癫痫脑片中及左乙拉西坦作用下多巴胺能神经元电生理特性的研究

发布时间：2018-01-16 02:12

本文关键词：癫痫脑片中及左乙拉西坦作用下多巴胺能神经元电生理特性的研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:癫痫相关的精神障碍(psychosis of epilepsy,POE)是指癫痫患者伴发精神障碍,如焦躁、抑郁、易激惹等症状,其在癫痫患者中的发病率要远远高于普通人群。近年来也越来越受到社会关注。既往研究表明,POE与癫痫状态下中脑边缘多巴胺系统中多巴胺(dopamine,DA)神经元超敏性有关。中脑腹侧被盖区(ventral tegmental area,VTA)是DA能神经元聚集的地方,参与自然奖赏与厌恶、药物成瘾以及一些精神疾病等。另外癫痫动物模型在体胞外记录发现癫痫状态下DA能神经元自发放电增多,而且可能是由腹侧下脚钩回-伏隔核-腹侧苍白球-VTA通路介导,但具体的机制尚不清楚。除了癫痫发作,服用抗癫痫药物(antiepileptic drugs,AEDs)也可以出现POE,临床常见的是左乙拉西坦(levetiracetam,LEV)。它是一种新型广谱抗癫痫药,主要作用于中枢神经系统中的突触囊泡蛋白2A(synaptic vesicle protein2A,SV2A),但临床上部分癫痫患者在服用LEV后会出现精神心理变化。而关于LEV的精神方面副作用,据报道与DA含量有关。但LEV对DA能神经元电生理的作用既往罕见报道。研究目的:本研究利用膜片钳技术,记录癫痫脑片模型中及加入LEV后VTA区DA能神经元自发放电、细胞内在兴奋性以及外部兴奋与抑制性突触输入等的改变来探索癫痫发作以及抗癫痫药物LEV导致POE的机制,为寻找新的治疗靶点提供新的思路和方向。研究方法:1.选用TH-GFP转基因型小鼠制备离体皮层-中脑VTA脑片。同时利用无镁离子人工脑脊液(artificial cerebrospinal fluid,ACSF)孵育脑片诱导皮层出现癫痫样放电制作离体癫痫模型(epileptic model,EP-model)。2.利用细胞贴附式、全细胞式膜片钳技术记录正常状态下VTA区神经元自发动作电位以及激发动作电位的特点,并通过Avidin以及免疫组化染色识别VTA区神经元的种类。同时将DA能神经元放电模式进行分类。3.癫痫脑片模型中,DA能神经元自发放电频率的改变以及与内在兴奋性的关系。记录正常状态下以及EP-model中,DA能神经元自发放电频率、膜输入阻抗、细胞膜电容、动作电位阈值、动作电位半宽、后超级化电位、基强度、导数最大值、导数最小值、发放频率曲线等的改变。4.癫痫脑片模型中,DA能神经元自发放电频率的改变与外在突触输入的关系。利用加入铯离子(Cs+)和QX-314的电极内液记录正常以及EP-model下DA能神经元自发兴奋性输入突触后电流(spontaneous excitatory postsynaptic currents,s EPSC)和抑制性突触后电流(spontaneous inhibitory postsynaptic currents,s IPSC),并分析其反应-反应间隔(inter-event interval IEI)、频率以及幅度的改变。给予局部胞外金属电极刺激,记录正常以及EP-model下DA能神经元激发后兴奋性突触后电流(evoked excitatory postsynaptic currents,e EPSC)和激发后抑制性突触后电流(evoked inhibitory postsynaptic currents,e IPSC),通过计算成对脉冲比(paired-pulse-ratio,PPR)了解癫痫状态下突触前递质释放概率的改变。5.癫痫脑片模型下,DA能神经元兴奋性改变与外部突触输入的关系的进一步验证。利用细胞贴附式技术记录正常ACSF、EP-model、EP-model加入兴奋性突触受体阻断剂CNQX(AMPA受体拮抗剂)和AP5(NMDA受体拮抗剂)后自发动作电位的改变进一步验证兴奋性突触输入在癫痫状态下对DA能神经元的作用。6.LEV对DA能神经元外部突触输入的作用。利用含有加入Cs+和QX-314的电极内液,分别记录正常ACSF以及100μM、200μM、400μM浓度LEV灌流液下DA能神经元s EPSC、s IPSC的IEI、频率及幅度的改变。另外通过加入TTX(电压依赖钠通道阻断剂)+PTX(GABAA受体阻断剂)+AP5记录正常及200μM LEV下的微小兴奋性突触后电流(minature excitatory postsynaptic currents,m EPSC)。通过加入TTX+CNQX+AP5记录正常及200μM LEV下微小抑制性突触后电流(minature inhibitory postsynaptic currents,m IPSC),并分析m EPSC和m IPSC的IEI、频率和幅度的改变。7.LEV对DA能神经元内在兴奋性的作用。利用全细胞式膜片钳技术,记录正常及200μM LEV灌流下DA能神经元内在兴奋性的改变,记录内容同方法3。8.LEV对DA能神经元自发放电的作用。利用细胞贴附式膜片钳技术,记录正常及LEV灌流下DA能神经元自发放电频率的改变。研究结果:1.TH-GFP小鼠中在中脑VTA区域,根据动作电位发放特点,结合免疫组化染色,其神经元主要可分为两大类,DA能神经元和GABA能神经元。前者有典型的起搏式放电特点,与GABA能神经元相比,其动作电位宽度相对较宽,发放频率相对较慢。根据其动作电位发放特点,可以分为三型,I型占37.5%;II型47.9%;III型14.6%。2.在癫痫脑片模型中,用贴附式膜片钳技术记录DA能神经元的自发放电频率显著增加,伴动作电位宽度显著变窄。全细胞膜片钳技术记录自发动作电位的结果与其一致。3.在癫痫脑片模型中,DA能神经元内在兴奋性显著增加,发放频率曲线在癫痫状态下左移,在0-280 p A下激发动作电位发放频率显著高于对照组。同时动作电位半宽变窄,动作电位导数最小值及面积均变小,而且有统计学差异。但膜输入阻抗及细胞膜电容未明显改变。4.在癫痫脑片模型中,DA能神经元s EPSC的IEI累积分布曲线左移伴频率显著增加,而s EPSC的幅度无明显改变。而s IPSC的频率和幅度未明显改变。利用胞外电刺激诱发出e EPSC和e IPSC,对比正常状态下发现e EPSC PPR和e IPSC PPR虽然没有统计学差异,但均有增加趋势。5.在癫痫脑片模型中,加入兴奋性谷氨酸受体拮抗剂CNQX和AP5之后,癫痫状态下增加的自发放电频率能够被抑制,而且介于正常及癫痫状态之间。6.加入不同浓度LEV后,发现在200μM LEV以及400μM LEV作用下,DA能神经元s EPSC IEI累积分布曲线较对照组左移,在400μM LEV下有统计学差异,但频率均值无统计学差异。另外s IPSC、m EPSC以及m IPSC的IEI、频率及幅度也未发生明显改变。7.LEV在一定范围内可以抑制DA能神经元的内在兴奋性。发放频率曲线在200μM LEV作用下右移,并在40-100 p A以及140、160 p A处激发动作电位发放频率显著低于对照组。同时伴细胞膜电容变大,细胞膜阻抗变小,且均有统计学差异。8.在200μM LEV及400μM LEV作用下DA能神经元的自发放电频率较对照组降低,但没有统计学差异。研究结论:1.利用无镁离子ACSF孵育30分钟可成功诱导皮层出现癫痫样放电。2.在TH-GFP品系小鼠中,可以根据动作电位发放特点及动作电位半宽来鉴定中脑VTA区域中DA能神经元。动作电位宽度大于1 ms的神经元为DA能神经元。根据斜坡的存在情况,DA能神经元的动作电位发放模式可分为三型。3.在癫痫脑片模型下,DA能神经元自发放电数目增多,与内在兴奋性的增加及外在接受的兴奋性突触后电流增加均有关系。而且癫痫状态下,DA能神经元动作电位半宽变窄,但突触前释放概率并不改变。4.LEV对DA能神经元的自发动作电位的频率没有明显调节作用,但是可以在一定范围内抑制DA能神经元的内在兴奋性。
