCC是否参与了LHb-DBS治疗抑郁症的过程,探讨LHb-DBS上调神经可塑性的具体机制。为DBS治疗抑郁症的临床运用提供可靠的实验依据,并针对其机制开发抗抑郁新药提供理论依据。【研究方法】LHb-DBS对LH大鼠的抑郁样行为的影响参照Daniela Schulz等的方法,建立大鼠习得性无助(LH,learned helplessness)抑郁模型。观察LH大鼠与正常大鼠行为差别,评价LH造模的可行性;对LH大鼠进行外侧缰核深部脑刺激,观察LHb-DBS对习得性无助大鼠抑郁样行为的影响。同时给予侧脑室注射L-VDCC阻断剂(硝苯地平,nifedipine),观察其对LHb-DBS抗抑郁作用的影响。LHb-DBS对mTOR磷酸化及其表达的作用参照Daniela Schulz等的方法,建立大鼠LH抑郁模型,通过手术安放电极于LHb,深部脑刺激为实验组,深部脑假刺激为阳性对照组,未接受LH造模的正常老鼠为阴性对照组。通过western-blot,观察外侧缰核深部脑刺激后,海马内mTOR磷酸化的表达变化。同时给予侧脑室注射L-VDCC阻断剂(硝苯地平,nifedipine),观察其能否改变外侧缰核深部脑刺激对mTOR磷酸化的调节。通过脑片模拟深部脑刺激观察其对突触可塑性影响急性分离LH大鼠冠状位脑片于人工脑脊液中孵育,采用体外模拟LHb-DBS技术(具体详见方法)刺激LHb,在海马CA1 stratum radiatum(SR)亚区记录由刺激联合纤维引起的兴奋性突触后场电位(Shaffer collateral-CA1 field excitatory postsynaptic potentials,SC-CA1 fEPSP),观察外侧缰核刺激对SC-CA1 fEPSP的影响;给予20uM硝苯地平灌流脑片,观察其对外侧缰核刺激引起SC-CA1 fEPSP变化的影响;给予冠状脑片模拟外侧缰核深部脑刺激情况下,记录海马CA1区椎体神经元的自发放电,探讨LHb-DBS激活L-VDCC的机制。【研究结果与结论】LHb-DBS能显著改善LH大鼠的抑郁样行为,而硝苯地平能有效阻断外侧缰核深部脑刺激的抗抑郁作用,提示L-VDCC参与了LHb-DBS的抗抑郁机制。同时LHb-DBS能激活海马mTOR并引起海马SC-CA1 fEPSP长时程升高,这一现象也能被硝苯地平阻断,证明LHb-DBS能通过激活mTOR、改善突触可塑性达到抗抑郁作用,且这一机制依赖L-VDCC的激活;LHb-DBS能显著提高海马CA1区椎体细胞自发动作电位的频率以及宽度,增加L-VDCC的开放次数与时间,进一步证明了L-VDCC参与其抗抑郁的机制。
[Abstract]:[background] depression seriously affects human health and social development. The WHO estimates that depression will become the second largest disease in the world by 2020. At present, the treatment of depression is mainly drug therapy and psychotherapy, but only 1/3 of the patients have a satisfactory effect. The patients with depression are called treatment-resistant depression (TRD). For these patients, the treatment of Deep Brain Stimulation (DBS) is often used in this kind of patients. The lateral habenular nucleus (Lateral Habenula, LHb) is a nucleus connecting the basal ganglia and the marginal system and participates in the regulation of reward system. And the secretion of monoamine neurotransmitters. Deep brain stimulation of the lateral habenular nucleus (LHb-DBS) is one of the main ways to treat refractory depression, but its mechanism is still not clear.Malinow.R, Hongmei Meng and so on. DBS can improve depressive symptoms by regulating the discharge of LHb neurons and raising the release of monoamine neurotransmitters in the brain. The hypothesis of aminergic neurotransmitters is not consistent with the clinical characteristics of DBS in patients with monoamine drug insensitivity and the rapid onset of DBS, suggesting that LHb-DBS has other mechanisms for depression. The current study found that the rapid antidepressant effect was induced by the mammalian rapamycin site (mTOR, mammalian target of rapamycin). Deep brain stimulation in the lower marginal cortex can increase the level of mTOR phosphorylation, suggesting that LHb-DBS may be activated by activating mTOR to treat depression.Duman, such as the L type voltage dependent calcium channel (L-type voltage-dependent calcium channel, L-VDCC) blocking agent can effectively block the activation of mTO R and the rapid antidepressant effect. L-VDCC may also be involved in the process of depression in the treatment of LHb-DBS. Other studies have shown that DBS can induce long synaptic changes, and the activation of mTOR is often accompanied by synaptic changes such as the occurrence of tree spines and synaptic remodeling. Further hints that LHb-DBS may improve the mechanism of synaptic plasticity to improve the antidepressant effect by activating mTOR. Immunoblotting, behavioural and patch clamp techniques were used to reveal and explore the mechanism of mTOR activation and related synaptic plasticity in the treatment of depression in LHb-DBS. [Objective] to verify that activation of mTOR and improvement of nerve plasticity is an important way to treat depression in the treatment of LHb-DBS, whether L-VDCC is involved in the process of LHb-DBS treatment of depression, and to explore LHb- DBS provides a reliable experimental basis for the clinical application of DBS in the treatment of depression, and provides a theoretical basis for the development of new antidepressants for its mechanism. [Methods] the effects of LHb-DBS on the depressive behavior of LH rats refer to the methods of Daniela Schulz, and establish the acquired helplessness of rats (LH, learned he) Lplessness) depression model. Observe the behavior difference between LH rats and normal rats, evaluate the feasibility of the LH model, and observe the effect of LHb-DBS on the depressive behavior of the Acquired Helplessness rats by the deep brain stimulation of the lateral habenular nucleus in LH rats. At the same time, a L-VDCC blocker (nifedipine, nifedipine) is injected into the lateral ventricle, and the anti depressive effect of LHb-DBS on LHb-DBS is observed. The effects of.LHb-DBS on the phosphorylation and expression of mTOR in the rat model of LH depression were established by using the methods of Daniela Schulz and so on. Through the operation, the experimental group was placed on the electrode at LHb, the deep brain stimulation was the experimental group, the deep brain stimulation was the positive control group, and the normal mice without the LH model were negative control group. Through Western-blot, the lateral habenon was observed. Changes in the expression of phosphorylation of mTOR in the hippocampus after deep brain stimulation. The L-VDCC blocker (nifedipine, nifedipine) was injected into the lateral ventricle to observe the changes in the regulation of mTOR phosphorylation in the deep brain stimulation of the lateral habenular nucleus. The effects of the synaptic plasticity on the coronary position of the acute LH rats were observed by the brain slices simulated deep brain stimulation. The brain slices were incubated in the artificial cerebrospinal fluid. In vitro simulated LHb-DBS technique (detailed method) was used to stimulate the LHb. The excitatory postsynaptic potential (Shaffer collateral-CA1 field excitatory postsynaptic potentials) was recorded in the CA1 stratum radiatum (SR) subregion of the hippocampus (Shaffer collateral-CA1 field excitatory postsynaptic potentials), and the lateral habenular nucleus was observed. The effects of irritation on SC-CA1 fEPSP, 20uM nifedipine perfusion brain slices were given to observe the effect of the external habenular nucleus stimulation on the changes of SC-CA1 fEPSP. The spontaneous discharge of the vertebral neurons in the hippocampal CA1 region was recorded and the mechanism of LHb-DBS activation of L-VDCC in the hippocampus of the lateral habenular nucleus was given, and the mechanism of LHb-DBS activation of L-VDCC was discussed. LHb-DBS can significantly improve the depressive behavior of LH rats, while nifedipine can effectively block the antidepressant effect of the deep brain stimulation of the lateral habenular nucleus, suggesting that L-VDCC participates in the antidepressant mechanism of LHb-DBS. LHb-DBS can activate hippocampus mTOR and cause the long time course of SC-CA1 fEPSP in the hippocampus. This phenomenon can also be blocked by nifedipine, proving LHb-DB S can improve the synaptic plasticity to achieve antidepressant effect by activating mTOR, and this mechanism depends on the activation of L-VDCC. LHb-DBS can significantly increase the frequency and width of spontaneous action potential of the vertebral body cells in the CA1 region of the hippocampus, increase the opening times and time of L-VDCC, and further prove that L-VDCC is involved in its antidepressant mechanism.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.4

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