[Abstract]:Background: mental disorders related to epilepsy (psychosis of, epilepsy, POE) refers to mental disorders, such as epilepsy patients with anxiety, depression, irritability and other symptoms, the incidence of epilepsy patients is much higher than the general population. In recent years, more and more social attention. Previous studies showed that dopamine the mesolimbic dopamine system and epilepsy in the state of POE (dopamine, DA) neurons hypersensitivity. The ventral tegmental area (ventral tegmental, area, VTA) is DA neurons gathering place, participate in natural reward and aversion, drug addiction and psychiatric problems. In addition the epilepsy animal model in vivo extracellular recording under the condition of DA can be found in epileptic spontaneous discharges of neurons increased, and may be caused by the ventral subiculum uncinate nucleus accumbens ventral pallidum mediated by -VTA pathway, but the mechanism is not clear. In addition to seizures, antiepileptic Drug (antiepileptic drugs, AEDs) can also appear POE, clinical common is levetiracetam (levetiracetam, LEV). It is a new broad-spectrum antiepileptic drug, the main role of synaptic vesicle in the central nervous system in the global 2A (synaptic vesicle protein protein2A, SV2A), but the clinical part of epilepsy patients will appear spirit psychological changes after taking LEV and LEV on the mental side effects, according to reports related with DA content. But LEV neurons function rarely reported on DA. Previous research objective: This study using patch clamp technique, recorded epileptic brain slices in the model after accession to the LEV and VTA DA to the spontaneous discharge of neurons cells, the intrinsic excitability and external excitatory and inhibitory synaptic input to explore the change of epilepsy and the anti epilepsy drug LEV to POE mechanism, provides new ideas and direction for the search for new treatment targets. Research methods: 1. selected TH-GFP transgenic mice were prepared in vitro midbrain cortex brain slices of VTA. At the same time using magnesium free ACSF (artificial cerebrospinal, fluid, ACSF) incubating brain slices induced cortex epileptiform discharges in vitro model of epilepsy (epileptic model, EP-model.2.) by using cell attached, whole cell patch clamp technique to record characteristics of VTA neurons in the normal state of spontaneous action potentials and excitation action potentials, and by Avidin and immunohistochemical staining of neurons in VTA region of species identification. At the same time, DA can classify the firing pattern of neurons.3. epileptic brain slice model, DA can change the spontaneous discharge rate of neurons and inner excitement the record under normal condition and EP-model, DA neurons spontaneous discharge frequency, membrane input impedance, membrane capacitance, action potential threshold, action potential half width, Afterhyperpolarization, medium intensity, the maximum of the derivative derivative, the minimum value, the change of firing frequency curve.4. epileptic brain slice model, DA can change the spontaneous discharge rate of neurons and synaptic input. By adding external cesium ion (Cs+) electrode and QX-314 fluid in the normal and EP-model DA record neurons spontaneous excitatory postsynaptic current input (spontaneous excitatory postsynaptic currents, s EPSC) and inhibitory postsynaptic currents (spontaneous inhibitory postsynaptic currents, s IPSC), and to analyze the response response interval (inter-event interval IEI), the frequency and amplitude changes. Local cell stimulation and recording of normal metal electrode. EP-model DA neurons excited by excitatory postsynaptic currents (evoked excitatory postsynaptic currents, e EPSC) inhibitory postsynaptic currents and excitation (evoked inhibit Ory postsynaptic currents, e IPSC), by calculating the paired pulse ratio (paired-pulse-ratio, PPR) of epileptic state presynaptic neurotransmitter release probability change.5. epilepsy brain slice model, DA relationship between neuronal excitability changes and external synaptic input to further verify. Using cell attached recording technique of normal ACSF, EP-model. EP-model joined the excitatory synaptic receptor antagonist CNQX (AMPA receptor antagonist) and AP5 (NMDA receptor antagonist) after spontaneous action potential changes to further verify the role of excitatory synaptic inputs to.6.LEV neurons of DA in epileptic state neurons outside the synaptic input effect on DA. By adding Cs+ and QX-314 containing electrode in the liquid, were recorded in normal ACSF and 100 M, 200 M, 400 M concentration of LEV perfusate DA neurons s EPSC, IPSC s IEI, the frequency and amplitude changes. In addition by adding TT X (voltage dependent sodium channel blocker +PTX (GABAA) receptor antagonist) +AP5 record normal and 200 M LEV miniature excitatory postsynaptic currents (minature excitatory postsynaptic currents, m EPSC). By adding the TTX+CNQX+AP5 record and 200 M LEV under normal miniature inhibitory postsynaptic currents (minature inhibitory postsynaptic currents m, IPSC, EPSC and m) and analysis of M IPSC IEI, frequency and amplitude changes of.7.LEV neurons intrinsic excitatory effect on DA. By using the whole cell patch clamp technique, log normal and 200 M LEV perfusion DA neurons intrinsic excitability changes, with the method of DA 3.8.LEV records effect of spontaneous discharges of neurons. Using cell attached patch clamp technique, normal and LEV perfusion DA can record the spontaneous discharge rate of neurons change. Results: in VTA region in 1.TH-GFP mice, according to the dynamic Potential distribution characteristics, combined with immunohistochemical staining, the neurons can be divided into two categories, DA neurons and GABA neurons. The former has the typical characteristics of pacing type discharge, compared with GABA neurons, the action potential width is relatively wide, relatively slow release frequency. According to the characteristics of action potentials. Can be divided into three types, I type accounted for 37.5%; II 47.9%; III 14.6%.2. in epileptic brain slice model, with the spontaneous discharge frequency attached patch recording technique of DA neurons increased significantly, with action potential width is significantly narrowed. The whole cell patch clamp technique to record the spontaneous action potential in agreement with the.3. in the epileptic brain slice model, DA significantly increased the intrinsic excitability of neurons, firing frequency curve in epileptic state left, spike frequency was significantly higher than the control group at 0-280 P excitation A. At the same time the half width of the action potential Narrow, the minimum value and derivative action potential area was smaller, but there were significant differences. But the membrane input resistance and membrane capacitance of.4. did not change significantly in epileptic brain slice model, s EPSC DA neurons IEI cumulative distribution curve to the left with a significant increase in the frequency, and no significant changes in the amplitude of EPSC and S. The frequency and amplitude of s IPSC did not change significantly. The extracellular evoked by electrical stimulation of E EPSC and E IPSC, compared with normal state EPSC PPR and E IPSC e PPR although not statistically significant, but the increasing trend of.5. in the epileptic brain slice model, after adding excitatory glutamate receptor antagonists CNQX and AP5. Spontaneous discharge increased frequency of epileptic state can be reduced, and between normal and epileptic.6. with different concentrations of LEV, found in 200 M LEV and 400 M LEV, s EPSC IEI DA neurons cumulative distribution curve Compared with the control group, there were significant differences in the left, 400 M LEV, but no significant difference between the mean frequency. In addition, s IPSC, m EPSC and m IPSC IEI, frequency and amplitude are not significantly change the intrinsic excitability of.7.LEV can inhibit DA neurons in a certain range of firing frequency curve to the right in the 200. M LEV and 40-100 P under A P and 140160 A excitation spike frequency was significantly lower than the control group. At the same time with the membrane capacitance change, cell membrane impedance becomes small, and there were significant differences in.8. at 200 M LEV and 400 M LEV function DA neural element self release the electric frequency is lower than control group, but the difference was not statistically significant. Conclusions: 1. the magnesium ion ACSF were incubated for 30 minutes can successfully induce cortex epileptiform discharges in.2. TH-GFP mice, according to the characteristics of action potentials and action potentials in half width Set in the VTA region of midbrain DA neurons. The action potential width of more than 1 ms neurons DA neurons. According to the existing situation of the slope can be divided into three types of.3. in epileptic brain slice model under the action potential firing pattern of DA neurons, DA neurons firing number, current increased between acceptance and the increase in intrinsic excitability and extrinsic excitatory postsynaptic and epileptic state, DA neurons action potential half width narrowed, but does not change the probability of presynaptic release of.4.LEV spontaneous action potentials of the DA neurons frequency has no obvious regulating effect, but can inhibit the intrinsic excitability of DA neurons in a certain range.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.1

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